New therapeutic approaches in myelodysplastic syndromes: Hypomethylating agents and lenalidomide

Accepted Manuscript New therapeutic approaches in myelodysplastic syndromes: hypomethylating agents and lenalidomide Clémence Loiseau, Ashfaq Ali, Raphael Itzykson PII:

S0301-472X(15)00197-6

DOI:

10.1016/j.exphem.2015.05.014

Reference:

EXPHEM 3272

To appear in:

Experimental Hematology

Received Date: 15 May 2015 Revised Date:

28 May 2015

Accepted Date: 29 May 2015

Please cite this article as: Loiseau C, Ali A, Itzykson R, New therapeutic approaches in myelodysplastic syndromes: hypomethylating agents and lenalidomide, Experimental Hematology (2015), doi: 10.1016/ j.exphem.2015.05.014. 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.

ACCEPTED MANUSCRIPT New therapeutic approaches in myelodysplastic syndromes: hypomethylating agents and lenalidomide

Clémence Loiseau1, Ashfaq Ali2, Raphael Itzykson1,2 1

Department of Hematology, Saint-Louis Hospital, Assistance Publique – Hopitaux de Paris, Paris

Diderot University, Paris, France 2

INSERM U944, Saint-Louis Institute, Paris France

Corresponding Author: Raphael Itzykson, MD PhD Adult Hematology Unit Hopital Saint-Louis 1 avenue Claude Vellefaux 75010 Paris, France Tel: +33142385127 Fax : +33142385128 Email : [email protected]

Abstract: 102 words Text: 5095 words References: 150 Figures: 3 Keywords: myelodysplastic syndromes; hypomethylating agents; azacitidine; decitabine; lenalidomide

ACCEPTED MANUSCRIPT Abstract Recent advances in the treatment of myelodysplastic syndromes have come from the use of the hypomethylating agents decitabine and azacitidine, and the immunomodulatory drug lenalidomide. Their clinical benefit has been demonstrated by randomized phase III clinical trials, mostly in higher risk and del(5q) myelodyslastic syndromes, respectively. However, neither drug appears to eradicate myelodysplastic stem cells, and thus do not currently represent curative options. Here we review data from both clinical and translational research on those drugs, in order to identify their molecular and cellular mechanisms of action, and to delineate paths for improved treatment allocation and further therapeutic advances in myelodysplastic syndromes.

Introduction MDS are clonal disorders arising in aging HSC as a result of cell-intrinsic genetic (and potentially epigenetic) insults, and micro-environmental alterations1. These alterations result in chronic cytopenias, with transfusion-dependent anemia being present in 80% of patients, and increased risk of progression to AML. Prognosis of MDS depends on the number and severity of cytopenias, percentage of bone marrow blasts, and cytogenetics2. MDS is often broadly categorized in lower-risk disease, where therapy mainly aims at coping with anemia, and higher-risk disease, where therapeutic interventions seek to delay progression to AML and improve survival. Hematopoietic stem cell transplantation (HSCT) remains the only curative option in MDS patients, though relapse remains frequent after HSCT. In recent years, two major advances in the treatment of myelodysplastic syndromes have occurred. The DNA methyltransferase (DNMT) inhibitors azacitidine and decitabine, also called ‘hypomethylating agents’ (HMA) have been shown to alter the natural course of higher-risk MDS, significantly prolonging survival of patients compared to other treatment options (aside from transplantation), by delaying progression to AML and improving cytopenias. The immunomodulatory agent lenalidomide durably, though not indefinitely, restores erythropoiesis and induces clonal suppression in lower-risk MDS patients with del(5q), and also improves anemia in a subset of patients without del(5q). Here we review the lessons learnt from clinical trials and translational studies performed with these drugs in MDS.

Hypomethylating agents Epigenetics of MDS The methylome of cancer cells is characterized by global DNA hypomethylation, specifically in heterochromatin, contrasting with focal aberrant hypermethylation of promoter CpG islands that

ACCEPTED MANUSCRIPT have been linked to repression of tumor suppressor gene expression and tumor progression. In euchromatin, the regulatory roles of DNA methylation extend outside of CpG islands. Intragenic DNA methylation is involved in RNA transcription processivity and splicing 3. DNA methylation at distal enhancers can also play an important role in transcriptional regulation 4. In MDS, aberrant promoter hypermethylation of the CDKN2B promoter is a prototypic driver ‘epimutation’ associated with disease progression 5, and HMA were thought to erase these epimutations and thereby delay MDS progression to AML. Comprehensive analyses of MDS methylomes are emerging, and have only recently extended beyond CpG islands, with the advent of whole genome bisulfite sequencing (WGBS)

