Mutations of myelodysplastic syndromes (MDS): An update

Accepted Manuscript Title: Mutations of myelodysplastic syndromes (MDS): An update Author: Bani Bandana Ganguly N.N. Kadam PII: DOI: Reference:

S1383-5742(16)30004-7 http://dx.doi.org/doi:10.1016/j.mrrev.2016.04.009 MUTREV 8164

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Mutation Research

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Please cite this article as: Bani Bandana Ganguly, N.N.Kadam, Mutations of myelodysplastic syndromes (MDS): An update, Mutation Research-Reviews in Mutation Research http://dx.doi.org/10.1016/j.mrrev.2016.04.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mutations of myelodysplastic syndromes (MDS): An update up-todate

Bani Bandana Ganguly1, NN Kadam2

1

MGM Center for Genetic Research & Diagnosis

MGM New Bombay Hospital, New Mumbai, India 2

MGM Medical College and Hospital, Navi Mumbai, India

Correspondence: Bani B. Ganguly, Ph.D, FICMCH MGM Center for Genetic Research & Diagnosis MGM New Bombay Hospital Vashi Plot 35, Sector 3, New Mumbai 400703, India Tel: 91 22 61526527, 91 9869214680 Fax: 91 22 27824618, 91 22 27820520 E-mail: [email protected], [email protected]

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Abstract:

The plethora of knowledge gained on myelodysplastic syndromes (MDS), a heterogeneous premalignant disorder of hematopoietic stem cells, through sequencing of several pathway genes has unveiled molecular pathogenesis and its progression to AML. Evolution of phenotypic classification and risk-stratification based on peripheral cytopenias and blast count has moved to five-tier risk-groups solely concerning chromosomal aberrations. Increased frequency of complex abnormalities, which is associated with genetic instability, defines the subgroup of worst prognosis in MDS. However, the independent effect of monosomal karyotype remains controversial. Recent discoveries on mutations in RNA-splicing machinery (SF3B1, SRSF2, ZRSR2, U2AF1, U2AF2); DNA methylation (TET2, DNMT3A, IDH1/2); chromatin modification (ASXL1, EZH2); transcription factor (TP53, RUNX1); signal transduction/kinases (FLT3, JAK2); RAS pathway (KRAS, NRAS, CBL, NF1, PTPN11); cohesin complex (STAG2, CTCF, SMC1A, RAD21); DNA repair (ATM, BRCC3, DLRE1C, FANCL); and other pathway genes have given insights into the independent effects and interaction of co-occurrence of mutations on diseasephenotype. RNA-splicing and DNA methylation mutations appeared to occur early and are reported as ‘founder’ mutations in over 50% MDS patients. TET2 mutation, through altered DNA methylation, has been found to have independent prognostic response to hypomethylating agents. Moreover, presence of DNMT3A, TET2 and ASXL1 mutations in normal elderly individuals forms the basis of understanding that accumulation of somatic mutations may not cause direct disease-development; however, cooperation with other mutations in the genes that are frequently mutated in myeloid and other hematopoietic cancers might result in clonal expansion through self-renewal and/or proliferation of hematopoietic stem cells. Identification of small molecules as inhibitors of epigenetic mutations has opened avenues for tailoring targeted drug development. The recommendations of a Clinical Advisory Committee is being considered by WHO for a revised classification of risk-groups of MDS, which is likely to be published in mid 2016, based on the new developments and discoveries of gene mutations.

Keywords: Myelodysplastic syndromes, mutations in pathway genes, complex chromosomal aberration, monosomal karyotype, EZH2/IDH inhibitors as drug targets

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1. Introduction

Myelodysplastic syndromes (MDS) represent heterogeneous clonal pre-malignant hematopoietic changes that are typically associated with peripheral blood cytopenia, ineffective hematopoiesis, hypercellular bone marrow with morphologically defined dysplasia of one or more lineages and increased risk of progression to acute myeloid leukemia (AML) [1, 2]. Among the chromosomal rearrangements, del(3q/5q/7q/11q/12p/20q), monosomy 5/7, trisomy 8/19, i(17q), -Y, have been frequently detected in MDS with variable frequencies [3-8]. A number of chromosomal anomalies indicate a presumptive diagnosis of MDS in the absence of morphological dysplasia, including del(5q), -7/del(7q), del(9q), -13/del(13q), del(11q)/t(11q), del(12p)/t(12p), 17/del(17p)/i(17q), +19/t(19) and idic(Xq13); whereas, +8, del(20q) and -Y are not considered as presumptive evidence of MDS because of their global presence in other myeloid neoplasms. Clinical evolution of MDS is characterized by acquisition of multiple genetic changes and subsequent development of clonal selection and subclonal development. Therapeutic response and karyotypic evolution of chromosomal rearrangements are taken into account for classification and risk assessment of progression of MDS [9]. However, normal chromosomal pattern in over 30% de novo MDS and 50% therapy related MDS (t-MDS) resulted in silent progression of MDS to AML in different age groups. The advent of modern technology has facilitated genome-wide analysis of genetic rearrangements and identified driver mutations such as RNA splicing, DNA methylation, chromatin modification, transcription regulation, DNA repair, signal transduction and cohesin complex genes in MDS with or without associated cytogenetic abnormalities.

Besides chromosomal alterations, sequencing of whole genome/exome and targeted deepsequencing have identified a landscape of mutated genes that encode signal transduction proteins (NRAS at 1p13, FLT3-ITD at 13q12, CBL at 11q23, JAK2 at 9p24, KIT at 4q12), transcription factors (RUNX1 at 21q22, TP53 at 17p13, ETV6 at 12p13), tumor suppressors (TP53, WT1), epigenetic modifiers (TET2 at 4q24, ASXL1 at 20q11, IDH1 at 2q34, IDH2 at 15q26, EZH2 at 7q36, DNMT3A at 2p23), RNA splicing machinery (SF3B1 at 2q33, U2AF1 at 21q22, SRSF2 at 17q25, ZRSR2 at Xp22) and components of the cohesin complex (STAG2, RAD21, SMC3, SMC1A) [10-14]. However, no specific mutation has been detected in ~20% of MDS patients. 3

Despite low mutation frequency and inter-patient variation, some of the mutations have demonstrated independent expression and prognostic value such as SF3B1 mutation with ring sideroblasts; reduced TET2 activity in stem and progenitor cells results in an increase in DNA methylation and contributes to clonal dominance and also augments the transformation to AML; DNMT3A+ve MDS patients reveal lower overall survival (OS) and faster progression to AML; association of TET2 mutation with an increased overall response to hypomethylating agent (HMA) 5-Azacitidine (AZA) and so on [15-18]. A diverse group of molecular tests, including array-CGH (aCGH), array-SNP (SNP-array), multiple ligation dependent probe amplification (MLPA), Next generation sequencing (NGS), etc. during the last decade have enabled molecular understanding of MDS with special emphasis on targeted therapy depending on the type of mutations present. However, though such investigations have identified mutations in several mechanistic pathways, they require a large data base for understanding the patho-mechanism and tailoring therapy for specific mutations accordingly. Lack of clinical validation further limits adoption of sequencing of these identified genes into real-time clinical practice. Kosmider et al. [19] have observed that TET2 mutations appear as a frequent molecular event in MDS and are an independent predictor of good prognosis leading to a longer OS than unmutated patients. The group has further postulated that TET2 mutations are observed during the early steps of the disease [20], and might influence the phenotype and clinical behavior of the disease. In vitro studies using shRNA-based approaches have suggested a role of TET2 in regulating myeloid differentiation [21, 22], and stem/progenitor cell proliferation [21]. The shRNAmediated knockdown led to stable reductions in TET2 expression by 50–70%. Characterization of conditional deletion of Tet2 in the hematopoietic compartment demonstrated a role of Tet2 in regulating hematopoietic stem cell renewal and differentiation at multiple stages. It has been elucidated that loss of Tet2, even through loss of a single allele, leads to progressive defects in hematopoiesis with increased self-renewal and myeloid commitment for transformation, and provides insight into how mutations in epigenetic modifiers contribute to malignancy [23]. In particular, 41.5% of TET2 mutations are reported in the first coding exon of TET2 (exon 3), including biallelic or monoallelic loss in leukemia patients [24].

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The present review will comprehensively address the landscape of molecular mutations of several pathway genes involved in leukemogenesis of MDS, and their clinical implication in prognosis and treatment outcome as independent effect or through biological interaction in cooccurrence and mutual exclusivity. The review has also addressed the inhibitory characteristics of small molecules in epigenetic modification as possible drug targets.

2. Genetic Mutations in MDS

The rapid technological development for understanding the genetic mutations over the last decade has changed the knowledge of MDS-pathogenesis and prognosis primarily based on the discovery of higher incidence of genetic abnormalities in MDS. Several DNA-methylation profiling studies have confirmed abnormal epigenome in MDS etiology [25, 26]. Complex chromosome aberrations (CK) detected by conventional cytogenetics, including monosomal karyotype (MK), also continue to be important in risk-stratification of MDS. Application of high throughput technologies such as array-based comparative genomic hybridization and single nucleotide polymorphism, and Next Generation Sequencing, including targeted deep-sequencing, have been established for collection of mutations involved in initiation of MDS to leukemic transformation.

Recent studies on 111 genes in 439 and 738 MDS patients by Bejar et al. [27] and Papaemmanuil et al. [28] respectively, and 104 genes in 944 patients by Haferlach et al. [10], by employing high throughput Next Generation Sequencing, have unveiled the landscape of gene mutations in initiation of MDS and its evolution to AML. A number of random non-pathogenic acquired mutations (‘passengers’) in hematopoietic stem cells have been described before acquisition of the initiating non-random ‘driver’ mutations involved in pathogenesis of MDS [29]. Till present, driver mutations in over 40 genes have been documented in MDS, and most importantly, at least one mutation with a median of two or three mutations are detected per patient in approximately 90% of MDS patients [10, 28, 30]. Functionally, these mutations have been categorized into 10 subgroups depending on their prevalence, including RNA-splicing machinery (64%): SF3B1, SRSF2, ZRSR2, U2AF1, U2AF2; DNA methylation (45%): TET2, DNMT3A, IDH1/2; chromatin 5

modification (27%): ASXL1, EZH2; transcription factor (15%): TP53, RUNX1, ETV1, GATA2; signal transduction/kinases (15%): FLT3, JAK2, MPL, GNAS, KIT; RAS pathway (12%): KRAS, NRAS, CBL, NF1, PTPN11; cohesin complex (13%): STAG2, CTCF, SMC1A, RAD21; DNA repair (10%): ATM, BRCC3, DLRE1C, FANCL; other mutation (10%): and no mutation (10%) [10, 31]. Among these, mutations in splicing machinery components were frequently described at least one mutation in over 50% MDS, indicating its involvement in MDS-pathogenesis. The landscape of molecular mutations is presented in Table 1.

2.1. Spliceosomal mutations in MDS

Spliceosome, a protein complex, is involved in removal of introns from pre-mRNA to generate a mature mRNA. Splicing factor somatic mutations, including SF3B1, U2AF1 (U2AF35), SRSF2, ZRSR2, PRPF40B, SF1, SF3A1 and U2AF2, have been identified through massive parallel sequencing as extremely common (45-85%) class of mutated genes in MDS that occur in a mutually exclusive manner at 3’splice site of mRNA-processing, and are predominantly heterozygous, missense and largely located in restricted regions of proteins [30, 32]. These mutations in mRNA-splicing machinery are also called MDS-specific mutations because of their rarity in AML and MPN, and the resultant differential cellular functions lead to manifestation of unique MDS-phenotype. Mutations in other splicing factors such as PRPF40B, SF3A1, SF1, and U2AF2 (U2AF65) have also been documented less frequently in 1-2% MDS. Spliceosome mutations could alter the intron-exon borders of the select transcribed genes by inappropriate incorporation of introns or exons into the specific RNA, which could further alter the ability of the cell to differentiate into fully mature blood cells giving rise to dysplastic phenotype [33]. Mutations in multiple spliceosomes and their mutual exclusivity could describe that MDS is driven by alterations in mRNA splicing machinery. The presence of mutational hot spots and absence of frameshift or nonsense mutations could be correlated with gain-of-function or change-of-function of splicing factor genes leading to MDS-pathogenesis. Extensive investigation on elucidation of the exact mechanistic significance of the mutant proteins, gain or loss of function and their downstream expression target new therapeutic agents in MDSmanagement. Frequency and specificity of spliceosomal mutations/dysfunction in MDS reveal a new leukemogenic pathway. 6

The spliceosomal mutations and their prognostic implications have been demonstrated with conflicting reports. While SF3B1 mutations have shown independent favorable outcome in most MDS, U2AF1 mutations have shown either insignificant impact or poorer survival in different studies [34, 35]. Spliceosome mutations, though mutually exclusive with one another, co-occur with mutations of specific epigenetic modifiers in MDS, indicating co-operations of these mutations result in specific MDS phenotype [34, 36]. Mutations in SRSF2 are concomitantly present with RUNX1, IDH2, ASXL1, TET2 and STAG2, where co-mutation of TET2 and SRSF2 was frequently predictive of a leukemic transformation of MDS, CMML and other monocytosis [37]. Co-occurrence of ZRSR2 with DNA methylation modifier TET2, SF3B1 with mutation of the methyltransferase DNMT3A, U2AF1 with ASXL1 or TET2 has been noted [28, 38].

