Analysis of the molecular genetics of acute promyelocytic leukemia in mouse models

Analysis of the Molecular Genetics of Acute Promyelocytic Leukemia in Mouse Models Eduardo M. Rego and Pier Paolo Panda& Acute promyelocytic leukemia (APL) is characterized by reciprocal chromosomal translocations that always involve the retinoic acid receptor-a (RARa) gene on chromosome 17. RARa! variably fuses to the PML, PLZF, NPM, NuMA, and STAT 5b genes (X genes), leading to the generation of X-RARcY and RARwX fusion genes. The aberrant X-RARa proteins retain the dimerization domains of their parental proteins and therefore can act as dominant negative oncogenic products on both RARcx/RXR and X pathways. Studies in transgenic mice harboring X-RAR~u and RARo+X fusion genes and in mice lacking X genes have helped unravel the molecular mechanisms underlying APL leukemogenesis, which lead to the development of novel therapeutic strategies. Moreover, transgenic mouse models of APL were useful to test in vivo the efficacy of these novel therapeutic approaches as well as of drug combinations such as retinoic acid and As,O, that were previously known to be effective as single agents in human APL. Semin Hematol38:54-70. Copyright 0 2001 by W.B. Saunders Company.

A

CUTE PROMYELOCYTIC leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by the expansion of leukemic cells blocked at the promyelocytic stage of the myelopoiesis. According to the French-American-British (FAB) classification of the acute leukemias, APL corresponds to the M3 and M3-variant subtypes’ and accounts for 10% to 15% of AML cases in adults.48,71 APL is distinct from other subtypes of leukemia because of (1) its invariable association with reciprocal chromosomal translocations always involving the retinoic acid receptor-a (RAR~Y) gene on chromosome 17 and (2) its exquisite sensitivity to the differentiating action of all-trdns retinoic acid (ATRA). In 1991, four different groups demonstrated that the APLassociated chromosomal translocation, t(l5; 17), caused the fusion between the RARo! gene locus and the promyelocytic leukemia (PML) gene locus located on chromosome 15,

From the Department of Human Genetics and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division, Graduate School of Medical Sciences, Cornell University, New York, NY. Address reprint requests to Pier Paolo Panda@, MD, PhD, Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Aue, New York, NY 10021. Copyright 0 2001 by W.B. Saunders Company 0037-1963/01/3801-0006$35.00/O doi:lo. 1 OS3lsbem.2001.20865

54

Seminars

in Hematology,

yielding to a fusion gene translated in an aberrant PML-RARa fusion protein.22,31J1,82 Besides PML, four other distinct RARo! partner genes in APL-associated chromosomal translocations have been identified to date: the promyelocytic leukemia zinc finger (PLZF) gene, the nucleophosmin (NPM) gene, the nuclear mitotic apparatus (NuMA) gene, and the signal transducer and activator of transcription 5 b (Stat5 b) located on chromosomes llq23, 5q32, llq13, and 17ql1, respectively.2,16,s7,117 The fact that the RARa locus was invariably involved in APL-specific translocations suggested that the disruption of retinoic acid (RA) pathway might represent the main mechanism leading to leukemia. However, data obtained in transgenic mice (TM) harboring the X-RAR~Y fusion genes, as well as in mutant mice in which the function of the X genes was inactivated by homologous recombination (knock-out mice), showed that the impairment of the X function caused by the X-RARa oncoproteins is also critical for the development of APL in all its subtleties. We review what has been learned by studying the function of RAR~K, its partners, and the aberrant fusion proteins of APL in vivo through the analysis of mouse models, and how this new understanding has allowed the development of novel therapeutic approaches.

Vol 38, No 1 (January),

2001:

pp 54-70

Molecda~ Genetics of APL in Mouse Moa2.h

RARa and Its Fusion Partners Retinoic acid receptors (RARs) are members of the nuclear hormone receptor superfamily that act as RA-inducible transcriptional activators in their heterodimeric form with retinoid X receptors (RXRs), a second class of nuclear retinoid receptor. l3 Among the RARs, the RARo isoform is the most relevant for myelopoiesis.23 Its crucial role in myeloid differentiation is demonstrated by the fact that the expression of RARcY mutants with a deletion in the ligandbinding domain (therefore no longer capable of transcriptional activation) in multipotential hemopoietic cells caused a switch in the lineage commitment from the granulocyticlmonocytic to the mast cell lineage.lo6 Furthermore, Collins et al demonstrated that the expression of a dominant negative mutant of RARa in HL-60 cells rendered these cells resistant to the differentiating activity of RA, which could be restored by the add-back of wild-type RARa, RARP, or RAR?. l9 In addition, the overexpression of wild-type RARo! in murine hemopoietic cells caused an accumulation of promyelocytes in the myeloid colonies in vitro.80,107 However, RARal and RARy knock-out mice present normal hemopoiesis in vivo and show only a modest maturation defect in vitro.57,70 In the vast majority of APL cases, RARa fuses to the PML as a consequence of a reciprocal and balanced translocation between chromosomes 15 and 17 .48,71 PML belongs to a family of proteins characterized by the presence of the RING-B-box-coiled-coil (RBCC) motif,9,93,126 which consists of a C,HC, zinc finger (RING finger) and one or two additional Cysrich regions (B-boxes) followed by a predicted leucine coiled-coil region. The PML RING finger is thought to mediate protein-protein interactions,9 whereas the coiled-coil domain is responsible for the formation of stable PML and PML-RARar homodimers and heterodimers.*> The heterodimerization between PML and PML-RARo results in the delocalization of the former from discrete speckled nuclear structures, the nuclear bodies (NB).*9 Consequently, in APL cells, PML acquires a microspeckled nuclear localization.126 This observation led to the hypothesis that the function of PML is