6

or reduced

representation bisulfite sequencing (RRBS) 7. Early methylome studies focusing on CpG islands identified a signature of promoter hypermethylation in MDS compared to normal hematopoietic stem/progenitors cells, with dynamic accumulation of aberrant hypermethylation marks during disease progression from low- to high- risk and AML 8,9. Intergenic methylation maps remain poorly characterized in MDS, with recent data emerging in the CMML subset 7. Detailed cell sorting experiments have confirmed that aberrant methylation marks arise in MDS ‘stem cells’

10

. Mutations in the DNMT3A gene that encodes one of the three DNA

methyltransferases involved in de novo (as well as maintenance) methylation are present in MDS, though at lower frequencies than in AML. Mutations in the TET2 gene, and to a lesser extent in the IDH1 and IDH2 genes whose many functions include regulation of TET2 enzymatic activity, are prevalent in MDS. TET2 catalyzes hydroxymethylation of methylated cytosines, leading to demethylation. There is limited data regarding the inter-relationship between aberrant methylomes and gene mutations in MDS. In CMML, aberrant methylation seems to be only partly dictated by mutations in epigenetic regulators such as TET2, DNMT3A or ASXL1 7,11. Mutations in splicing factors, which are specific of MDS or secondary AML compared to other subtypes of myeloid neoplasms, also have a distinct methylation signature, presumably because these mutations induce mis-splicing of epigenetic regulators 7. An alternative mechanism of acquisition and selection of aberrant methylation is aging of HSC itself, which leads to accumulation of DNA hypermethylation in polycomb-regulated genes

12

. TET2 mutations provide pre-leukemic HSC with a competitive

advantage over normal HSC in murine models

13,14

, and probably in healthy aging subjects

15,16

. The

HSC ‘self-renewal’ pathways involved in this clonal advantage remain elusive, although methylome studies have pointed to HOX transcription factors 17 and Wnt signaling pathways as candidates 8.

Lessons learnt from clinical trials with HMA

ACCEPTED MANUSCRIPT Azacitidine and decitabine have been compared to conventional care, mostly supportive care or lowdose cytarabine, in several large randomized clinical trials of higher-risk MDS patients: both drugs were shown to induce significant overall response rate, mostly correction of cytopenias (‘hematological improvement’) contrasting with a limited 15-20% complete remission rate, and to delay progression of MDS to AML higher-risk MDS

19

18-21

. Azacitidine was also shown to prolong overall survival in

and AML with lower blast count

22

. No survival benefit was demonstrated for

decitabine, most likely because of difference in study designs 20,21, though it remains possible that the two drugs have different biological activities in MDS. Several lessons have been taken from detailed analyses of these trials that shed light on the possible mechanism of action of these drugs. First, several (4-6) months of exposure to HMA are required to achieve a response, and this response can improve over time

23

. Second, continued exposure to HMA is required to maintain response, and

relapse occurs within months of HMA withdrawal in most cases

24

. Achievement of complete

remission is not a prerequisite for prolonged survival with HMA, as a survival benefit is also seen in patients with hematological improvement despite persistence of blast excess

25,26

. Cytogenetic

response is not a better surrogate for survival benefit 27. Response is difficult to predict with routine clinical variables, though these can identify subsets of patients with different survival with HMA 26, stressing the need for biomarkers of HMA. Finally, outcome of patients after HMA failure is dismal 28.

Metabolism of hypomethylating agents The hypomethylating agents (HMA) azanucleotides 5-azacytidine (azacitdine, AZA) and 5azadeoxycytidine (decitabine, DAC) are cytosine analogs with unmodified sugar moiety, therefore with reduced cytotoxicity upon incorporation into DNA compared to cytarabine. At high doses, these compounds nonetheless display significant cytotoxic activity in AML cells. At lower doses, incorporation of azanucleotides leads to cell-cycle dependent global demethylation, in part because the aza- moiety inhibits maintenance DNA methyl- group transfers during replication, but mostly because azanucleotides covalently bind DNMTs and induce their proteosomal degradation 29. After several rounds of cycling, the DNMT pool is depleted, leading to a profound and global hypomethylation, which is rapidly reversible upon HMA withdrawal. DNA incorporation-independent DNMT depletion has also been described in vitro at high doses of DAC 30. The relevance of such cellcycle independent- DNA hypomethylation at clinically relevant DAC concentrations has not yet been determined. Increasing evidence suggest that inter-individual differences in intra-cellular uptake and metabolism of HMA are responsible for the variable, and hardly predictable clinical activity of these compounds in patients. In vitro experiments