2.1.1. SF3B1 Papaemmanuil et al. [32] and Yoshida et al. [30] have demonstrated SF3B1 mutation in low-risk RARS-MDS using whole exome sequencing technique. Despite the mutual exclusivity of mutations in spliceosomal members, mutations in SF3B1 appeared to be markedly high in RARS-MDS with an incidence of 60%-80% of these patients, and 10-20% of unselected MDS. Multiple studies have described that SF3B1-mutated RARS patients, a relatively favorable subset of MDS, may have better outcome compared with their wild-type partners [27, 39]. Heterozygous SF3B1 mutations are clustered at amino acids 622-700, especially K700E [35]. Haploinsufficiency of SF3B1 as the mode of action of SF3B1 in MDS has been suggested based on ring sideroblast evidenced in heterozygous Sf3b1 knockout mouse [40]; however, other studies could not find this association [41, 42], and finally there was no MDS in these haploinsufficient mice.

Recently, Savage et al. [43] have identified spliceosomal proteins, including SF3B1 and PRP8, and BCLAF1 in a DNA damage-induced BRCA1 protein complex. The protein complex regulates pre-mRNA splicing of genes involved in DNA damage signaling and repair, and thus the altered activity of this complex results in impaired DNA repair and genetic instability. In MDS patients with SF3B1 mutations, such impaired function of this complex is demonstrated to be associated with possible downstream effects of its DNA repair potential [43, 44].

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2.1.2. SRSF2 and ZRSR2

Mutations in SRSF2 (12-15%) and ZRSR2 (3-11%) were reported in other d-MDS phenotypes. SRSF2 mutation is more common in CMML [30, 38, 45]. In SRSF2, heterozygous mutations occur exclusively at P95 for forming extensive contact with the target RNA through stacking [35]. Higher risk of SRSF2 mutations in MDS predicted worsened OS indicating role of SRSF2 in AML transformation [27]. SRSF2 mutations are associated with neutropenia and pronounced thrombocytopenia. Mutations of ZRSR2, reported in 3-11% of MDS with isolated neutropenia, are widely distributed along the entire protein without a specific location [30].

2.1.3. U2AF1 U2AF1 (U2AF35) mutations, documented in 5-12% of MDS, result in downregulation of many genes, including splicing and RNA recognition motif (RRM) genes following alterations of two amino acid residues such as S34 in the zinc finger 1 domain or Q157 in the zinc finger 2 domain [35]. U2AF1 mutations induce global abnormalities in RNA splicing, resulting in elevation of transcripts with unspliced intronic sequences and reduced cellular proliferation, or influence splicing and exon skipping [30, 46]. Co-mutation of U2AF1 and ASXL1 has been reported in MDS [10].

2.1.4. PRPF8 Recently, somatic mutations and deletions on the other splicing gene PRPF8 have been reported in MDS and AML [10, 47]. This gene encodes the largest and the most evolutionarily conserved protein of the spliceosome [34]. PRPF8 mutations and deletions were observed in 1-4% of MDS, and associated with ring sideroblast phenotype. The association of ring sideroblast phenotype with PRPF8 and SF3B1 suggests a common pathogenetic mechanism in MDS development. Cells carrying PRPF8 mutations are also responsible for abnormal splicing of several transcripts involved in hematopoiesis and iron metabolism in mitochondria [47].

2.2. Epigenetic alterations

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Deregulation of CpG island methylation within gene promoters is a major epigenetic transcriptional control mechanism in human solid tissue and hematologic tumorigenesis [48-50]. Alterations in DNA methylation patterns affect CpG islands, and inter- and intragenic regions, especially enhancers. Such epigenetic changes contribute to altered gene expression in the absence of sequence mutations in genomic DNA. Several regulatory genes of DNA methylation (DNMT3A, IDH1/2, and TET2) are frequently mutated in MDS. In MDS, epigenetic alterations are frequently reported causing hypermethylation of genes that control proliferation, adhesion and disease-specific changes in hematopoietic stem cells (HSC). Aberrant silencing of tumor suppressor and DNA-repair genes through hypermethylation of promoter-associated CpG islands have been frequently reported in MDS [51, 52]. The facility of genome-wide epigenetic studies have connected methylation status with response to DNAmethyltransferase (DNMT) inhibitors in MDS with marked improvement of hematopoiesis and overall survival, and delay in AML transformation, when treated with hypomethylating agents viz. 5-azacytidine (AZA) and decitabine (DEC) [16, 53, 54]. However, conflicting reports on aberrant epigenetic profiles and onset of MDS raises possibility of other pathogenic mechanisms. In low-risk MDS, 6% of analyzed genes involved in a series of functions, including DNA-repair, cell cycle control, and regulation of development, differentiation and apoptosis, display promoter methylation in contrast to 12% in high-risk group. Hypermethylation of ALOX12, GSTM1, HIC1, FZD9 and HS3ST2 was present in 70% of high risk MDS patients [26]. In the DNA-methylation machinery, the role of DNMT family (DNMT3A, DNMT3B and DNMT1), Ten-Eleven Translocation (TET) proteins (TET2, TET2, TET3) and Isocitrate Dehydrogenase (IDH1/IDH2) are mutually exclusive in MDS and results in methylation at 5’position of cytosine nucleotides in a context of CpG dinucleotide to 5-mthyl cytosine and 5-hydroxyl methylcytosine [14]. Mutations in DNMT3A (3-13%), TET2 (15-27%) and IDH1/2 (4-12%) have been considered as ‘drivers’ in pathogenesis of MDS causing demethylation and thus, have potential as therapeutic markers [27, 24, 55, 56 ].

2.2.1. DNMT3A (2p23) DNMT3A is a methyltransferase enzyme that catalyzes the transfer of methyl groups to cytosines in CpG dinucleotides of DNA. Hence, DNMT3A is an important component for efficient maintenance of methylation of active chromosome domains [57]. DNMT3A mutation 9

occurs at low frequency in MDS with preponderance at older age. These mutations are heterozygous and mostly missense or nonsense that cluster at the methyltransferase (MTase) domain. The mutation at arginine 882 (R882) results in disruption of methyltransferase domain leading to reduced enzyme activity. The presence of DNMT3A mutations in nearly all bone marrow cells of MDS patients, irrespective of blast count, has led to hypothesize that this is an early genetic event in disease-initiation process that may result in a clonal advantage of cells harboring this mutation [58]. Challen et al. [59] have demonstrated that a conditional silencing of DNMT3A in the hematopoietic system led to progressive impairment of hematopoietic stem cell differentiation with simultaneous expansion of stem cell compartment, and thus, plays crucial role in silencing of regulatory genes. DNMT3A mutations are frequent in AML and rare in MDS (3-13%) with no specific association with any cytogenetic subgroup, but are found to correlate with poor survival rate and a faster AML-transformation [55, 60-63].

2.2.2. TET2 (4q24) TET proteins are Fe(II)- and 𝛼–ketoglutarate (𝛼KG) dependant oxygenase enzymes that catalyze hydroxylation of 5-methylcytosine (5mC) to hydroxymethylcytosine (5hmC) on DNA leading to loss of DNA methylation, and thus play major roles in cytosine methylation and epigenetic gene regulation [22]. Its knockdown effects result in loss of 5hmC with a possible concomitant increase in 5-mC. TET2 is a crucial regulator of HSC-homeostasis [64]. TET2 mutations in MDS are thought to result in loss of catalytic activity. Abnormalities in TET2 with impaired TET function are observed in 15-27% MDS, including deletions, loss of heterozygosity and missense/nonsense/frameshift mutations leading to impaired DNA demethylation [65, 66]. Conditional or unconditional TET2 deletion results in leucocytosis due to monocytosis and marked splenomegaly at ~3 months latency. Somatic TET2 mutations in MDS are associated with advanced age with clonal hematopoiesis, and normal karyotype suggesting that TET2 mutation is an aging-associated factor of hematopoietic cells or an initiating mutation [60, 67]. Associated risk of TET2 mutation and its clinical impact and patient survival are matters of debate [19, 60]. Mutations in TET2 are reported independently associated with shorter OS in MDS patients after HSC transplantation [68]. Reduced TET2 activity due to mutation in HSC and its progenitor cells contribute to self10

renewal and clonal dominance in HSC, and also augments AML transformation [23]. Tet2deficient mice developed myeloid malignancies, resembling characteristics of human MDS and CMML, due to increased HSC self-renewal and altered cell differentiation skewing toward monocytic/granulocytic lineages [69, 70]. Reduced TET2 expression in progenitors, erythroid precursors and granulocytes of MDS patients has been reported with no detectable TET2 mutations, which suggests alternative mechanisms of TET2 regulation, and highlights the physiological importance of putative tumor suppressor function of TET2 protein [20]. Collectively, these data indicate that mutations that impair 5-hydroxymethylation represent a novel mechanism of transformation in myeloid malignancies. In addition, it is possible that therapies that modulate hydroxymethylation levels might be of benefit in malignancies characterized by loss of TET enzyme function by inhibiting malignant stem cell self-renewal [23, 68].

The French MDS study group demonstrated the association of TET2 mutations with an increased overall response to AZA treatment; however, this response was not associated with responseduration and OS [71]. It has been established that mutations in the metabolic enzymes IDH1 and IDH2 are mutually exclusive with TET2 mutations [72], and that production of 2hydroxyglutarate by neomorphic IDH1/2 mutant proteins inhibits TET2 catalytic activity [21, 7274]. In summary, the published data demonstrates TET2 as a master regulator of normal and malignant hematopoiesis. It is hypothesized that TET2 may have distinct roles in other hematopoietic lineages, and may contribute to transformation into lymphoid malignancies or even epithelial tumors. Dysregulation of hydroxymethylation by mutations in the TET family of enzymes and by other somatic mutations may contribute to malignant transformation in other contexts. However, subsequent studies are essential for identification of additional mutations in epigenetic modifiers, which contribute to neoplasia by similar mechanisms.

2.2.3. IDH1/2 (2q33.3/15q26.1)

Isocitrate dehydrogenase (IDH) catalyzes the conversion of isocitrate to alpha ketoglutarate (𝛼KG) in an NADP-dependant manner. Of the three isoforms of IDH such as IDH1 (located in 11

the cytoplasm), IDH2 (located in the mitochondria), and IDH3 (functions as part of the tricarboxylic acid (TCA) cycle), mutations occur in the active site of IDH1 at position R132, and at R140 and R172 in IDH2 [72]. The 𝛼KG metabolite, generated by IDH1/2, regulates the activity of TET2. Missense heterozygous mutations at R132 (IDH1) and R140 and R172 (IDH2) result in a change in substrate-specificity, thereby IDH1/2 enzymes are no longer capable of converting isocitrate into 𝛼KG, instead using 𝛼KG as a substrate and catalyze its conversion into beta-hydroxylglutarate (2-HG) in a reaction that consumes NADPH [72, 73]. 2-HG inhibits TET proteins directly and thus links these mutations in epigenetic machinery through hypermethylation of target genes. Thus, mutated IDH inhibits the hydroxylation of 5methylcytosine catalyzed by TET2, and expression of IDH2 mutation and repression of TET2 function result in impaired hematopoietic cell-differentiation in vitro with an increase in HSCmarker expression suggesting a shared pro-leukemogenic effect [21]. A marked elevation of the onco-metabolite 2-HG was reported in patients carrying IDH1/2 mutations that may directly inhibit TET2 function with a concomitant increase in HOXA expression [74]. Mutations in IDH1/2 are seen in 4-12% of MDS and 10-15% of AML with an unfavorable clinical outcome. The two heterozygous missense mutations are mutually exclusive to each other with TET2 mutations in AML. 2.3. Histone – modifying enzymes

Epigenetic regulation of posttranslational modifications of histone proteins at specific residues, including acetylation, methylation and ubiquitination is catalyzed by a group of histone modifying enzymes with distinct specificity, some of which have been observed with mutations in MDS (EZH2, ASXL1, UTX) [14, 49].

2.3.1. EZH2 (7q35-36) and PRC2

Enhancer of Zeste Homolog 2 (EZH2) is the main catalytic member of the Polycomb Repressive Complex 2 (PRC2) that also includes putative loss-of-function mutations in EED, SUZ12 and EZH1 (all at less than 5% frequency). EZH2 encodes a histone methyltransferase that is responsible for mono-, di- and trimethylation of lysine 27 of histone 3 (H3K27me1, me2, m3, 12

respectively). Histone modifications are associated with transcriptional repression, and recombinant enzyme with these point mutations possesses higher catalytic activity for the H3K27 methylation reaction resulting in a dramatic increase in H3K27 trimethylation (H3K27me3) in malignant cells. EZH2 also interacts with DNMT3A, affecting DNA-methylation via H3H27me3 for the involvement of DNMT proteins. Somatic loss-of-function mutations in EZH2 have been reported in 6-12% d-MDS with worsened OS [62], and it is considered to act as a tumor suppressor in myeloid malignancy [75, 76]. These mutations were associated with UPD7q and del7q, and reduced expression of EZH2 in CD34+ cells [77]. Truncation mutations were reported throughout the gene and missense mutations at the C-terminus SET domain and cysteine-rich domain that affects the binding of other components to PRC2. Disruption of SET domain, in turn, reduces the levels of H3K27me2/me3 [78]. Mutation at PRC2 complex (EED and SUZ12) reduces methyltransferase activity and demonstrates the importance of PRC2 activity in myeloid malignancies [79]. Bejar et al. [62] has shown that combining the Lower-Risk Prognostic Scoring System stratification and EZH2 mutation status identified a group of patients with lower-risk MDS with a worse-than-expected prognosis that may benefit from earlier initiation of disease-modifying therapy. Notably, EZH2 mutations are rarely seen in de novo AML, suggesting that these mutations may be important hallmark of disease progression in MDS. Mutations in BCOR/BCORL1, another polycomb complex component, are detected in MDS patients associated with an unfavorable outcome [10].