55

deregulated in the presence of the PML-RARcv fusion oncoprotein.127,128 PML knock-out (PML-‘-) mice (Table 1) have increased susceptibility to spontaneous botryomycotic infections and a reduced number of circulating monocytes and granulocytes, although their hematopoietic precursors retain normal myeloid differentiation capacity.llO These mutant mice, as well as PML-‘cells, are protected from multiple caspase-dependent apoptotic signals such as Fas, tumor necrosis factor (TNF), ceramide, interferons (IFN), and ionizing radiations,l12 demonstrating that PML is essential for multiple apoptotic pathways. mice are more susceptible Moreover, PML-Ito papillomas and carcinomas of the skin when treated topically with dimethybenzanthracene (DMBA) followed by the tumor pro13moting agent 12-O-tetradecanoylphorbolacetate (TPA).liO Furthermore, when injected mice developed B and T with DMBA, PML-‘lymphomas and fibrohistiocytomas at significantly higher frequencies than the wild-type controls.ll” Therefore, in vivo PML acts as a putative tumor suppressor. These results corroborate previous studies in vitro showing that PML expression can inhibit transformation induced by neu (c-erbB2, ERBB2), Ha-ras, mutant ~53, and Ha-ras plus c-myc in NIH3T3 and rat fibroblasts.67,74,75 However, it remains to be established whether in vivo PML would also antagonize the oncogenic activity of these molecules. In addition to controlling programmed cell death, PML inactivation may render cells susceptible to neoplastic transformation, causing genomic instability.l*’ This is suggested by the fact that PML-‘cells have a higher frequency of sister-chromatid exchange than PML+‘+ controls, as reported in cells from patients with Bloom syndrome, who lack the function of the Bloom gene (BLM).12’ In this respect, it is interesting that PML, BLM, and p53 colocalize in the PML-NB.43*83J27 In fact, PML physically interacts with ~53, acting as a p5 3 coactivator. *3 PML plays a role in controlling both p53-induced cellular senescence upon oncogenic transformation by Ras and p53induced apoptosis upon DNA damage.*3ss3 In rare cases, APL is associated with the t(l1;17)(q23;q2 l), which causes the fusion be-

56

Rego and Panab&

Table Genetic

1. Effects

Modification

PML inactivation

PLZF inactivation

RARo-PML

RARo-PLZF

of X Gene

Inactivation

and of RARo-X

Phenotype Increased susceptibility to opportunistic infections and to the development of tumors when challenged with mutagens. PML-/mice are protected from multiple p53dependent and -independent apoptotic stimuli (yirradiation or Fas). PML-/cells are unresponsive to RA and IFN. PML-/cells do not senesce upon oncogenic transformation by Ras and are genomically unstable. Defects in limb and skeletal patterning. Impaired spermatogenesis. PLZF-/mice do not develop leukemia. However, PLZFFcells are protected from apoptosis and display survival advantage. Moreover, there is deregulation of Hox gene expression in PLZF-‘cells. Normal hemopoiesis.

Slow and progressive accumulation of myeloid cells in bone marrow and spleen. Absence of block in myeloid differentiation. Do not develop leukemia.

tween the RARa and PLZF genes.l’J* The latter encodes a transcription factor with repressive activity and is a member of the POK (PO2 and Kriippel) family of proteins, which shares an N-terminal PO2 motif and a C-terminal DNA-binding domain made by Kriippel-like C2-H2 zinc fingers. *~~a~*,~* PLZF is a nuclear protein that accumulates in nuclear speckles, which partially overlap with the PML-NB.54 PLZF has a myeloid growth-suppressive activity. a6 Its expression is accompanied by accumulation of cells in the GO/G1 compartment of the cell cycle and an increased incidence of apoptosis.g6 PLZF knock-out mice have limb and skeletal defects caused by aberrant patterning.5 PLZFFcells in the limb have a greater proliferative capacity and have a reduced rate of apoptosis.5 Moreover, the expression of entire Hox gene complexes such as the AbdB Hox gene cluster, as well as genes encoding bone morphogenetic proteins (BMPs), is altered in the developing limb of PLZF knock-out mice.5