31

and in vivo correlative studies

7,32,33

suggest that sensitivity to

ACCEPTED MANUSCRIPT azanucleotides mostly depends on the level of its incorporation in DNA, which can be modulated by differences in intracellular metabolism or rate of DNA replication. These phenomena as summarized on Figure 1. Similar to other cytosine analogs, intracellular uptake of azanucleotides depends on equilibrative nucleoside transporters hENT1 and hENT2 34. The first step of decitabine intracellular metabolism is conversion to 5-aza-dCMP, which is catalyzed by deoxycytidine Kinase (dCK), whereas uridinecytidine kinase (UCK) is responsible for initial activation of AZA to 5-aza-CMP. Conversely, cytidine deaminase (CDA) catalyzes inactivation of 5-aza-dCTP to 5-aza-dUTP, which can no longer be incorporated into DNA. In vitro resistance to DAC in leukemia cell lines has been assigned to insufficient DNA incorporation, through low expression of DCK and hENT1, and high expression of CDA 31. In different studies, patients who fail to respond to DAC have been reported to have lower hENT1 expression 35, or a higher ratio of CDA/DCK expression than responders 32. One study found lower DCK expression at relapse after achieving response to DAC

35

, but this result has not been

confirmed 32. Lower expression of UCK at baseline predicts resistance to AZA, whereas SNP in CDA have not been reported to impact the outcome of MDS patients treated with AZA 33. Finally, slow DNA replication also increases the rate of DAC incorporation 36, and high expression of genes involved in DNA replication and cell cycle predict clinical response to DAC 7. Hyperexpression of the chemokines CXCL4 and CXCL7 have been associated to resistance to DAC, and this is thought to result from induction of MDS stem cell quiescence 7. Only 20-30% of intracellular 5-azacytidine is incorporated into DNA, and this requires an additional enzymatic step of reduction of 5-aza-CDP to 5-aza-dCDP catalyzed by ribonucleotide reductase (RNR). The majority of AZA is converted to 5-aza-CTP and is incorporated into RNA, with potential consequences on protein synthesis and cell viability that have been incompletely explored in MDS 37,38

. One of the key proteins downregulated through this pathway could be the RNR enzyme itself,

leading to depletion of nucleotide pools and to further cellular stress 39. Recent data have implicated AZA in the reversion of nonsense mediated RNA decay (NMD) in a MYC-dependent fashion 40. The NMD pathway targets transcripts that contain premature stop codons for degradation. Mis-spliced transcripts frequently accumulate in MDS as a result of mutations in components of the spliceosome machinery

41

. Further efforts are required to explore the role of AZA on reverting the post-

transcriptional consequences of splice mutations in MDS, as technological advances now render feasible the monitoring of RNA incorporation of AZA 42.

Epigenetic modulation induced by HMA

ACCEPTED MANUSCRIPT Incorporation of HMA into DNA has been reported to trigger the DNA damage response pathway, which in turn can lead to apoptosis 43. DNA damage resulting from HMA incorporation is repaired by the BER machinery 44, which can be blocked by PARP inhibitors, suggesting possible synergy between both drug classes. Most of the DNA damaging properties of HMA have been reported in in vitro experiments performed in AML cells lines at micromolar concentrations

43-45

, whereas

pharmacokinetic studies indicate that peak plasmatic concentrations of AZA are in the range of 50 – 500 nM 46. The relevance of the genotoxic activity of HMA on the current clinical activity in MDS or AML is thus dubious, although DNMT1 depletion per se has been reported to induce mismatch repair defects in human embryonic cells 47. At clinically active doses, HMA exposure leads to genome-wide uniform demethylation, as assessed by WGBS 6,48. Several studies have also reported genome-wide modifications in gene expression upon AZA or DAC exposure

37,49

. Of note, the patterns induced by the two drugs only partly overlap,

presumably because AZA incorporation into RNA can affect the stability of certain transcripts. However, genome-wide studies focusing on promoter CpG islands failed to identify a clear correlation between promoter demethylation patterns and gene expression modifications upon HMA exposure, whether in vitro 48,50 or in vivo 51. Several non-mutually exclusive hypotheses may account for this puzzling observation. First, overall promoter methylation may not be the best surrogate for methylation ‘locking’ of gene expression. Application of a sophisticated algorithm to WGBS data for instance identified a subset of upregulated genes with a shared hypomethylation signature at a very precise location, near the TSS, suggesting that demethylation of these specific TSS-flanking CpGs by HMA was directly responsible for gene expression upregulation 6. Another explanation is that demethylation outside of promoter CpG islands, notably in distant enhancers, can account for the gene expression modifications induced by HMA 7. In particular, demthylation of the PU.1 upstream regulatory element (URE) may account for the differentiating effect of AZA 52. Finally, demethylation of CpG promoters may also not be sufficient to induce gene expression without concomitant modification of repressive histone marks

53

. This key observation has set the ground for the

combination of HMA with other classes of ‘epidrugs’, in particular HDAC inhibitors.