2.3.2. ASXL1 (20q11.1) Addition of Sex Combs Like 1 (ASXL1) encodes a chromatin-binding protein involved in epigenetic regulation in hematopoietic cells and plays a crucial role in recruiting PRC2 to specific loci [80, 81]. ASXL1 belongs to a 3-member family of enhancers of trithorax and polycomb proteins (ASXL1/2/3), which is involved in maintenance of activation and the silencing of development-related Hox genes through chromatin remodeling. Mutations in ASXL1 were originally identified by Gelsi-Boyer et al. [82] in MDS with del20q11 through sequencing of the focal deletion in MDS. Mutations in ASXL1 involve somatic nonsense and out-of-frame insertion/deletion nonsense mutations at the 5’ end of the exon 12 of the gene that results in loss of ASXL1 protein expression [83]. ASXL1 is common in MDS (14-21%), CMML and 38-43% 13

MDS/MPN overlap syndromes, and is found to be associated with worsened OS among MDS patients transforming to AML independently of other clinical features, including age, cytogenetics and cytopenias [27, 84]. Role of ASXL1 mutations for disease progression to myeloid transformation are thought to be linked to PRC2-mediated gene expression [80]. In mouse model, Asxl1 mutation caused progressive, multilineage cytopenias, dysplasia with increased numbers of hematopoietic stem/progenitor cells, and occasional progression to overt leukemia similar to that of human MDS [85, 86]. This phenotype is linked to depression of Hoxa9 and microRNA-125a. Impaired myeloid differentiation of human hematopoietic cells by ASXL1 knockdown was reported in vitro [81].

There is evidence that suggests that mutations in DNMT3A, ASXL1, and TET2 are independently associated with better response in MDS treated with hypomethylating drugs [68, 71, 87]. Furthermore, TET2 mutations resulted in the highest rate of response to hypomethylating agents (HMAs) in MDS without clonal ASXL1 mutations [15, 68]. Based on massively parallel sequencing of 40 mutated genes from 213 HMA-treated MDS patients, Bejar et al. [68] hypothesized that mutations of individual genes may serve as biomarkers for evaluating response of HMAs. TET2 mutations have resulted in altered methylation, and caused self renewal of HSCs leading to leukemic transformation. Response to HMAs was observed in patients with mutations that confer even a very poor prognosis. However, the acquisition of ASXL1 as a secondary mutation could lead to more clonally progressive disease, which subsequently might result in resistance to hypomethylating treatment. Thus, further evaluations are suggested to identify mutations predictive of acquired resistance or relapsed disease for MDS-treatment at multiple time points, and also for differential response of mutations in other epigenetic regulators to epigenetic therapies.

2.4. Signal transduction and cell surface receptors:

Unlike other myeloid malignancies, mutations in tyrosine kinase signaling pathways are described in 5-10% of MDS only, with mutations in NRAS and KRAS being the most frequent during transformation to AML. The common mutations of receptor tyrosine kinases include FLT3 and KIT (<5% MDS, commonly seen in de novo AML), and other members of 14

RAS/RAF/MEK pathway viz. NF1, BRAF, PTPN11 are rarely mutated. CBL mutations in dMDS (~5%) are involved in negative modulation and aberrant tyrosine kinase signaling [88, 89]. The V617F mutation in the JAK2 gene, encoding a cytoplasmic tyrosine kinase, has been described in 5% of MDS contributing to megakaryocytic proliferation, and ~50% of MDS/MPN overlapping refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) [34, 37, 90]. Mutations in signal transduction pathways occur in a mutually exclusive manner in individual MDS [27]. Genes involved in this category are frequently involved in pathogenesis of MDS/MPN, especially juvenile myelomonocytic leukemia (JMML), and one of the mutations induces hypersensitive proliferation of HSC contributing to development of MDS [46].

2.5. Transcription factors, cell cycle regulators and tumor suppressors Mutations of the transcriptional factors have been reported to cause impairment of differentiation and maintenance of HSCs of MDS. Germ-line mutations of these genes are responsible for familial MDS/AML [91].

2.5.1. RUNX1/AML1 (21q22) RUNX1 mutations have been reported frequently in various types of hematological malignancies, and play pivotal roles in the pathogenesis of MDS/AML in both mouse and human CD34+ cells [46]. Mutations (translocation and amplification) in RUNX1 (also known as AML1 or CBFA2) are reported in 10-20% MDS, more commonly in t-MDS and high-risk MDS, at the C-terminus of the protein resulting in haploinsufficiency of the gene [92, 93]. It is frequently observed in 7/del7q and thus associated with severe thrombocytopenia resulting in poor prognosis. The highly conserved N-terminal

domain (RUNT domain) of RUNX1 protein

causes

heterodimerization with the B-subunit of CBF, and the C-terminal transactivation domain recruits transcriptional cofactors. The rearrangement of EVI1-RUNX1 (t(3;21))

has been

reported in MDS. Amplification of RUNX1 occurs through polysomy of 21 or through true highlevel amplification [94]. Functional consequences of RUNX1 amplification on MDS-pathology are still not clear [95]. One of the familial platelet disorder (FPD), also known as familial MDS, is caused by a heterozygous germ line mutation in RUNX1 with a predisposition to AML [91].

15

2.5.2. ETV6 (12p13) Somatic mutations and heterozygous deletions of ETV6 (ETS variant gene 6, also known as TEL) gene occur in 2-5% of MDS [27, 96]. Isolated 12p-deletions are placed in the ‘good’ cytogenetic risk group of IPSS-R [9]. However, adverse prognosis has been reported after adjustment for IPSS in a large multi-centre study [27]. Common rearrangements, including ETV6-RUNX1 (TELAML1) translocations and deletion of ETV6 are frequently reported in B-ALL. ETV6 encodes for a transcription factor, which is required for hematopoietic stem cell maintenance. ETV6 mutations involve either homodimerization or DNA-binding domain in heterozygous mutations, which results in truncated or altered protein that is incapable of repressing transcription and shows dominant negative effects [97].

2.5.3. TP53 (17p13) TP53 is a tumor suppressor and also a transcription factor that responds to cellular stress by activating various protective pathways for inducing apoptosis, cell cycle arrest and DNA repair. Deletion of TP53 develops resistance to apoptosis and other cellular death mechanisms. TP53 mutations have been reported in ‘high risk’ subtypes and predominantly in t-MDS [98]. Although TP53 alterations have been described in about 10% of MDS patients, 30-50% of these MDS with loss of TP53 have complex karyotype [27]. Thus, TP53 mutation is always associated with adverse prognosis with relatively high risk of leukemic transformation in MDS, even after adjustment of other prognostic variables [12, 27]. TP53 mutations in association with isolated del(5q) at early stage of the disease disturbs treatment outcome due to loss of p53 protein function and predisposes to evolution to AML. Normal TP53 function is essential for erythroid apoptosis, which is promoted by RPS14 haploisufficiency in del(5q) MDS [99]. Presence of TP53 mutation has been reported in 18% of MDS with isolated del(5q) and <5% blasts, which were present at low level at diagnosis and years prior to initiation of Lenalidomide therapy leading to suboptimal response or resistance to therapy [100]. Patients with TP53 mutation had a paucity of mutations in other genes implicated in myeloid malignancies with poor survival in high-risk MDS and AML treated with azacitidine [12, 36, 101].

2.5.4. NPM1/CEBPA/WT1/GATA1/SPI1 16

Mutations in other transcription factors, including NPM1/CEBPA/WT1/GATA1/GATA2/SPI1 have been frequently observed in AML and also in <5% of MDS [102, 103]. Germline mutations in RUNX1, CEBPA and GATA2 have been reported in familial predisposition to AML and MDS [104, 105]. Simultaneous presence of two different mutations in CEBPA shows a favorable outcome in most AML, whereas only a single mutation in the gene coupled with a variety of gene mutations shows poor prognosis in MDS [106].

2.6. Mutations of cohesin complex

Reports have been documented on disturbance of the cohesin of sister chromatids, postreplicative DNA repair and transcriptional regulation in MDS and other myeloid disorders by recurrent mutations and deletions in the cohesin complex factors, including STAG2, RAD21, SMC1A and SMC3 [107]. These mutually exclusive mutations have been reported in 1-10% MDS with loss-of-function indicating poor OS, particularly with STAG2. Mutations in spliceosome subunits, particularly in PRPF8 and SRF3B1 have also been thought to disrupt sister chromatid cohesin in human cells and HeLa Kyoto cells [108]. Such phenotypic link between splicing and sister chromatid cohesin indicates a possible alteration of cohesin on sister chromatids by SF3B1 mutations resulting in genetic instability. Significant association of cohesin complex mutations with RUNX1 mutations is frequently reported in high-risk MDS [107]. However, cohesin complex mutations did not affect survival of AML patients [109].

2.7. Other mutations

The role of miRNA has been illustrated in mouse MDS model with deleted Dicer1 as well as the effects of MIR145 on del5q MDS [110, 111]. The post-transcriptional protein expression is affected by miRNA through RNA-induced silencing complex (RISC), which targets RNA sequences for destruction mediated by an endoribonuclease Dicer that recognizes double stranded RNA. The complementary binding of miRNA to single stranded RNA causes Dicermediated destruction and modulation of protein expression. The role of miRNA on hematopoiesis and epigenetic modification of protein expression in MDS has facilitated detection of miRNA products in MDS and its progression to AML, which could direct targeted 17

drug development. However, assay system of miRNA investigation in clinical setting has yet to be established [13].

Mutations in mitochondrial DNA find a link with MDS resulting in decreased expression of iron transporter ABCB7. Mutations of ABCB7 are associated with inherited ring sideroblast formation. These mutations in mitochondrial DNA of iron transport lead to iron deposition in ring sideroblast. Although mitochondrial DNA mutations have been reported in 56% of the MDS patients and correlated with age and advanced state of disease, no prognostic or therapeutic implications, and no testing strategies of mitochondrial mutations in MDS have been directed till date [13, 112].

Somatic mutations in SETBP1(18q21.1) have recently been demonstrated in MDS and several other myeloid malignancies [113, 114]. SETBP1 encodes SET-binding protein 1, and is a regulator of SET nuclear protein, which is an oncogene involved in cell division. The mutations mainly affect 858-874 residues. SETBP1 binds directly to the promoters of HOXA9 and HOXA10 resulting in increased expression, and overexpression of SETBP1 causes stabilization of SET protein and inhibition of PP2A (protein phosphatases), leading to cell proliferation.

Co-

occurrence of SETBP1 has been noticed with -7/del(7q) and i(17q). Although del(7q) and i(17q) are associated with intermediate prognosis, co-occurrence of SETBP1 with these chromosomal rearrangements and monosomy 7 might indicate a poorer prognostic impact of SETBP1 in MDS than its wild type. Large scale screening of MDS and CMML has established that SETBP1 is acquired during leukemic transformation. Hence, this mutation is a key function towards disease progression.

3. Mutational complexity in MDS

Acquisition of somatic genetic mutations in MDS-stem cells have been possible to be backtracked through recent advancement of technologies. Mutational landscape collected in the recent studies in MDS-development involves mainly RNA-splicing genes (SF3B1, SRSF2, ZRSR2, U2AF1, etc.) and epigenetic modification (TET2, DNMT3A, ASXL1) genes. Acquiring a clonal abnormality such as del(5q) further adds on diverse recurrent mutations developing sub18

clones such as mutations in chromatin modification and signaling, which may coexist, and thus the RNA splicing mutations are presumed to decide the disease types and clinical phenotypes of MDS [28]. However, presence of TET2 mutations in HSCs of normal elderly individuals, DNMT3A in pre-leukemic HSCs in AML, and DNMT3A, TET2, JAK2, ASXL1, TP53, BCORL1 and SF3B1 mutations in peripheral blood of non-hematopoietic cancer patients strongly suggests that these mutations are associated with advance age [115-117]. The information also supports that the therapeutic drugs used for treatment of other cancer in elderly people favors development of t-MDS through acquisition of clonal and subclonal mutations [117].

Molecular profiling of gene mutations also demonstrated the specificity of genotype-phenotype association such as ring sideroblast in over 75% of this MDS subtype carrying SF3B1 mutations, which also indicates favorable clinical outcome. Mutations in the PRPF8 spliceosome gene are also strongly associated with ring sideroblast phenotype; however, it indicates more aggressive phenotypic expression. Co-occurrence of pathway mutations and their biological interactions, and mutual exclusivity (TET2 and IDH1/2) of genes have been described with clonal evolution and likelihood for AML transformation. Mutation in SF3B1, which indicates a good prognosis, was mutually exclusive with ASXL1 and IDH2 mutations, which confer poor prognosis. Mutations in spliceosomes, however, rarely co-occur. In the high-risk group of WHO morphologic categories, higher number of average mutations was consistent with higher degree of clonal expansion, particularly in RAEB1 and RAEB2 subtypes [10, 118]. Clinical significance of clonal and subclonal events within the same genes appeared to be similar, and thus emphasizes the clinical importance of mutations in minor subclones, and pivotal role of targeted deep-sequencing of these mutations for understanding the independent and co-existent effects [119]. Disease-free OS appeared to be inversely proportional to the number of mutations, although some of the oncogenic mutations are already understood with independent prognostic implications [28].

Presence of somatic TET2 mutations in hematologically normal older individuals forms the understanding of such random genetic events prior to disease initiation [67]. In light of this finding and also based on the fact that MDS is a disease of the elderly population, attempts in quest of preclinical initiation of clonal hematopoiesis by exome sequencing in peripheral blood 19

DNA of participants of varying age groups has detected clonal hematopoietic expansion with somatic mutations in 5-18.4% individuals aged over 65 years to 108 years [115-117]. Presence of clonal hematopoietic mutations in elderly individuals has established the fact that age-related clonal hematopoiesis has increased risk for subsequent malignant development with associated risk of co-morbidities and increased mortality wherein age is the largest contributor. Among 805 [116], 3111 [115] and 77 [117] somatic mutations, DNMT3A, ASXL1 and TET2 were frequently mutated with indications of clonal hematopoiesis, which was consistent with earlier reports [29, 120, 121]. In these study groups of older individuals, DNMT3A mutations were prevalent over ASXL1 and TET2, and prevalence of transition of cytosine to thymine (C->T) was considered as the somatic mutational signature of aging [29]. Somatic mutations, irrespective of being driver (contributes to clonal expansion) or ‘passengers’ (which do not contribute), could indicate presence of a hematopoietic clone for subsequent development of cancer. Accumulation of bundles of ‘initiating’ somatic mutations in healthy elderly people may not cause direct disease-development; however, cooperation with other mutations in the genes that are frequently mutated in myeloid and other hematopoietic cancers (as chromosomal deletions or insertions or indels) might result in clonal expansion through selfrenewal and/or proliferation, and can cause hematopoietic malignancies [67, 115-117, 121]. Thus, it is apparent that initiating mutations in DNMT3A, TET2 and ASXL1 occur many years before the development of cancer and remain in subclinical states for long periods. Clonal mosaicism with complex chromosomal abnormalities reflects expansion of a specific cellular clone and increases the risk of malignancy. Cooperating expression of disruptive mutations in DNMT3A, TET2 and ASXL1, missense mutations in DNMT3A and other recurring cancer-causing mutations have comprised a set of 327 candidate ‘driver’ mutations for clonal hematopoiesis [115]. These three epigenetic regulators have a strong tendency to disrupt the protein-coding sequences by introducing frame-shift, nonsense or splice-site mutations. Significant excess of missense mutations in DNMT3A, being localized in exons 7 to 23 and enriched for cystineforming mutations, have potential to exert a dominant-negative effect on the tetrameric DNMT3A protein-complex [115]. Functional studies have suggested that loss of DNMT3A impairs differentiation of HSCs resulting in increase in the number of such cells in the bone

20

marrow [59], and loss of TET2 results in increased self-renewal of HSC and a competitive growth advantage [67].

Multiple genetic mutations of RNA-splicing machinery are believed to be the driving factors of MDS. Transformation to AML of ~30% MDS patients could occur through mutation acquisition and clonal selection. It is thought that management of the non-random driver mutations in MDS could eliminate the disease clone. In MDS-transformed AML, acquisition of multiple cytogenetic and molecular alterations is generally unspecific; however, worsen the prognosis.

4. Complex chromosomal aberrations (CK) and monosomal karyotype (MK)

Recurrent chromosomal abnormalities, including del(5q), -7, del(7q), +8, del(20q) as the most common ones [3-7], have been demonstrated to be secondary genetic events, originating from genomic instability caused by the early mutations [122]. However, happloinsufficiency for RPS14 and miR-145 mapping to the common deleted regions of 5q represents the pathophysiological basis of the MDS subgroup with del5q [99, 122]. Prognostic impact of cytogenetic risk groups on outcome of allo-HSC transplantation has been reported in MDS patients; especially very poor outcome of transplantation in patients with complex karyotype (CK) of ‘very poor’ cytogenetic subgroup [123]. However, a non-informative cytogenetics result having a normal karyotype or inadequate chromosome morphology might lead to a difficult situation for risk estimation.

Cytogenetic finding of MK (2 or more autosomal monosomies or one monosmy with at least one additional structural rearrangement), which are also detected in AML and MPN, have significant importance on prognostication of MDS [124, 125]. Conflicting reports on impact of monosomal karyotype (MK) as an independent predictor of survival of MDS draws attention for considering the prognostic weight of complex aberrations in association with autosomal monosomies [126128]. Schanz et al. [7] demonstrated prognostic significance of MK in patients with ≤4 aberrations; however, in a situation of ≥5 abnormalities, MK didn’t distinguish the prognostic subgroups. In advanced childhood MDS, MK status was not evidenced as an independent prognostic factor. The most frequent involvement of 5, 7, 17 and 18 was noticed in MK. Number 21

of chromosomal aberrations in MK positive (MK+) subgroups of MDS was directly related to OS, and thus, MK was not reflected as an independent prognostic factor.

However, MK was associated with worse OS in MDS independent of the number of cytogenetic abnormalities, and retained prognostic utility even in presence of CK in multivariate analysis carried out on Victorian MDS population [128]. A negative status for MK reflected favorable OS in patients having <3 aberrations or without CK in Korean MDS population treated with AZA, whereas MK value was not significant in patients with 3 or more aberrations in another study [126]. This study has stated that MK status of at least the IPSS intermediate cytogenetic risk group may have a predictive value of survival of patients treated with AZA. However, contradictory reports on MK as worse predictor of clinical outcome might have been influenced by heterogeneous treatment protocols followed and variable methodological approaches considered in different studies on both MDS and AML [7, 129, 130].

In AML, the number of monosomies directly correlates with a poor prognosis [129, 131]. A single monosomy has resulted in better prognosis and PFS, and thus, remained as a significantly independent prognostic factor for patients both treated or untreated with allo-HSC transplantation [127]. However, biological mechanism of single MK could be different from 2 or more MK, which needs further evaluation on both AML and MDS. Breems et al. [129] reported a dismal prognostic outcome in AML associated with MK than CK, which was profound with multiple monosomies or one MK in association with at least one structural abnormality. CK lost its prognostic significance when MK was taken into consideration for prognostic evaluation. In concordance to Breems et al. [129], negative effect on OS of single monosomy has been demonstrated by Yang et al. [132] in Chinese AML population.

In general, MK is associated with poor prognosis and worse with advanced age compared to nonMK (MK-) patients [124, 125]. Also MK+ conditions in MDS patients are at higher risk to AML transformation [133]. In adult MK+ MDS, 2-year survival is reported in 6% vs 23% for CK without MK, and 1-year leukemia-risk is 32% vs 14% for patients with CK and without MK groups. Independently MK has not been considered in the recent IPSS-R classification, due mainly to the fact that the missing chromosome can be present as a marker or rearranged on 22

seemingly normal chromosomes. Though independent effect of MK on MDS-risk is not streamlined, such abnormal status leads to genomic instability when it appears in association with CK [7]. Deeg et al. [123] proposed an additional discriminatory power of MK in predicting relapse and survival in an allogenic stem cell transplant setting. However, further studies are essential to demonstrate the independent effect of autosomal monosomies on pathogenesis of MDS.

Thus, chromosome abnormalities will continue to carry its clinical relevance even after the discovery of landscapes of gene mutations. Molecular karyotyping by comparative genomic hybridization (CGH), array-CGH (aCGH) for copy number variation (CNV), and single nucleotide polymorphism (SNP) arrays (SNP-array) have resulted in superior resolution over conventional metaphase analysis, and thus, aCGH and SNP-array eliminates use of metaphases in malignant samples. A combined approach of SNP-array and conventional cytogenetics showed convincing results for prediction of OS, EFS and PFS [134]. Thus, karyotype analysis will continue to be essential, as abnormal karyotypes, including CK and MK, support clonality, and the consideration of specific karyotypic abnormalities in morphologically subtle cases will most likely remain unchanged in the proposed risk-classification of MDS, which is likely to be published in mid 2016 [135].

5. Drug targets

A rapidly expanding knowledge has been gathered on epigenetic dysregulation in cancer through the technological advancement during the past several years [136]. Of this, histone and DNA modifications play critical roles in tumor growth and survival. The plethora of knowledge gained following the outcome of the ‘first generation’ hypomethylating agents such as Azacitidine (Vidaza) and Decitabine (Dacogen) and histone deacetylase (HDAC) inhibitors (Vorinostat, Romidepsin) has sensitized drug discovery and development community for formulation of ‘second generation’ targeted epigenetic agents [27, 137, 138]. Similarly, efficacy of lenalidomide (LEN) on del5q in MDS is another example [139]. The high throughput investigative result on epigenetic genes, which encode enzymes, may represent novel tractable therapeutic targets. Primarily, higher priority is being paid to histone methylation and acetylation, and targeting 23

somatic alterations in histone-modifying enzymes or epigenetic proteins as the starting point in drug discovery. The recurrent point mutations that activate or alter protein function are targeted as the subset of early biological validation for drug development. Epigenetic drug discovery is at its incipient stage. Understanding the complexities of the ‘histone code’ and associated scientific and pragmatic challenges are daunting task in the recent emerging field of drug discovery and identification of small molecules that directly and specifically silence the oncogenes or increase expression of tumor suppressors. Identification of drug-targets based on chromosomal translocations, insertions, deletions and point mutations of unknown biological consequences for tumor growth and survival is difficult. Interestingly, inactivation of an epigenetic gene makes tumor more sensitive to a second druggable target. A mechanistic understanding of epigenetic dysregulation and subsequent perturbation of biological function needs to be elucidated. There are underlying genetic and/or epigenetic mechanism that predisposes some tumors to EZH2 enzyme inhibition. Contextual role, acting as oncogenes in one cancer and tumor suppressor in another, of EZH2 and other epigenetic proteins can lead to cross talk between histone modifications and that can affect protein recruitment and biological response [76, 140]. The clinical utility of DNMT3A inhibitors and pan-HDAC inhibitors as well as the rapid pre-clinical advancement of the second generation of epigenetic modulators guides for future epigenetic drug discovery and development.

5.1. EZH2 inhibitors The epigenetic regulation of PRC2 that catalyzes trimethylation of lysine 27 on histone H3 via EZH2 methyltransferase, confers stemness and regulates differentiation during embryonic development. Given these activities of EZH2 and H3K27me3, plastic and dynamic features of cancer cells, especially cancer stem cells, were thought to be closely associated with this epigenetic mechanism [141]. These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in malignant cells. Therefore, EZH2 has been considered as a novel target for identification of small candidate compounds that can inhibit EZH2 activity. PRC2 complex have been used on a variety of substrates for identification of direct EZH2 inhibitors those bind directly to the protein and inhibit its enzyme activity through biochemical assays [142, 143]. The reaction product, that is H3K27me3 peptide, allosterically binds to PRC2 and increases catalytic activity of the complex where recognition of H3K27me3 24

by PRC2 is proposed to maintain repressed chromatin domains by re-establishing H3K27me3 onto naked nucleosomes being incorporated during DNA synthesis [144]. Some of EHZ2 inhibitors have displayed greater biochemical potency and a longer enzyme-inhibitor residence time when H3K27me3 peptide is bound to PRC2 [145]. Optimization of the EZH2 inhibitors has been considered for clinical trial. Some of the EZH2 inhibitors are described briefly below, and in table 2. GSK126 molecules have highly selective biochemical and cellular on-target potency, and are assessed by decrease in H3K27 trimethylation. Its pre-clinical testing on DLBCL cell lines and DLBCL xenografts in mice has resulted in tumor growth inhibition with improvement in survival [146]. GSK343 has been tested as a specific inhibitor of the histone H3-lysine 27 (H3K27) methyltransferase EZH2. The compound displays 60 fold selectivity for EZH2 vs. EZH1, and 1000 fold or greater selectivity against other histone methyltransferases. EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various malignant cells, which results in apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered [142]. EPZ-6438 is a potent and selective S-adenosyl-methionine-competitive small molecule and a first-in-class inhibitor of EZH2. EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both wild-type EZH2 and mutant lymphoma cells, leading to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations [147].

PRC2 contains either EZH1 or EZH2 as its catalytic subunit, with EZH1 being found in both dividing and non-dividing cells, whereas EZH2 is found only in actively dividing cells. UNC1999 is an orally bioavaliable selective inhibitor of both EZH2 and EZH1 lysine methyltransferases. 25

UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged cells [148]. UNC1999 is competitive with the cofactor S-adenosylmethionine (SAM) and non-competitive with the peptide substrate. Because it inhibits both EZH2 and EZH1, UNC1999 has potential advantages over EZH2 selective inhibitors in the disease settings where both PRC2 – EZH2 and PRC2 – EZH1 contribute to the methylation of H3K27 [148]. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to de-repression of polycomb targets including Cdkn2a. Gene de-repression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9 [148].

UNC2400 is an inactive analog compound useful for assessment of off-target effect that allows pharmacologic manipulation of EZH2 and EZH1. Collectively, Xu et al. [148] demonstrated the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of PRC2-EZH2 and PRC2-EZH1 by small-molecule compounds as novel therapeutics for MLL-rearranged leukemia. EPZ-5676 is a small molecule inhibitor of DOT1L, another histone methyltransferase, developed for the treatment of patients with acute leukemia in which the MLL gene is rearranged due to a chromosomal translocation (MLL-r) or a partial tandem duplication (MLL-PTD) (by Epizyme in collaboration with Celgene Corporation). Due to these rearrangements, DOT1L is misregulated, resulting in the increased expression of genes causing leukemia. Similarly there are a number of small molecule inhibitors considered in clinical trial for establishing them as compelling drug targets.

5.2. Inhibitors of IDH1/2

Mutations in IDH1/2 are known driver mutations in MDS, AML and many other types of cancer. IDH2 mutations are enriched in cytogenetically normal AML, whereas 20-30% patients have an abnormal karyotype of intermediate or unfavorable risk group. Moreover, there are marked differences in CR and OS between patients with an R172 and an R140 mutation of IDH2, 70% of R140 achieved CR compared to only 38% of R172 patients [149]. Clinically R172 patients 26

present lower WBC count than R140, have intermediate cytogenetic risk, and are much less likely to have co-occurring mutations. The frequency of IDH2 mutations increases with age in AML. Treatment outcome with high dose chemotherapy and allo-HSC transplantation in elderly patients above 60 years is poor. Outcome of IDH2 mutant patients with relapsed disease, age above 60 years, and patients treated up-front with a low dose of hypomethylating agents or low dose Ara-C remains unknown [150]. IDH1/2 mutations impair cellular differentiation in various cell lineages and promote tumor development in cooperation with other cancer genes. Thus, IDH1/2 mutations are targeted as important biomarkers for drug development, and the first generation inhibitors of IDH1/2 mutations have entered into clinical trials, which have shown promising efficacy of inhibiting mutations in AML [150-152].