Cellular consequences of demethylation The cellular consequences of clinically-relevant HMA exposure in MDS or AML remain elusive, notably because of the lack of a suitable in vivo murine model for this study. Most hypotheses are thus based on in vitro experiments or on correlative studies investigating potential biomarkers of HMA activity. They are summarized in Figure 2.

ACCEPTED MANUSCRIPT Many groups have focused on investigating cell death induced by HMA, mostly in AML cell lines. Autophagic cell death has been reported in a MDS cell line upon AZA exposure, and hyperexpression of the anti-apoptotic protein BCL2L10 correlates with resistance to AZA in patients

54,55

. Studies

investigating the induction of apoptosis (or autophagic cell death) in the recently identified ‘MDS stem cells’

10,56

in vivo are eagerly awaited. It is however unlikely that HMA induce significant MDS

stem cell eradication in the clinical setting: not only is the complete remission rate low with HMA, but response typically requires several months, compared to the few weeks required to achieve remission after intensive chemotherapy. Finally, cytogenetic response is infrequent in patients treated with HMA, and poorly correlates with long term survival 27, suggesting that clonal eradication at the stem cell level is not the main cause of improved outcome in MDS treated by HMA. Other studies, also relying on cell lines, have suggested that HMA exposure, though not inducing MDS/AML stem cell death, may reduce their self-renewal advantage 57,58, in keeping with studies in mice where reduced Dnmt1 expression impaired MLL-AF9 leukemic stem cell self-renewal

59

.

Interestingly, other studies performed in vivo suggest that HMA have an opposite effect on healthy HSC 60,61, although this property is not recapitulated in a Dnmt1+/- haplo-insuficient mouse model 62. These observations raise the intriguing possibility that HMA, rather than eradicating MDS stem cells, reverse their competitive advantage over normal HSC in contributing to hematopoiesis. This model could be addressed more directly by in vivo exposure to HMA in competitive transplant experiments in Tet2 knockout mice, because TET2-mutated MDS have a higher response rate to AZA because Tet2 invalidation provides a competitive advantage to murine HSC

63-65

, and

13,14

. A model of HMA-

induced MDS stem cell quiescence fits better with the transient reduction in leukemic stem cell frequency observed in patients by flow cytometry 66, the delay in progression to AML, and relatively rapid relapse after withdrawal of HMA 24. A third, non-mutually exclusive, direct mechanism of action of HMA could be to enhance the differentiation of MDS progenitor cells. This mechanism could in particular account for the frequent correction of cytopenias noted in clinical trials, which seem to convey part of the survival benefit of HMA

25,26

, where the restored hematopoiesis is often clonal

67,68

. HMA exposure can in particular

modulate the expression level of several key myeloid transcription factors, including PU.1, through its upstream regulatory element

52

, and the CEBP family

69

. In vitro data suggest synergism of these

differentiation properties with growth factors such as G-CSF 52, while limited clinical data indicate that addition of recombinant EPO can synergize with HMA in improving anemia, even in higher-risk MDS patients who have very limited sensitivity to EPO alone 70.

ACCEPTED MANUSCRIPT Whether the response to HMA is TP53-dependent is a matter of controversy. In vitro data suggest that HMA-induced cell death may be P53-independent 71, and in vivo experiments support the notion that HMA-induced differentiation is also P53-independent 58. The latter can be reconciled with the finding that response rates to HMA appear independent of TP53 status or expression 72,73. However, the survival of TP53 patients remains poor under HMA

72,73

, and there is no direct evidence from

randomized trials that the poor prognosis of TP53 is even partly alleviated by HMA. In the AZA-001 trial, the outcome of patients with complex karyotype, half of whom harbor TP53 mutations 74, was dismal regardless of treatment 19,75.