Enzyme assay in presence of saturating NADPH identified a series of heterocyclic urea sulfonamides as small molecule inhibitors of IDH, which have shown promising pre-clinical and early phase clinical activity with an efficacy to decrease the levels of 2-HG and reverse the block in cellular differentiation. AGI-6780 (Agios Pharmaceuticals) has the property of slow-tight binding and non-competitive inhibition. Ex vivo treatment of human AML cells with AGI-6780 has shown a burst of cellular differentiation with a decrease in myeloblasts and increase in mature monocytes and granulocytes [153]. Allosteric inhibition of IDH1 mutation was tested in primary AML cells with clinically relevant R132 mutations of IDH1 ex vivo, which led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells [154, 155]. Allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including cell cycle, G1/S transition, cellular growth and proliferation, and cell death and survival. Ex vivo treatment of AML cells with IDH1 inhibitor AGI-5198 revealed no change on production of reactive oxygen (ROS) and alleviated the redox potentials of IDH inhibitors [153]. AG-221 is a reversible inhibitor of mutant IDH2, which has led to dramatic decrease (upto 98% with R140Q and 88% for R172K mutations) in both plasma and the oncometabolite 2-HG in bone marrow to baseline levels in MDS and other myeloid malignancies [150, 156].

27

Altogether, novel insights into the cellular and molecular effects of inhibition of mutant IDH1/2 have established proof-of-concept for the molecular and biological activity of inhibitors for targeting different mutations in leukemia and opens up new avenues for targeting drug development. The time required for first response to IDH2 inhibitors has led to speculation that the inhibitors may be most effective in an up-front setting before the patients develop complications. IDH1/2 inhibitors are not expected to have side effects as those of anti-cancer agents because the role of mutant IDH is not necessary for normal cells [154]. Thus, as a differentiating agent, IDH2 inhibitors are expected to avoid issues with aplasia, neutropenia, and thrombocytopenia associated with salvage chemotherapy. Molenaar et al. [153] suggested that inhibitors of IDH1/2 can be safely used as adjuvant to standard of care drugs such as Daunorubicin and Cytarabine, or whole body irradiation in the context of HSC transplantations.

5.3. Spliceosomal modulators Mutations in spliceosomal genes occur at highly restricted amino acid residues, are always heterogeneous, and rarely co-occur with one another. The point mutations in the spliceosomal genes can result in the loss of mRNA splicing fidelity and/or the usage of novel cryptic splice sites that leads to the generation of proteins with alternative new aberrant functions resulting in contribution to a broad spectrum of human diseases including hematologic malignancies. Transcriptome sequencing has identified somatic hot spots and point mutations in SF3B1, U2AF1, SRSF2, ZRSR2, and many other splicing factor genes, which are mutually exclusive with one another, and co-occur with specific epigenetic modifiers in MDS [28, 38]. Early appearance of mutations in RNA-splicing machinery is thought to result in initiation of disease onset. It was presumed that modulation of RNA splicing would be a highly deleterious event, which may result in potential loss of critical proteins and expression of aberrant function and initiation of apoptosis [157]. This understanding of splicing modulation is leading to development of novel targeted drugs. Identification of small molecule inhibitors of spliceosomal mutations are in fast pace in the recent era [157-159].

Several natural bacterial fermentation products such as Pladienolides, FR901464, Herboxidiene have shown efficacy to modulate the function of spliceosome in tumor xenograft models [140]. However, their highly complex structures do not lend themselves well to standard medicinal 28

chemistry manipulations. To meet the challenges of cost-effective and quantitative production, development of facile synthetic derivatives was targeted and some of which are being considered in pre-clinical trial [160]. However, these natural modulators were presented with significant cytotoxicity in all cases.

In Mx1-Cre Srsf2P95H/KO mouse model with MDS-like features (due to altered mRNA recognition), heterozygous Srsf2P95H mutation caused severe bone marrow failure due to complete loss of hematopoietic stem/progenitor cells [159]. Based on this fact, it was hypothesized that leukemias with spliceosomal mutations might display greater sensitivity to pharmacologic modulation of splicing than the normal counterparts. A spliceosomal modulator, E7107, resulted in decreased disease burden and survival benefit in Srsf2P95H/+ mice but not in similarly treated Srsf2+/+ mice in vivo [159]. Similar significant reduction in human leukemic burden was observed in response to E7107 in patient-derived xenografts in mice [159]. Thus, genetic and pharmacologic evidences of sensitivity of splicing factor mutant leukemias to splicing modulators are guiding for further understanding of therapeutic implications of splicing modulators.

Finally, targets with small molecule inhibitors have brought an important breakthrough in drug discovery in the present era. Indeed, the identification of new efficacious inhibitors is a great challenge targeting molecular mutations involved in pathway machineries and disease consequences. Thus, better understanding of epigenetic modification, splicing mechanism and genes/enzymes involved would be important with pharmaceutical value [118]. The sensory activity of TP53 in splicing mechanism demonstrates its importance in mRNA splicing. Novel compounds of signal pathways and targeted mutant genes or their products have already been established in CML, and won’t be far for MDS therapy. Identification of promising and clinically active IDH inhibitors as pharmaceutical biomarkers against leukemic clones is under extensive investigation. Further extensive investigation on new pathway genes would be essential for extraction of information towards pharmaceutical development for refinement of treatment modalities of MDS management in complex mutational scenario with coexistence of multiple mutations. However, more information on interplay of mutations on larger database would provide newer modalities of drug development with specific target agents. Inter-individual 29

variability in a scenario of complex chromosomal rearrangements and multiple genetic alterations further necessitates development of personalized medicines in near future for treatment of MDS.

6. Conclusion

MDS is a heterogeneous pre-malignant disease of hematopoiesis in older adults with a median age of 71 at diagnosis. MDS was differentiated based on cell morphology, blast count and presence or absence of ring sideroblasts in the French-American-British (FAB) classification system [66]. The World Health Organization (WHO) included Auer rod and del(5q), in addition to FAB-cytological characteristics [1]. Greenberg et al. [2] incorporated cytogenetic abnormalities in the International Prognostic Scoring System (IPSS) for risk-stratification (Good: normal, -Y, del5q, del20q; intermediate: other abnormalities; poor: complex with ≥3 abnormalities (CK), del7q/-7), along with bone marrow blasts and multiple cytopenias [2]. Riskstratification from ‘good’ to ‘very poor’ is further carried out based on a more detailed and prognostically higher weighted cytogenetic system empowering chromosomal aberrations in the revised IPSS (IPSS-R) [9]. Grouping of complex aberrations, which are not considered in IPSSR for prognostic stratification, has further been demonstrated by Schanz et al. [7] and GerciaManero [161]. Increased frequency of CK, which is associated with genetic instability, defines the subgroup of worst prognosis in MDS [7, 9].

The classification of MDS and other myeloid malignancies is of significantly importance owing to recently acquired knowledge on biological variation of genetic mutations in pre- and postinitiation of disease state, and for targeted drug development using small molecules as inhibitors of mutated genes. A Clinical Advisory Committee of ~100 hematologists, pathologists and cytogeneticists from all over the world has proposed incorporation of recent knowledge on cytogenetic and gene mutations detected in MDS [135]. In the proposed classification, MK is not considered as a specific MDS-subtype because of its controversial risk in presence or absence of CK [130, 162]. Although the five-tier IPSS-R classification has incorporated most MK in the ‘very poor’ risk–group [9], further study is needed to determine its impact on prognosis independent of other cytogenetic and molecular mutations. 30

Recent understanding of diversified pathway genes on MDS pathogenesis and its evolution to AML has thrown molecular insight towards MDS-prognostication based on point mutations [10, 27]. A prognostic impact of shorter OS has been documented in low-risk MDS group (LR-PSS) associated with some of these mutations [161]. Cooperation of mutations and their interplay by changing the expression of individual mutation affect treatment-response or develop resistance to therapeutic drugs. Therefore, a comprehensive molecular profiling of pathway mutations in MDS would be important for diagnosis, prognostic stratification, and ultimately lead to the development of targeted therapeutic agents. Furthermore, cytogenetic picture, which has already been established as the most critical factor in determining survival in 5-tier IPSS-R, would play pivotal role alongside mutation analysis. The importance of cytogenetic aberrations would persist for better monitoring of treatment outcome, especially with allogenic bone marrow transplantation, which is expected to eradicate the malignant hematopoietic clone [118]. Cytogenetically poor-risk group appeared with higher rate of relapse reflecting lower OS probability and mortality in GITMO co-operative study [123, 163]. Therefore, the burgeoning knowledge on molecular and cytogenetic complexity could provide novel insights into the patho-biology of MDS. The exercise on clinical application and refinement of prognostic risk-models is progressing at fast pace. The understanding of biological interactions of mutations through co-occurrence and combining that with the cytogenetic picture could be translated into targeted therapies with potential to change the existing history of the disease process [164]. Further focus on developing simple methods for detection of mutations would be important since use of sequencing techniques for identification of driver mutations at bedside is far in the future. Although NGS is a combination of conventional cytogenetics and aCGH and SNP-array, and a comprehensive platform for genome-wide investigation of asprecise-as low frequency mutations, analysis of sequencing data with low–frequency or noncoding mutations, and cost and time required for NGS are the limiting factors for routine clinical work-up. Moreover, cytogenetic anomalies detectable by conventional karyotyping could probably be underestimated by NGS in clinical setting. However, detection of key mutations could be accomplished through targeted sequencing of small groups of genes of diagnostic and therapeutic interest (10, 68). NGS could also be an important tool for diagnosis of otherwise diagnostically challenging cases. Therefore, a combination of molecular karyotyping and mutation analysis would further guide to design individualized therapeutic program. 31

Moreover, detection of somatic mutations associated with clonal hematopoiesis in blood-DNA of healthy individuals has raised concerns of DNA sequencing for screening of at-risk individuals/families for early detection and prevention of blood cancers like cholesterolscreening for cardiovascular disease. Sequence screening would also ascertain the high-risk states, monitor progression or remission of high-risk condition and monitor mutations before their clinical manifestation as illness. Early detection of clonal hematopoiesis would facilitate periodic screening for the presence of cooperating mutations at low allele frequencies that could favor malignancy subsequently. Therefore, dynamic screening of somatic mutations of clonal hematopoiesis via DNA sequencing over the course of an asymptomatic person’s life could be predictive of clinical disease and death, and that may eventually replace the traditional dichotomy between illness and fitness with a continuum of ascertainable genomic states that are associated with elevated risks of future illness.

Additionally, the emerging discovery of small molecules as inhibitors of mutations in genes of epigenetic DNA methylation, chromatin modeling and RNA-splicing mechanism has established significant hope in management of heterogeneous hematological malignancies. Identification of drug targets and elucidation of expression of mutated genes at pre-clinical and disease states would further lead to better understanding and control of the stages of disease progression. Individualized gene expression and genome wide association study would further promise to develop personalized therapeutic modules with pharmaceutical tailoring of targeted drugs.

7. Acknowledgements

The authors wish to acknowledge Shameek Ganguly, Ph.D. student, Stanford University, CA, for editing the manuscript.

8. Conflict of Interest There is no conflict of interest.

32

9. References

[1] WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues. In Myelodysplastic syndromes. Edited by Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. Ed 4, Lyon, France: IARC Press, 2008, pp 87-108. [2] Greenberg P, Coc C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D, Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J. International scoring system for evaluating prognosis in myelodysplastic syndromes [Erratum appeared in Blood 1998, 91:1100], Blood 1997, 89: 2079 – 2088. [3] Ganguly BB, Dolai TK, De R, Kadam NN. Spectrum of complex chromosomal aberrations in a myelodysplastic syndrome (MDS) and a brief review. J Cancer Res Ther 2016 (in press). [4] Haase D, Germing U, Schanz J, et al. New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: Evidence from a core dataset of 2124 patients. Blood 2007, 110:4385-4395. [5] Olney HJ, Le Beau MM. Evaluation of recurring cytogenetic abnormalities in the treatment of myelodysplastic syndromes. Leuk Res 2007, 31:427-434. [6] Schanz J, Tüchler H, Solé F, Mallo M, Luňo E, Cervera J, Granada I, Hildebrandt B, Slovak ML, Ohyashiki K, Steidl C, Fonatsch C, Pfeilstőcker M, Nősslinger T, Valent P, Giagounidis A, Aul C, Lubbert M, Stauder R, Krieger O, Garcia-Manero G, Faderl S, Pierce S, Le Beau MM, Bennett JM, Greenberg P, Germing U, Haase D. New comprehensive cytogenetic scoring system for primary myelodysplastic syndromes (MDS) and oligoblastic acute myeloid leukemia after MDS derived from an international database merge. J Clin Oncol 2012, 30:820-829. [7] Schanz J, Tüchler H, Solé F, Mallo M, Luňo E, Cervera J, Grau J, Hildebrandt B, Slovak ML, Ohyashiki K, Steidl C, Fonatsch C, Pfeilstőcker M, Nősslinger T, Valent P, Giagounidis A, Aul C, Lubbert M, Stauder R, Krieger O, Le Beau MM, Bennett JM, Greenberg P, Germing U, Haase D. Monosomal karyotype in MDS: explaining the poor prognosis? Leukemia 2013, 27:1988-1995. [8] Heim S, Mitelman F. Cancer Cytogenetics: Chromosomal and Molecular Genetic Aberrations of Tumor Cells. 4th Ed, 2015, Wiley-Blackwell. [9] Greenberg PL, Tuechler H, Schanz J, Sanz G, Garcia-Manero G, Solé F, Bennet JM, Bowen D, Fenaux P, Dreyfus F, Kantarjian H, Kuendgen A, Levis A, Malcovati L, Cazzola M, Cermal 33

J, Fonatsch C, Le Beau MM, Slovak ML, Krieger O, Luebbert M, Maciejewski J, Magalhaes SM, Miyazaki Y, Pfeilstöcker M, Sekeres M, Sperr WR, Stauder R, Tauro S, Valent P, Vallespi T, van de Loosdrecht AA, Germing U, Hasse D. Revised International Prognostic Scoring System for myelodysplastic syndromes. Blood 2012, 120:2454-2465. [10] Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G, Schnittger S, Sanada M, Kon A, Alpermann T, Yoshida K, Roller A, Nadarajah N, Shiraishi Y, Shiozawa Y, Chiba K, Tanaka H, Koeffler HP, Klein H-U, Dugas M, Aburatani H, Kohlmann A, Miyano S, Haferlach C, Kern W, Ogawa S. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia, 2014, 28(2): 241-247. [11] Lukackova R, Bujalkova MG, Majerova L, Mladosievicova B. Molecular genetic methods in the diagnosis of myelodysplastic syndromes. A review. Biomed Pap Med Fac Univ Palacky Olomouc Repub, 2014, 158(3): 339-345. [12] Kulasekararaj AG, Mohamedali AM, Mufti GJ. Recent advances in understanding the molecular pathogenesis of myelodysplastic syndromes. B J Hematol, 2013, 162:587-605. [13] Nybakken GE, Bagg A. The genetic basis and expanding role of molecular analysis in the diagnosis, prognosis and therapeutic design for myelodysplastic syndromes. The Journal of Molecular Diagnostics, 2014, 16:145-158. [14] Abdel-Wahab O, Figueroa ME. Interpreting new molecular genetics in myelodysplastic syndromes. Advances in the Pathogenesis and Treatment of Myelodysplastic Syndromes. Hematology 2012, pg. 56-64. [15] Bejar R, Stevenson KE, Caughey B, et al. Somatic mutations predict poor outcome in patients with myelodysplastic syndrome after hematopoietic stem cell transplantation. J Clin Oncol 2014a; 32: 2691-2698. [16] Kantarjian H, Issa JP, Rosenfeld CS, et al. Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer 2006; 106: 17941803. [17] Valencia A, Masala E, Rossi A, et al. Expression of nucleoside-metabolizing enzymes in myelodysplastic syndromes and modulation of response to azacitidine. Leukemia, 2014, 28(3)” 621-628.

34

[18] Saunthararajah Y. Key clinical observations after 5-azacitidine and decitabine treatment of myelodysplastic syndromes suggest practical solutions for better outcomes. Hematology, 2013, 2013: 511-521. [19] Kosmider O, Gelsi-Boyer V, Cheok M, et al. TET2 mutation is an independent favorable prognostic factor in myelodysplastic syndromes (MDSs). Blood 2009; 114:3285-3291. [20] Langemeijer SM, Kuiper RP, Berends M, Knops R, Aslanyan MG, Massop M, StevensLinders E, van Hoogen P, et al. Acquired mutations in TET2 are common in myelodysplastic syndromes. Nature Genet 2009; 41: 838-842. [21] Figueroa ME, Abdel-Wahab O, Lu C, et al. Leukemic IDH1 and IDH2 mutations result in hypermethylation phenotype, disrupt TET2 function and impair hematopoietic differentiation. Cancer Cell 2010; 18(6): 553-567. [22] Ko M, Huang Y, Jankowska AM, et al. Impaired hydroxylation of 5-methylcytosine in yeloid cancers with mutant TET2. Nature, 2010, 468(7325):839-843. [23] Moran_Crusio K, Reavie L, Shih A, et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell, 2011, 20(1): 11-24. [24] Abdel-Wahab O, Mullally A, Hedvat C, et al. Genetic characterization of TET1, TET2, and TET3 alterations in myloid malignancies. Blood, 2009, 114(1): 144-147. [25] Figueroa ME, Skrabanek L, Li Y, et al. MDS and secondary AML display unique patterns and abundance of aberrant DNA methylation. Blood 2009, 114(16): 3448-3458. [26] Jiang Y, Dunbar A, Gondek LP, Mohan S, Rataul M, O’Keefe C, Sekeres M, Saunthararajah Y, Maciejewski JP. Aberrant DNA methylation is a dominant mechanism in MDS progression to AML. Blood 2009, 113: 1315-1325. [27] Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med, 2011, 364(26): 2496-2506. [28] Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood 2013; 122: 3616-3627. [29] Welch JS, Ley TJ, Link DC, Miller CA, Larson DE, Koboldt DC, Wartman LD, et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 2012, 150: 264-278. [30] Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature, 2011, 478(7367): 64-69.

35

[31] Cazzola M, De La Porta MG, Travaglino E, Malcovati L. Classification and prognostic evaluation of myelodysplastic syndromes. Semin Oncol 2011; 38: 627-634. [32] Papaemmanuil E, Cazzola M, Boultwood J, Malcovati L, Vyas P, Bowen D, Pellagatti A, et al. Somatic SF3B1 mutation in myelodysplasia with rinf sideroblasts. N Eng J Med 2011; 365: 1384-1395. [33] Abrahamsson AE, Geron I, Gotlib J, Dao KH, Barroga CF, Newton IG, Giles FJ, et al. Glycogen synthase kinase 3beta missplicing contributes to leukemia stem cell generation. Proc Natl Acad Sci USA 2009, 106: 3925-3929. [34] Pellagatti A, Boultwood J. The molecular pathogenesis of the myelodysplastic syndromes. E J Hematol 2015; 95: 3-15, doi:10.1111/ejh.12515. [35] Makishima H, Visconte V, Sakaguchi H, Jankowska AM, Abu Kar S, Jerez A, Przychodzen B, et al. Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis. Blood 2012; 119:3203-3210. [36] Mian SA, Smith AE, Kulasekararaj AG, et al. Spliceosome mutations exhibit specific associations with epigenetic modifiers and proto-oncogenes mutated in myelodysplastic syndrome. Hematologica 2013, 98(7): 1058-1066. [37] Malcovati L, Papaemmanuil E, Ambaglio I, et al. Driver somatic mutations identify distinct disease entities within myeloid neoplasms with myelodysplasia. Blood 2014; 124: 1513-1521. [38] Thol F, Kade S, Schlarmann C, Loffeld P, Morgan M, Krauter J, Wlodarski MW, Kolking B, Wichmann M, Gorlich K, Gohring G, Bug G, Ottmann O, Niemeyer CM, Hofmann WK, Schlegelberger B, Ganser A, Heuser M. Frequency and prognostic impact of mutations in SRSF2, U2AF1 and ZRSR2 in patients with myelodysplastic syndromes. Blood 2012; 119: 3578-3584. [39] Malcovati L, Papaemmanuil E, Bowen DT, Boultwood J, Della-Porta MG, Pascutto C, Travaglino E, et al. Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms. Blood 2011; 118: 6239-6246. [40] Visconte V, Rogers HJ, Singh J, et al. SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes. Blood 2012; 120: 3173-3186. [41] Matsunawa M, Yamamoto R, Sanada M, et al. Happloinsufficiency of Sf3b1 leads to compromised stem cell function but not to induce myelodysplacia. Leukemia 2014; 28: 18441850. 36

[42] Wang C, Sashida G, Saraya A, Ishiga R, Koide S, Oshima M, Isono K, Koseki H, Iwama A. Depletion of Sf3b1 impairs proliferative capacity of hematopoietic stemm cells but is not sufficient to induce myelodysplasia. Blood 2014; 123: 3336-3343. [43] Savage KI, Gorski JJ, Barros EM, et al. Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability. Mol Cell 2014; 54: 445459. [44] Dolatshad H, Pellagatti A, Fernandez-Mercado M, et al. Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells. Leukemia 2014; doi:10.1038/leu.2014.331. [45] Itzykson R, Kosmider O, Renneville A, et al. Prognostic score including gene mutations in chronic myelomonocytic leukemia. J Clin Oncol 2013; 31: 2428-2436. [46] Harada H, Harada Y. Recent advances in myelodysplastic syndromes: Molecular pathogenesis and its implications for targeted therapies. Cancer Sci 2015, 106(4):329-336. [47] Kurtovic-Kozaric A, Przychodzen B, Singh J, et al. PRPF8 defects cause missplicing in myeloid malignancies. Leukemia 2015; 29: 126-136. [48] Claus R, Lubbert M. Epigenetic targets in hematopoietic malignancies. Oncogene 2003; 22: 6489-6496. [49] Issa JP. Epigenetic changes in the myelodysplastic syndrome. Hematol Oncol Clin North Am 2010; 24: 317-330. [50] Toyota M, Issa JP. Epigenetic changes in solid and hematopoietic tumors. Semin Oncol 2005; 32: 521-530. [51] Brakensiek K, Lӓnger F, Schlegelberger B, Kreipe H, Lehmann U. Hypermethylation of the suppressor of cytokine signaling-1 (SOCS-1) in myelodysplastic syndrome. Br J Haematol, 2005, 130(2): 209-217. [52] Christiansen DH, Anderson MK, Pedersen-Bjergaard J. Methylation of p15INK4B is common, is associated with deletion of genes on chromosome arm 7q and predicts a poor prognosis in therapy-related myelodysplasia and acute myeloid leukemia. Leukemia, 2003, 17(9): 1813-1819. [53] Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al. Efficacy of azacytidine compared to that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomized, open-label, phase III study. Lancet Oncol, 2009, 10(3): 223-232. 37

[54] Silverman LR, McKenzie DR, Peterson BL, et al. Further analysis of trials with azacitidine in patients with myelodysplastic syndrome: studies 8421, 8921 and 9221 by the Cancer and Leukemia Group B. J Clin Oncol, 2006, 24 (24):3895-3903. [55] Thol F, Winschel C, Lüudeking A, et al. Rare occurrence of DNMT3A mutations in myelodysplastic syndromes. Hematologica, 2011a, 96(12): 1870-1873. [56] Shen L, Kantarjian H, Guo Y, et al. DNA methylation predicts survival and response to therapy in patients with myelodysplastic syndromes. J Clin Oncol, 2010, 28(4): 605-613. [57] Raddatz G, Gao Q, Bender S, Jaenisch R, Lyko F. Dnmt3a protects active chromosome domains against cancer-associated hypomethylation. PLoS Genet 2012; 8: e1003146. [58] Walter MJ, Shen D, Shao J, et al. Clonal diversity of recurrently mutated genes in myelodysplastic syndromes. Leukemia, 2013, 27(6): 1275-1282. [59] Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stemm cell differentiation. Nat Genet, 2012, 44(1):23-31. [60] Smith AE, Mohamedali AM, Kulasekararaj A, Lim Z, Gaken J, Lea NC, Przychodzen B, et al. Next-generation sequencing of the TET2 gene in 355 MDS and CMML patients reveals lowabundance mutant clones with early origins, but indicates no definite prognostic value. Blood 2010, 116: 3923-3932. [61] Lin J, Yao DM, Qian J, Chen Q, Qian W, Li Y, Yang J, et al. Recurrent DNMT3A R882 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome. Plos One 2011, 6: e26906. [62] Bejar R, Stevenson KE, Caughey BA, et al. Validation of a prognostic model and the impact of mutations in patients with lower-risk myelodysplastic syndrome. J Clin Oncol, 2012, 30(27): 3376-3382. [63] Walter MJ, Ding L, Shen D, et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia 2011; 25(7): 1153-1158. [64] Nakajima H, Kunimoto H. TET2 as an epigenetic master regulator for normal and malignant hematopoiesis. Cancer Sci 2014; 105: 1093-1099.

[65] Mohamedali AM, Smith AE, Gaken J, Lea NC, Mian SA, Westwood NB, Strupp C, Gattermann N, Germing U, Mufti GJ. Novel TET2 mutations associated with UPD4q24 in myelodysplastic syndrome. J Clin Oncol 2009, 27: 4002-4006. 38

[66] Gondek LP, Tiu R, O’Keefe CL, Sekeres MA, Theil KS, Maciejewski JP. Chromosomal lesions and uniparental disomy detected by SNP arrays in MDS, MDS/MPD, and MDS-derived AML. Blood, 2008, 111(3): 1534-1542. [67] Busque L, Patel JP, Figueroa ME et al. Recurrent somatic TET2 mutations in normal elderly individuals with clonal hematopoiesis. Nat Genet 2012; 44: 1179-1181. [68] Bejar R, Lord A, Stevenson K, Bar-Natan M, Perez-Ladaga A, Zaneveld J, Wang H, Caughey B, Stojanov P, Getz G, Garcia-Manero G, Kantarjian H, Chen R, Stone RM, Neuberg D, Steensma DP, Ebert BL. TET2 mutations predict response to hypermethylating agents in myelodysplastic syndrome patients. Blood, 2014b, 124:2705-2712. [69] Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer 2012; 12: 599-612. [70] Li Z, Cai X, Cai CL, Wang J, Zhang W, Petersen BE, Yang FC, Xu M. Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies. Blood 2011; 118: 4509-4518. [71] Itzykson R, Kosmider O, Cluzeau T. Mansat-De Mas V, Dreyfus F, Beyne-Rauzy O, Quesnel B, Vey N, Gelsi-Boyer V, Raynaud S, Preudhomme C, Ades L, Fenaux P. Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemia. Leukemia 2011; 25: 1147-1152. [72] Ward PS, Patel J, Wise DR, et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2hydroxyglutarate. Cancer Cell, 2010, 17(3): 225-234. [73] Dang L, White DW, Gross S, et al. Cancer-associated IDH1 mutations produce 2hydroxyglutarate. Nature, 2009, 462(7274): 739-744. [74] Xu F, Li X, Wu L, et al. Overexpression of the EZH2, RING1 and BMI1 genes is common in myelodysplastic syndromes: relation to adverse epigenetic alteration and poor prognostic scoring. Ann Hematol 2011; 90: 643. [75] Ernst T, Chase AJ, Score J, et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet 2010; 42: 722-726. [76] Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER, van der Heijden A, Scheele TN, et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet 2010; 42: 665-667. 39