HMA as immunomodulatory drugs Non cell-autonomous mechanisms of action of HMA are also an area of investigation. The bone marrow niche has been involved in the pathogenesis of MDS 76,77, but there is so far limited data on the activity of HMA on the stromal environment of MDS stem and progenitor cells (Götze et al., 13th International Symposium on Myelodysplastic Syndromes, Washington DC, May 2015). Conversely, several authors have studied the consequences of HMA on the immune system of patients with MDS or AML, notably in the context of ASCT. Clinical evidence supports an immunomodulatory role of HMA in MDS: treatment with HMA alleviates auto-immune MDS-related manifestations

78

, and

patients treated with a combination of HMA and donor lymphocyte infusions (DLI) after HSCT seem to be less frequently subject to aggressive GVHD models

68,79

. These findings, as well as data from murine

80,81

, support the contention that HMA have immunomodulatory properties against GVHD yet

preserve allogeneic graft-versus-leukemia effects. This model will be addressed by ongoing trials of HMA maintenance after HSCT. The molecular underpinnings of such dissociated immunomodulatory role remain unclear, but could involve enhanced generation of regulatory T cells (Tregs) through demethylation of the FOXP3 receptor

80,81

. A similar induction of Tregs has been noted in patients

receiving HMA after HSCT 82,83, though the functionality of these expanded Tregs is debated 84,85. HMA may also induce anti-tumoral immune responses outside of the transplant setting, notably by restoring the expression of tumor antigens at the surface of MDS cells. HMA have for instance been shown to induce a CD8+ T-cell response to cancer testis antigens in MDS/AML patients

86,87

.

Prospective studies investigating immune parameters as biomarkers of HMA in higher-risk MDS are thus eagerly awaited, as HMA can be double-edged swords in anti-tumoral immune responses. For instance, recent data suggest that azacitidine demethylates the promoter of PD-1 of on T cells, restoring expression of this auto-inhibitory receptor, potentially leading to exhaustion of antitumoral T-cell mediated responses 88,89.

ACCEPTED MANUSCRIPT

Predicting the response to HMA There have been numerous attempts at validating biomarkers of response or survival benefit in MDS or AML patients treated with HMA. Baseline global methylation levels do not predict response to HMA 7,51,90,91, and, in studies where global methylation has been found to impact overall survival, this impact was also found in cohorts not receiving HMA 91. Conversely, demethylation under HMA has repeatedly been found to predict outcome

90,91

, again stressing the role of variable DNA

incorporation of HMA as a key parameter. Gene expression profiles may predict sensitivity to HMA, and most profiles appear directly or indirectly related to cell cycle or to global regulation of methylation 7, such as mir-29b, which regulates DNMT expression

92

, or MLL5, which has been

proposed to interact with TET2 and thus with active demethylation 93. Mutations in TET2 have been shown by several groups to predict superior response rates to HMA 6365

, whereas evidence regarding mutations in other epigenetic regulators such as DNMT3A or ASXL1 is

more conflictual 7,64,65,94,95. The molecular underpinning of the higher response rate in TET2-mutated patients is currently unknown, and, as previously discussed, is unlikely to reflect differences in global methylation levels. However, the superior response rate of TET2-mutated patients does not translate in a significant survival benefit. TET2 mutations do not have a strong effect on MDS prognosis outside of HMA treatment 96, thus ruling out a potential ‘Simpson’s paradox’ as explanation to this lack of survival benefit, and stressing the need for a better understanding of HMA mode of action to identify relevant biomarkers predicting survival rather than response with HMA.

Improving the therapeutic benefit of HMA Current clinical research aiming at improving the outcome of MDS patients with HMA follow two different but non-exclusive routes: achieving more continuous hypomethylation without undue toxicity, and combining HMA with other drug classes. Current administration schemes of both AZA and DAC are discontinuous (7 and 5 days per monthly cycles in the most frequently used schedules, respectively), and thus only induce transient hypomethylation, with complete genomic remethylation before the start of the next HMA cycle 90. More continuous administration of lower, non-genotoxic doses of DAC seem to yield similar clinical efficacy in MDS 97. Oral azacitidine, though it has variable inter-individual pharmacokinetics, can also potentially achieve more continuous hypomethylation than parenteral administration 46. SGI-110, a dinucleotide of decitabine and deoxyguanosine, is a new HMA that increases in vivo exposure of