[77] Jerez A, Sugimoto Y, Makishima H, et al. Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic patghogenesis. Blood 2012; 119: 6109-6117. [78] Margueron R, Li G, Sarma K, Blais A, Zavadil J, Woodcock, CL, Dynlacht BD, Reinberg D. Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Molecular Cell 2008, 32:503-518. [79] Score J, Hidalgo-curtis C, Jones AV, Winkelmann N, Skinner A, Ward D, Zoi K, Ernst T, Stegelmann F, Dohner K, Chase A, Cross NC. Inactivation of polycomb repressive complex 2 components in myeloproliferative and myelodysplastic/myeloproliferative neoplasms. Blood 2012, 119: 1208-1213. [80] Abdel-Wahab O, Adli M, LaFave LM, et al. ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression. Cancer Cell 2012; 22: 180-193. [81] Davies C, Yip BH, Fernandez-Mercado M, Woll PS, Agirre X, Prosper F, Jacobsen SE, Wainscoat JS, Pellagatti A, Boultwood J. Silencing of ASXL1 impairs the granulomonocytic lineage potential of human CD34+ progenitor cells. Br J Hematol 2013; 160: 842-850. [82] Gelsi-Boyer V, Trouplin V, Adelaide J, et al. Mutations of polycomp-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukemia. Br J Haematol, 2009, 145(6): 788-800. [83] Abdel-Wahab O, Pardanani A, Patel J, et al. Concomitant analysis of EZH2 and ASXL1 mutations

in

myelofibrosis,

chronic

myelomonocytic

leukemia

and

blast-phase

myeloproliferative neoplasms. Leukemia, 2011, 25(7):1200-1202. [84] Thol F, Friesen I, Damm F, et al. Prognostic significance of ASXL1 mutations in patients with myelodysplastic syndromes. J Clin Oncol, 2011b, 29(18): 2499-2506. [85] Abdel-Wahab O, Gao J, Adli M, et al. Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo. J Exp Med 2013; 210: 2641-2659. [86] Inoue D, Kitaura J, Togami K, et al. Myelodysplasti syndromes are induced by histone methylation-altering ASXL1 mutations. J Clin Invest 2013; 123: 4627-4640. [87] Triana F, Visconte V, Elson P, et al. Impact of molecular mutations on treatment response to DNMT3 inhibitors in myelodysplasia and related neoplasms. Leukemia 2014; 28:78-87. [88] Bacher U, Haferlach T, Kern W, Haferlach C, Schnittger S. A comparative study of molecular mutations in 381 patients with myelodysplastic syndrome and in 4130 patients with acute myeloid leukemia. Hematologica 2007, 92: 744-752. 40

[89] Christiansen DH, Andersen MK, Desta H, Pedersen-Bjergaard J. Mutations of genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal transduction pathway in therapy-related myelodysplasia and acute myeloid leukemia. Leukemia 2005, 19: 2232-2240. [90] Ceesay MM, Lea NC, Ingram W, et al. The JAK2 V617F mutation is rare in RARS but common in RARS-T. Leukemia 2006, 20: 2060-2061. [91] Liew E, Owen C. Familial myelodysplastic syndromes: a review of the literature. Hematologica 2011, 96: 1536-1542. [92] Imai Y, Kurokawa M, Izutsu K, Hangaishi A, Takeuchi K, Maki K, Ogawa S, Chiba S, Mitani K, Hirai H. Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis. Blood 2000; 96: 3154-3160. [93] Christiansen DH, Andersen MK, Pedersen-Bjergaard J. Mutations of AML1 are common in therapy-related myelodysplasia following therapy with alkylating agents and are significantly associated with deletion or loss of chromosome arm 7q and with subsequent leukemic transformation. Blood 2004, 104: 1474-1481. [94] Streubel B, Valent P, Lechner K, Fonatsch C. Amplification of the AML1 (CBFA2) gene on ring chromosomes in a patient with acute myeloid leukemia and a constitutional ring chromosome 21. Cancer Genet Cytogenet 2001, 124: 42-46. [95] Roumier C, Fenaux P, Lafage M, Imbert M, Eclache V, Preudhomme C. New mechanisms of AML1 gene alteration in hematological malignancies. Leukemia 2003, 17: 9-16. [96] Haferlach T. Molecular genetics in myelodysplastic syndromes. Leuk Res 2012, 36: 14591462. [97] Barjesteh van Wallwiik van Doorn-Khosrovani S, Spensberger D, de Knegt Y, Tang M, Lowenberg B, Delwel R. Somatic heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid leukemia. Oncogene 2005, 24: 4129-4137. [98] Kita-Sasai Y, Horiike S, Misawa S, Kaneko H, Kobayashi M, Nakao M, Nakagawa H, Fuji H. Taniwaki M. International prognostic scoring system and TP53 mutations are independent prognostic indicators for patients with myelodysplastic syndrome. Br J Hematol 2001, 115: 309312. [99] Barlow JL, Drynan LF, Hewett DR, Holmes LR, Lorenzo-Abalde S, Lane AL, Jolin HE, Pannell R, Middleton AJ, Wong SH, Warren AJ, Wainscoat JS, Boultwood J, McKenzie AN. A

41

p53-dependent mechanism underlies macrocytic anemia in a mouse model of human 5qsyndrome. Nature Medicine 2010; 16: 59-66. [100] Jӓdersten M, Saft L, Smith A, et al. TP53 mutations in low-risk myelodysplastic syndromes with del(5q) predict disease progression. J Clin Oncol 2011; 29: 1971-1979. [101] Bally C, Adés L, Renneville A, Sebert M, Eclache V, Preudhomme C, Mozziconacci MJ, de The H, Lehman-Che J, Fenaux P. Prognostic value of TP53 gene mutations in myelodysplastic syndromes and acute myeloid leukemia treated with azacitidine. Lek Res 2014, 38(7): 751-755. [102] Zhang Y, Zhang M, Yang L, Xiao Z. NPM1 mutations in myelodysplastic syndromes and acute lyeloid leukemia with normal karyotype. Leuk Res 2007, 31: 109-111. [103] Laricchia-Robbio L, Premanand K, Rinaldi CR, Nucifora G. EVI1 impairs myelopoiesis by deregulation of PU.1 function. Cancer Res 2009, 69: 1633-1642. [104] Hahn CN, Chong CE, Carmichael CL, Wilkins EJ, Brautingan PJ, Li XC, Babic M, et al. Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia. Nature Genet 2011, 43: 1012-1017. [105] Smith ML, Cavenagh JD, Lister TA, Fitzgibbon J. Mutation of CEBPA in familial acute myeloid leukemia. New Eng J Med 2004, 351: 2403-2407. [106] Kato N, Kitaura J, Doki N, et al. Two types of CEBPA mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models. Blood 2011; 117: 221-233. [107] Thota S, Viny AD, Makishima H, et al. Genetic alterations of the cohesin complex genes in myeloid malignancies. Blood 2014; 124: 1790-1798. [108] Sundaramoorthy S, Vazquez-Novelle MD, Lekomtsev S, Howell M, Petronczki M. Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion. EMBO J 2014; 33: 2623-2642. [109] Thol F, Bollin R, Gehlhaar M, et al. Mutations in the cohesion complex in acute myeloid leukemia: clinical and prognostic implications. Blood 2014; 123: 914-920. [110] Raajimakers MH, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA, Ebert BL, Al-Shahrour F, et al. Bone progenotir dysfunction induces myelodysplasia and secondary leukemia. Nature 2010, 464: 852-857.

42

[111] Kumar MS, Narla A, Nonami A, Mullally A, Dimitrova N, Ball B, McAuley JR, Poveromo L, Kutok JL, Galili N, Raza A, Attar E, Gilliland DG, Jacks T, Ebert BL. Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q- syndrome. Blood 2011, 118: 4666-4673. [112] Wulfert M, Küpper AC, Tapprich C, Bottomley SS, Bowen D, Germing U, Haas R, Gattermann N. Analysis of mitochondrial DNA in 104 patients with myelodysplastic syndromes. Exp Hematol 2008; 36(5):577-586. doi: 10.1016/j.exphem.2008.01.004. [113] Fernandez-Mercado M, Pellagatti A, Di Genua C, et al. Mutations in SETBP1 are recurrent in myelodysplastic syndromes and often coexist with cytogenetic markers associated with disease progression. Br J Hematol 2013; 163: 235- 239. [114] Hou HA, Kuo YY, Tang JL, et al. Clinical implications of the SETBP1 mutation in patients with primary myelodysplastic syndrome and its stability during disease progression. Am J Hematol 2014; 89: 181-186. [115] Genovese G, Kähler AK, Handsaker RE, Lindberg J, Rose SA, Bakhoum SF, Chambert K, et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med 2014; 371: 2477-2487. [116] Jaiswal S, Fontanillas P, Flannick J, Manning A, Gaurman PV, Mar BG, Lindsley RC, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med 2014; 371: 2488-2498. [117] Xie M, Lu C, Wang J, McLellan MD, Johnson KJ, Wendl MC, McMichael JF, et al. Agerelated mutations associated with clonal hematopoietic expansion. Nat Med 2014; 20:1472-1478. Doi:10.1038/nm.3733. [118]

Odenike

O,

Onida

F,

Padron

E.

Myelodysplastic

syndromes

and

myedysplastic/myeloproliferative neoplasms: An update on risk stratification, molecular genetics, and therapeutic approaches including allogenic hematopoietic stem cell transplantation. Asco Educational Book 2015, American Society of Clinical Oncology,. Asco.org.edbook. [119] Gerstung M, Pellagatti A, Malcovati L, et al. Combining gene mutation with gene expression data improves outcome prediction in myelodysplastic syndromes. Nat Commun 2015; 6: 5901.

43

[120] Jan M, Snyder TM, Corces-Zimmerman MR, et al. Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia. Sci Transl Med 2012; 4: 149ra18. [121] Shlush LI, Zandi S, Mitchell A, et al. Identification of preleukemic hematopoietic stem cells in acute leukemia. Nature 2014; 506: 328-333. [122] Lindsley RC, Ebert BL. The biology and clinical impact of genetic lesions in myeloid malignancies. Blood 2013; 122(23):3741-8. doi: 10.1182/blood-2013-06-460295. Epub 2013 Aug 16. [123] Deeg HJ, Scott BL, Fang M, Shulman HM, Gyurkocza B, Myerson D, Pagel JM, Platzbecker U, Ramakrishnan A, Radich JP, Sandmaier BM, Sorror M, Stirewalt DL, Wilson WA, Storb R, Appelbaum FR, Gooley T. Five-group cytogenetic risk classification, monosomal karyotype, and outcome after hematopoietic cell transplantation for MDS or acute leukemia evolving from MDS. Blood 2012, 120:1398-1408. [124] Patnaik MM, Hanson CA, Hodnefield JM, Knudson R, van Dyke DL, Tefferi A. Monosomal karyotype in myelodysplastic syndromes, with or without monosomy 5 or 7, is prognostically worse than an otherwise complex karyotype. Leukemia 2011, 25: 266-270. [125] Belli CB, Bengió R, Aranguren PN, Sakamoto F, Flores MG, Watman N, Nucifora E, Prates MV, Arbelbide J, Larripa I. Partial and total monosomal karyotypes in myedysplastic syndromes: comparative prognostic relevance among 421 patients. Am J Hematol 2011, 86:540545. [126] Hwang KL, Song MK, Shin HJ, Na HJ, Shin DH, Kim JK, Moon JH, Ahn JS, Song IC, Hong J, Lee GW, Chung JS. Monosomal and complex karyotypes as prognostic parameters in patients with International Prognostic Scoring System higher risk myelodysplastric syndrome treated with azacitidine. Blood Research 2014; 49: 234-240. [127] Jian JE, Min YH, Yoon J, Kim I, Lee JH, Jung CW, Shin HJ, et al. Single monosomy as a relatively better survival factor in acute myeloid leukemia patients with monosomal karyotype. Blood Cancer J 2015; 5, e358; doi: 10.1038/bcj.2015.84. [128] McQuilten ZK, Sundararajan V, Wood EM, Curtis DJ, Polizzotto MN, Campbell LJ, Wall M. Monosomal karyotype is associated with worse survival independent of complex karyotype in patients with myelodysplastic syndrome. Blood 2013; 122 (21): 1523. DOI:http://dx.doi.org.

44

[129] Breems DA, Van Putten WLJ, De Greef GE, Van Zelderen-Bhola SL, Gerssen-Schoorl KB, Mellink CH, Nieuwint A, et al. Monosomal karyotype in acute myeloid leukemia: A better indicator of poor prognosis than a complex karyotype. J Clin Oncol 2008; 26: 4791-4797. [130] Valcarcel D, Adema V, Sole F, et al. Complex, not monosomal, karyotype is the cytogenetic marker of poorest prognosis in patients with primary myelodysplastic syndromes. J Clin Oncol 2013; 31: 916-922. [131] Medeiros BC, Othus M, Fang M, Roulston D, Appelbaum FR. Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia: the Southwest Oncology Group (SWOG) experience. Blood 2010; 116: 2224-2228. [132] Yang XF, Sun AN, Yin J, Cai CS, Tian XP, Qian J, Chen SN, Wu DP. Monosomal karyotypes among 1147 Chinese patients with acute myeloid leukemia: prevalence, features and prognostic impact. Asian Pacific J Cancer Prev 2012; 13: 5421-5426. [133] Perdigão J, Gomes da SM. Monosomal karyotype (MK) in myeloid malignancies. Atlas Genet Cytogenet Oncol Hematol 2011; 15: 890-891. [134] Xu X, Johnson EB, Leverton L, Arthur A, Watson Q, Chang FL, Raca G, Laffin JJ. The advantage of using SNP-array in clinical testing for hematological malignancies – a comparative study of three genetic testing methods. Cancer Genetics 2013; 206: 317-326. [135] Arber DA, Hasserjian RP. Reclassifying myelodysplastic syndromes: What’s where in the new WHO and why. Hematology Am Soc Hematol Educ Program. 2015;2015:294-8. doi: 10.1182/asheducation-2015.1.294. [136] Stein EM, Tallman MS. Emerging therapeutic drugs for AML. Blood 2015; DOI: http://dx.doi.org/10.1182/blood-2015-07-604538. [137] Mahfouz RZ, Jankowska A, Ebrahem Q, et al. Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacitidine or decitabine therapy. Clin Cancer Res, 2013, 19(4): 938-948. [138] Qin T, Castoro R, El Ahdab S, et al. Mechanisms of resistance to decitabine in the myelodysplastic syndrome. Plos ONE, 2011, 6(8):e23372. [139] Mallo M, Del Rey M, Ibanez M. et al. Response to lenalidomide in myelodysplastic syndromes with del(5q): influence of cytogenetics and mutations. Br J Hematol 2013; 162: 7486.