ACCEPTED MANUSCRIPT decitabine by preventing deamination of DAC by CDA. Because of its prolonged half-like, SGI-110 could allow more stable hypomethylation, with a favorable toxicity profile due to its lower peak concentration compared to DAC 98. Another approach to improve the efficacy of HMA is to combine it with another drug class. HDAC inhibitors have been thoroughly investigated, because of the abundant in vitro data showing synergism in restoring silenced gene expression. HDAC inhibition can also restore the expression of mir-29b, which in turn downregulates DNMT expression. The levels of expression of mir-29b have indeed been shown to impact response to HMA 92. This observation advocates for the use of HDACi as priming before HMA administration 99. However, this strong pre-clinical rationale has been faced by disappointing clinical data. Randomized trials have yet to demonstrate superiority of a HMA / HDACi combination 100. Several reasons can explain this lack of clinical efficacy. First, detailed in vitro studies have shown that the timing of combination is of critical importance 101. Second, HDACi are in fact most often deacetylase inhibitors, with incomplete specificity for histones. Off-target effects may thwart their activity in leukemic cells, and probably accounts for the limiting GI toxicity of this drug class. Combination of HMA with other classes of ‘epidrugs’ are also being investigated to restore gene expression. These drugs either inhibit the histone demethylase LSD1 involved in erasing activating histone marks such as H3K4me3 102, or instead inhibit the histone methyltransferase EZH2 that writes the inactivating mark H3K27m3

103

. Like HDACs, LSD1 also has non-histone substrates, including

DNMT1. Interestingly, methylation of DNMT1 by LSD1 stabilizes DNMT1, and combination of HMA with LSD1 inhibitors could synergize in inducing global hypomethylation

102

. Combination of HMA

with myeloid differentiating agents such as vitamin D or vitamin A derivatives including RAR and RXR agonists has also been investigated

104-106

, again without clinical evidence for strong synergism.

Finally, the most promising combination of HMA may be with the lenalidomide, though strong biological rationale for such combination is currently lacking. These empirically designed combinations take advantage of the non-overlapping extra-hematological toxicity of these drugs, and of the specific activity of LEN in MDS 107.

Lenalidomide Lenalidomide is a thalidomide derivative with immunomodulatory (IMiD) and anti-neoplastic activities in some solid tumors, in myeloma, lymphoma, and in MDS. IMiDs including lenalidomide, regulate the E3 ubiquitin ligase complex CRBN-CRL4 through the DNA damage-binding protein-1 (DDB1) and cereblon (CRBN) subunits, both blocking the degradation of endogeneous substrates of

ACCEPTED MANUSCRIPT this ubiquitin ligase, and recruiting novel targets for degradation (Figure 3) 108-110. In myeloma and T cells, lenalidomide interaction with the CRBN-CRL4 complex leads to selective ubiquitination and degradation of the lymphoid transcription factors IKZF1 and IKZF3 109.

LEN in 5q- syndromes In early trials of LEN in the treatment of MDS, its clinical activity appeared prominent in patients with lower-risk disease (ie. with predominant anemia and no blast excess) and del(5q) 111,112. In later trials of LEN focusing on del(5q) patients, LEN was found to achieve prolonged RBC transfusion independence in 50-60% of patients, potentially improving survival 113,114. Cytogenetic responses are also noted in half of cases but do not seem to delay transformation to AML or prolong overall survival 115. In MDS, del(5q) induces haploinsufficiency of a commonly deleted region (CDR) that encompasses 40 genes and several miRNAs. A functional screen has identified that haploinsufficiency of the RPS14 gene involved in ribosomal biogenesis plays a major role in the macrocytic anemia of 5q- syndromes 116

. Ribosomal stress resulting from reduced RPS14 gene dosage is thought to result in degradation of

MDM2, in turn activating p53 117. This activation of the P53 pathway, which is also evident in GEP of del(5q) patients

118

, is predominant in the erythroid compartment and it is not certain that it is

involved in the stem cell advantage of del(5q)

117

. Haploinsufficiency of two microRNA contained in

the CDR, mir-145 and mir-146, accounts for the thrombocytosis found in these patients, in part through non cell-autonomous mechanisms, and may as well contribute to the clonal dominance of del(5q)

119

. However, the exact mechanisms leading to clonal advantage of del(5q) MDS stem cells

remains to be fully determined. CSNK1A1, which encodes casein kinase 1A1 and SPARC, a gene involved in cellular adhesion to extracellular matrix, are potential haploinsufficient tumor suppressor genes located in the CDR that may contribute to clonal dominance 120,121. There is conflictual evidence as to whether LEN restores expression of RPS14 122, mir-145 123 or SPARC 120

in del(5q) cells. In particular, in vivo pre/post-treatment comparisons may be influenced by the

reduction in del(5q) cells and concomitant expansion of non-clonal progenitors. In vitro, one group found a statistical link between induction of mir-145 expression by LEN in CD34+ del(5q) stem/progenitor cells and clinical response to LEN 123. A first model of LEN activity in del(5q) MDS cells involves stabilization of MDM2 through inhibition of of two haploinsufficient phosphatases, PP2A and CDC25C. Stabilization of MDM2 in turn leads to P53 degradation, cell cycle reentry, and G2/M arrest of del(5q) cells

124,125

. In keeping with this model,

resistance to LEN has been shown to be associated with restoration of P53 accumulation, and

ACCEPTED MANUSCRIPT inhibition of TP53 expression by an antisense oligonucleotide modestly enhances del(5q) erythropoiesis

126

. Finally, sub-clonal TP53 mutations are associated to lower rates of cytogenetic

responses to LEN, though they do not influence the restoration of erythropoiesis 127. A second, not mutually exclusive mechanism has been reported, so far only in abstract form

128

. In

this study, high throughput proteomic screening identified CSNK1A1 as targeted for degradation by ubiquitination by the cereblon complex upon LEN treatment. The cellular consequences of CSNK1A1 degradation remain to be determined but preliminary data suggest that it induces a p21- and p53mediated response, in keeping with previous observations on the role of TP53 in resistance to LEN 128

. Because these mechanisms require happloinsufficiency of PP2A, CDC25C, or CSNK1A1, they can

account for the selective kill of del(5q) MDS progenitors compared to non-del(5q) progenitors induced by LEN, in keeping with the important rate of cytogenetic response noted in these patients, and with the finding that LEN-induced hematological ‘toxicity’ is correlated with achievement of response. LEN hematological ‘toxicity’ likely reflects suppression of clonal hematopoiesis

129

.

However, LEN does not seem to eradicate MDS stem cells, as demonstrated in elegant studies combining detailed immunophenotyping and FISH

130

. This finding can be reconciled with the

observation that CSNK1A1 haploinsufficiency drives clonal dominance of del(5q) cells, because the same study found that complete abrogation of Csnk1a1 in mice, mimicking the potential action of LEN on del(5q) HSC, conversely impairs HSC self-renewal

121

.

There has been initial controversy as to whether LEN treatment accelerates progression of 5qsyndromes to higher-risk MDS or AML, reports that may have been obscured by the greater propensity of progression in the heavily transfused del(5q) MDS enrolled in the LEN trials

131,132

.

Achievement of erythroid response with LEN prolongs survival, and may even delay progression to AML

114

. Detailed analyses on a limited number of patients suggest that LEN does not prevent the

accumulation of driver mutations in del(5q) stem cells 56. It will be necessary to better understand the selection pressure that LEN exerts on the subclonal TP53 mutations found in ~20% of lower-risk del(5q) MDS patients that often pre-date LEN exposure and confer a poor prognosis 133. High dose LEN induces some CR in higher-risk MDS and AML with del(5q)

134,135

. In higher-risk MDS

and AML, del(5q) is larger, frequently associated to a complex karyotype, and detailed genetic studies have identified an additional CDR in those patients

136

. Whether PP2A, CDC25C or CSKN1A1

are involved in these responses remains to be determined. Pretreatment of AML cells with LEN has been shown to increase their chemosensitivity through upregulation of mir-181a 137, in keeping with preliminary clinical reports of LEN-chemotherapy combinations 138.

ACCEPTED MANUSCRIPT Non-cell autonomous mechanisms of LEN therapy on MDS clones are also under investigation. Preliminary reports suggest that LEN has a modest effect on MDS MSC

139

. Thalidomide derivatives

including LEN have important immunomodulatory properties (thus their denomination as IMiDs), inducing activation of effector T cells and NK cells, whereas inhibiting Tregs. Such properties may also be involved in LEN activity in MDS. Indeed, one study found that LEN can overcome anergy in MDS T cells 140.

LEN in non-del(5q) anemia LEN also induces 20-25% erythroid responses in lower-risk MDS without del(5q), including in patients resistant or unlikely to respond to EPO

141,142

. In those patients, the mechanism of action of LEN is

likely different, as no cytogenetic response has been noted in informative cases and hematological toxicity is milder than in del(5q) MDS. Only a subset of non-del(5q) MDS have reduced expression of RPS14 and there is to date limited evidence that this confers sensitivity to LEN in non-del(5q) lowerrisk MDS 143. A gene expression signature indicative of impaired erythroid differentiation was initially reported to predict LEN sensitivity in non-del(5q) patients later trials

144

, but has not received independent validation in

142

. In vitro, LEN promotes erythropoiesis and induces fetal hemoglobin expression 145. LEN

acts cell-autonomously at the CFU-E stage of differentiation, even in the absence of ribosomal stress 146