45

[140] Campbell RM, Tummino PJ. Cancer epigenetics drug discovery and development: the challenge of hitting the mark. J Clin Invest 2014; 124: 64-69. [141] Kondo Y. Targeting histone methyltransferase EZH2 as cancer treatment. J Biochem 2014; 156: 249-257. [142] Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, Sacks JD, Raimondi A, Majer CR, Song J, Scott MP, Jin L, Smith JJ, Olhava EJ, Chesworth R, Moyer MP, Richon VM, Copeland RA, Keilhack H, Pollock RM, Kuntz KW. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol 2012; 8(11):890-896. doi: 10.1038/nchembio.1084. Epub 2012 Sep 30. [143] Qi W, et al. Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Proc Natl Acad Sci USA 2012; 109: 21360-21365. [144] Margueron R, et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 2009; 461: 762-767. [145] Van Aller GS, Pappalardi MB, Ott HM, Diaz E, Brandt M, Schwartz BJ, Miller WH, Dhanak D, McCabe MT, Verma SK, Creasy CL, Tummino PJ, Kruger RG. Long residence time inhibition of EZH2 in activated polycomb repressive complex 2. ACS Chem. Biol 2014;9 (3): pp 622–629. DOI: 10.1021/cb4008748. [146] McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, Liu Y, Graves AP, Della Pietra A 3rd, Diaz E, LaFrance LV, Mellinger M, Duquenne C, Tian X, Kruger RG, McHugh CF, Brandt M, Miller WH, Dhanak D, Verma SK, Tummino PJ, Creasy CL. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 2012; 492(7427):108-112. doi: 10.1038/nature11606. Epub 2012 Oct 10. [147] Knutson SK, Kawano S, Minoshima Y, Warholic NM, Huang KC, Xiao Y, Kadowaki T, et al. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitutmor activity in EZH2-mutant non-Hodgkin lymphoma. Mol Canncer Ther 2014; 13: 842-854. [148] Xu B, On DM, Ma A, Parton T, Konze KD, Pattenden SG, Allison DF, Cai L, Rockowitz S, Liu S, Liu Y, Li F, Vedadi M, Frye SV, Garcia BA, Zheng D, Jin J, Wang GG. Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLLrearranged leukemia. Blood 2015; 125(2):346-357. doi: 10.1182/blood-2014-06-581082. Epub 2014 Nov 13.

46

[149] Marcucci G, Maharry K, Wu YZ, Radmacher MD, Mrozek K, Margeson D, et al. IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol 2010; 28: 2348-2355. [150] Stein EM. IDH2 inhibition in AML: Finally Progress? Best Pract Res Clin Hematol 2015; 28: 112-115. [151] Clark O, Yen K, Mellinghoff IK. Molecular pathways: isocitrate dehydrogenase mutations in cancer. Clin Cancer Res 2016; 22: 1-7. [152] Liu X, Ling ZQ. Role of isocitrate dehydrogenase ½ (IDH1/2) gene mutations in human tumors. Histol Hematol 2015; 30: 1155-1160. [153] Molenaar R, Patel BJ, Khurshed M, Przychodzen B, Sanikommu Reddy S, Carraway HE, Radivoyevitch T, et al. Ex vivo experiments show that IDH1/2 mutant inhibitors can be safely used as adjuvants to regular chemotherapy in IDH1/2 mutated acute myeloid leukemia. Blood 2015; 126: 3788. [154] Ogawara Y, Matsunaga H, Seki T, Machida Y, Araki K, Kitabayashi I. The role of IDH mutants, which are promising therapeutic targets for acute myeloid leukemia. Blood 2015; 126: 3796. [155] Okoye-Okafor CU, Bartholdy B, Cartier J, Gao E, Pietrak B, Rendina AR, Rominger C, et al. New allosteric inhibitors of mutant IDH1 in acute myeloid leukemia. Blood 2015; 126: 787. [156] DiNardo C, Stein EM, Altman JK, Collins R, DeAngelo DJ, Fathi AT, et al. AG-221, an oral, selective, first-in-class, potent inhibitor of the IDH2 mutant enzyme, induced durable responses in a phase 1 study of IDH2 mutation-positive advanced hematologic malignancies. Hematol Eur Hematol Assoc Annu Meet 2015; 100: 569. [157] Webb TR, Joyner AS, Potter PM. The development and application of small molecule modulators of SF3B1 as therapeutic agents for cancer. Drug Discov Today 2013; 18: 43-49. [158] Bonnal S, Vigevani L, Valcarcel J. The spliceosome as a target of novel antitumor drugs. Nat Rev Drug Discov 2012; 11: 847-859. [159] Lee SC-W, Dvinge H, Kim E, Cho H, Chung YR, Micol J-B, Durham B, et al. Therapeutic targeting of spliceosomal mutant myeloid leukemias through modulation of splicing catalysis. Blood 2015; 126: 4.

47

[160] German S, Gratadou L, Dutertre M, Auboeuf D. Splicing programs and cancer. J Nucleic Acids 2012; 2012:269570. doi: 10.1155/2012/269570. Epub 2011 Oct 24. [161] Garcia-Manero G. Myelodysplastic syndromes: 2015 update on diagnosis, riskstratification and management. Am J Hematol 2015; 90(9): 831-841. Doi: 10.1002/ajh.24102. [162] Gangat N, Patnaik MM, Benga K, et al. Evaluation of revised IPSS cytogenetic risk stratification and prognostic impact of monosomal karyotype in 783 patients with primary myelodysplastic syndromes. Am J Hematol 2013; 88: 690-693. [163] Della-Porta MG, Alessandrino EP, Bacigalupo A, et al. Predictive factors for the outcome of allogenic transplantation in patients with MDS stratified according to the revised IPSS-R. Blood 2014; 123: 2333-2342. [164] Zhang X, Lancet JE, Zhang L. Molecular pathology of myelodysplastic syndromes: new developments and implications for diagnosis and treatment. Leukemia & Lymphoma 2015; 56(11):3022-3030. doi:10.3109/10428194.2015.1037756.

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Table 1. Somatic mutations in MDS

Gene function

Gene symbol

Gene name

location

Mutation; effect

Prognostic implication

Protein function

Signaling

NRAS

Neuroblastoma RAS viral (v-ras) oncogene homolog

1p13.2

Missense; activation

GTPase signal transducer controlling cell growth

FLT3-ITD

Fms-related kinase 3

13q12

CBL

Cblproto-oncogene, E3 ubiquitin pritein ligase

11q23.3

JAK2

Januse kinase 2 V617F

9p24

Insertion; activation. Missense kinase domain; activation Missense; inactivation possibly dominant negative Somatic mutation

Poor (? Increased risk of AML evolution) Unclear

KIT

V-kit HardyZuckerman 4 feline sarcoma viral oncogene homolog Runt-related transcription factor 1

4q11-12

Missense; activation

21q22.3

TP53

Tumor protein p53

17p13.1

ETV6

Ets variant 6

CEBPA

NPM1

Transcription factors

RUNX1

tyrosine

Frequenc y %

~ 10

Common attributes Frequent in CMML, increased risk of leukemic transformation

FMS-like receptor tyrosine kinase, class III Tyrosone kinaseassociated ubiquitin ligase Tyrosine kinases that are associated with cytokine receptors

~2

unclear

Receptor kinase

~1

Missense in the runt domain; dominant negative. Nonsense/indel/splice site distal; nonfunctional Missense/indel; nonfunctional

Negative

Member of transcription protein complex

~ 15

Thrombocytopenia, high risk MDS, common in tMDS, increased risk of AML

Negative

Multiple: DNA repair, apoptosis

~ 10

12p13

Missense/indel; nonfunctional

Negative

ETS family transcription factor

~2

CCAAT/enhancerbinding protein (C/EBP), alpha

19q13.1

Indel/nonsense; nonfunctional

Basic leucine zipper (bZIP) transcription factor; cell cycle regulation

~1-4

Nucleophosmin (nucleolar phosphoprotein

5q35.1

Indel; localization, inactivation

Unclear; favourable in AML when 2 mutations are present Unclear

Asspciated with complex cytogenetics and isolated 5q-, poor prognosis Heterozygous mutations alter protein that is incapable of repressing transcription and shows dominant negative effects Familial predisposition to AML and MDS

Phosphoprotein, nuclear and cytoplasmic

~2

B23,

cytoplasmic p53

Unclear

Negative

tyrosine

~1

~5MDS, ~50% MDS/M PN

Uncommon in MDS, increased frequency in MDS progressing to AML Associated with UPD 11q; frequent in CMML and MD/MPN overlap Increased platelet count in MDS, megakaryocytic proliferation, RARS-T, and AML; associated with a subgroup of del(5q31) High risk, involved in leukemic transformation

Ribosome centrosome protein

biogenesis, duplication, chaperoning,

49

numatrin) BCOR

BCL6 Corepressor, a POZ/zinc finger transcription repressor GATA binding protein 2

Xp11.4

Subclonal driver mutation

Negative

3q21.3

Somatic mutation

Unclear; ? risk of progression to AML

TET2

Tet methylcytosine deoxygenase 2

4q24

Unclear; possibly positive

IDH1

Isicitrate dehydrogenase 1 (NADP+), soluble Isocitrate dehydrogenase 2 (NADP+), mitochondrial DNA (cytosine-5-)methyltransferase 3 alpha Additional sex combs like 1 (Drosophila)

2q33.3

Nonsense/indel throughout; nonfunctional, missense in catalytic domain; nonfunctional Missense; altered function

15q26.1

Missense; altered function

None

2p23

Missense; negative

dominant

20q11

EZH2

Enhancer of homolog (Drosophila)

7q35-36

SF3B1

Splicing factor 3b, subunit 1, 155 kDa

2q33.1

U2AF1

U2small nuclear RNA auxillary factor 1

21q22.3

SRSF2

Serine/arginine-rich splicing factor 2

17q25.1

ZRSR2

Zinc finger (CCCG type), RNA-binding motif and serine/argentine rich 2 Pre-mRNA processing factor 8

Xp22.1

GATA2

Epigenetic modifiers

IDH2

DNMT3A

Histone modification

RNA splicing

ASXL1

PRPF8

zeste 2

17p13.3

A transcription repressor; may influence apoptosis Zinc-finger transcription factors; development and proliferation of hematopoietic cells Alpha ketoglutaratedependent dioxygenase

<5

histone assembly, cell proliferation Associated with RCMD or RAEB

<5

Germline mutations causes familial predisposition to AML and MDS; monocytopenia

~ 20

UPD or microdeletion in 4q associated with advanced age and normal karyotype

NADP-dependent isocitrate dehydrogenase NADP-dependent isocitrate dehydrogenase

~2

Mutually exclusive to TET2 mutations, associated with normal karyotype, IDH 1 mutations impact adverse prognosis

Negative

DNA methyltransferase

~8

Nonsense/indel; dominant negative or activation

Adverse

Chromatin-binding protein

~ 10-20

Missense in the SET domain; non-functional, nonsense/indel; nonfunctional Missense; possible dominant negative or gain of function Missense; possibly dominant negative or gain of function Missense; possible dominant negative or gain of function Nonsense/indel/splice sites; non-functional

Negative

Histone-methylating protein

~7

Favorable

RNA-splicing factor 3b subunit 1, part of U2 U2 small nuclear RNA splicing factor

~ 20

Serine/arginine-rich pre RNA splicing factor Zinc finger RNAbinding associated with U2

~ 12

Missense/Deletions; defects in proof-reading functions

Unclear (poor in AML)

Catalytic step II in pre-mRNA splicing; sister chromatid cohesion

~1-4

Negative

Unclear

Negative

Unclear

~2

~7

~3

Not associated with normal karyotype, increased risk of AML Excess of blasts, intermediate risk IPSS, shorter OS, increased risk of AML UPD or microdeletion in 7q, worse outcome in lowrisk MDS Common in RARS-RARST, coexistence with DNMT3A High risk, enriched in patients with del20q11 and ASXL1 mutations Male gender, advanced age and coexistence of RUNX1, IDH1, ASXL1 mutations Male predilection, isolated neutropenia and clustering with TET2 mutations Associated with ring sideroblast phenotype in common with SRF3B1; retinitis pigmentosa

50

Cohesin complex

STAG2

Cohesin factor

complex

Other mutations

SETBP1

SET binding protein 1

Xq25

18q21.1

Indel; loss of function; subclonal mutation Missense in SKIhomologous domain, Impairs degrdation

Negative

Negative; high risk of leukemic evolution

Regulates separation of sister chromatids during cell division Binds SET, unclear function

1-10%

Associated with RCMD or RAEB

~2-5

Co-occur with –ASXL1 and BL mutations, 7/del(7q); mutual exclusiveness to TP53

Table. 2. EZH2 inhibitors

GSK 126

GSK343

EPZ005687

EPZ-6438

UNC1999

UNC2400

51