. LEN can also have non-cell autonomous effects on differentiation, notably by modulating

macrophages from the erythroblastic island

146

. EPO receptor (EpoR) signaling requires specialized

membrane microdomains called ‘lipid rafts’ as signaling scaffold, which are partly deficient in MDS erythroid progenitors. Formation of these rafts requires the activity of Rho and Rac GTPases, which can be activated by IMiDs 147. Indeed, treatment with LEN improves raft assembly, enhancing EpoR signaling and erythropoiesis in vitro 148. This model is in keeping with the finding that LEN synergizes with EPO in the treatment of non-del(5q) lower-risk MDS patients who have failed EPO alone 149. LEN activity in non del(5q) MDS is likely to be CRBN-dependent, as a polymorphism located at tyrosine 244 has been shown to predict response, independent of CRBN expression 150.

Conclusion The discovery of HMA and LEN activity in MDS owes more to serendipity and to the dedication of clinician researchers than to rational drug design. In spite of the significant advances made over the recent years, a lot remains to be done to bring together evidence from clinical and biological studies performed with these drugs and shed light on their mechanism of action, in order to allocate them

ACCEPTED MANUSCRIPT more adequately and to improve their activity. This will require relevant murine or xenotransplant models. As none of these drug classes leads to clonal eradication, which probably remains the only road to a cure in MDS, renewed efforts in understanding MDS biology, and ceaseless empirical clinical research will be required to design novel therapeutic approaches for our patients, for whom approval of HMA and LEN only represents the “end of the beginning” in the therapeutic management of MDS.

Figure Legends Figure 1: Metabolism of hypomethylating agents. hENT: equilibrative nucleoside transporters, CDA: cytidine deaminase, RNR: ribonucleotide reductase, UCK: uridine-cytidine kinase, dCK : deoxycytidine kinase. Dotted arrows indicate molecular consequences of HMA that occur at higher doses of HMA.

Figure 2: Potential Mechanisms of action of hypomethylating agents. In vitro or in vivo properties of HMA are italicized. Clinical benefits of HMA are underlined.

Figure 3: Potential Mechanisms of action of lenalidomide. Top panels summarize the presumed targets of LEN in MDS with del(5q), including induction of degradation of CSNK1A1, and inhibition of the PP2A and CDC25C phosphatases. Bottom right panel indicates the synergism of LEN with EPOR in non-del(5q) MDS, through modulation of lipid rafts, and direct modulation of erythropoiesis genes. Bottom left panel recapitulates the cellular consequences of these mechanisms on del(5q) hematopoiesis (inhibition of MDS stem cell self-renewal, eradication of clonal progenitors), and on non-del(5q) MDS progenitors (promotion of erythroid differentiation).

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Figure 1

DAC

hENT

SC

hENT

RI PT

AZA

5-aza-CR UCK 5-aza-CMP

M AN U

5-aza-dCR dCK 5-aza-dCMP

5-aza-CDP

5-aza-dCDP

RNR

5-aza-CTP

D

dNTP pools Depletion?

AC C

EP

Impaired Protein Synthesis

Replication

TE

RNA

5-aza-dCTP

DNMT depletion

5-aza-dUTP DNA

DNA damage?

HSC

M AN U

Enhanced differentiation

MDS

Improvement of cytopenias

AML

AC C

EP

TE

D

Clonal Extinction

Delayed Progression To AML

SC

Healthy Differential Self-renewal

Precursors

RI PT

Progenitors

Enhanced Immunosurveillance

Figure 2

DDB1 CUL4CRBN

RPS14

CRBN

PP2A

CDC25C

SC

MDM2

M AN U

Ub

Ub Ub

2

D

1

EP

AC C

G2/M arrest

EpoR

2

1

HSC

P53 activation

LEN

TE

LEN del(5q)

P53

Ub

CSNK1A1

non del(5q)

LEN

P

LEN

5q32q33

RI PT

Figure 3

Lipid rafts

Precursors

Progenitors

LEN LEN 3

3

STAT HbF…

ACCEPTED MANUSCRIPT

New therapeutic approaches in myelodysplastic syndromes: hypomethylating agents and lenalidomide

Clémence Loiseau, Ashfaq Ali, Raphael Itzykson

Highlights:

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Hypomethylating agents alter self-renewal and differentiation of MDS stem/progenitors.

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Metabolism of hypomethylating agents and cell cycling govern the depth of hypomethylation.

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Azacitidine incorporates into RNA and regulates protein synthesis.

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Lenalidomide induces del(5q) MDS progenitor eradication, sparing del(5q) stem cells.