Proteins

on the Leukemogenesis

in APL TM Models

Effect on Leukemogenesis in APL TM Model

References

Increases frequency and accelerates the onset of leukemia in hCG-PML/ RARo TM

Wang et al, 199811o Wang et al, 19981U Guo et al, 200043 Pearson et al, 200083 Zhong et al, 19991z7 Rego et al, submitted

Metamorphoses the CMLlike leukemia typical of hCG-PLZF/RARa TM in an APL-like leukemia

Barna et al, 20005 He et al, 200044

Increases frequency and accelerates the onset of leukemia in hCG-PML/ RARol TM Metamorphoses the CMLlike leukemia typical of hCG-PLZF/RARa TM in an APL-like leukemia

Pollock

et al, 1999ec

He et al, 200044

Because these genes have been implicated in the control of proliferation and differentiation of hemopoietic ce11s,8,1o9 it is conceivable that aberrant PLZF function in PLZF-RARa leukemic cells might lead to survival advantage through the deregulated expression of Hox genes and BMPs.~ In t(5;17)-associated APL, the RARa gene is translocated and fused to the NPM gene, which encodes for a major nonribosomal nucleolar phosphoprotein. 29 NPM is also found involved in t(2;5) and t(3;5) translocations associated with Ki- 1+ anaplastic large-cell lymphoma73,90 and myelodysplasia/AML-M6,1z2 respectively, suggesting that this protein plays an important role in tumorigenesis. NPM has been implicated in the control of ribosome biogenesis, in the shuttle of proteins between cytoplasm and nucleolus, and in the modulation of the transcriptional effects of IRF-1 and YY1.10,56J24 NPM levels are increased in proliferating cells, and overexpression of NPM is able to transform

Moledar

Genetics of APL in Mozrse MoaUs

3T3 cells. Recently, NPM has been shown to be a target of CdK2/CliclinE phosphorylation, which is essential for effective centrosome duplication during cell division.79 Moreover, through its capacity to bind to the tumor suppressor IRF-1, NPM can inhibit the antiproliferative effect of IFN,56 and the binding of NPM to the transcription factor YYl switches its activity from a transcriptional repressor to an activator.loO Unfortunately, this analysis has not yet been corroborated by in vivo studies in NPM knock-out animals, and thus the physiologic function of NPM and how it relates to leukemogenesis remain elusive. In the t( 11; 17)(ql3;q2 l)-associated APL, the fusion partner of RARa is the NuMA gene, 117a1ia which encodes for a ubiquitous expressed nuclear matrix-associated protein.20,21 NuMA is critical for the coordination of mitosis and formation of the nuclei in postmitotic daughter cells.20 NuMA interacts with the DNA matrix attachment regions (MAR), which are important for chromatin compaction and isolation of transcriptionally active loops of DNA. 69~108 Moreover, NuMA may also act as a proapoptotic factor. It is cleaved by caspase 3 and caspase 6, yielding a proteolytic product lacking a C-terminal effector domain, which may interfere with the normal nuclear structure.*l Nevertheless, as for NPM the in vivo role of NuMA is not known, and little is understood of its function in mammalian cells. Recently, a fifth fusion partner of the RARa gene was identified in an APL case presenting a t( 17; 17):Stat5 b, which encodes a transcription factor belonging to the janus kinase (JAK)-Stat signaling pathway.* Several signaling polypeptides have been shown to trigger Stat5 proteins activation, among those relevant to hemopoiesis are: interleukin (IL) 2, IL-3, IL-7, granulocyte colony-stimulating factor (G-CSF) and erythropoi&n.24,34,121 ,$ tat5a and Stat5b knock-out mice present deficiencies in prolactin and growth hormone functions, but normal peripheral blood (PB) counts and normal in vitro colony formation from bone marrow hematopoietic progenitors.ro4 The double-mutant StatSa -‘-b-lhave a profound deficiency in peripheral T-cell proliferation accompanied by

57

reduced numbers of IL-3-, IL-5-, and G-CSFinduced colony-forming units in bone marrow cells.lo4 The critical role of Stat5a and Stat5b in myeloid leukemogenesis has recently been defined.61,95s120 In fact, the leukemia-associated fusion proteins TELIPDGFPR, TELiJAK2, and TEL/ABL all activate the Stat5 proteins, suggesting that this may be a common pathway leading to leukemia by fusion oncoproteins with tyrosine kinase activity.61,120 Furthermore, Stat5alb-deficient mice do not develop the myeloproliferative and lymphoproliferative disorder that wild-type mice develop when their bone marrow cells are exposed to a retrovirus expressing TEL-JAK2, thus pointing at the essential role of these proteins in leukemogenesis.95 Thus, the partners of RARo in APL-specific translocation are structurally diverse and can exert distinct biochemical functions. However, they have all been implicated in the control of cell survival and proliferation, and they are all nuclear proteins, which normally function in the nucleus in homodimericioligomeric compleXes~~,6W%~17 B ecause the aberrant X-RAR~Y proteins retain the dimerization domains of their parental X proteins, they could act as dominant oncogenic products on X pathways (Fig 1). Thus, the interference of X-RARa fusion protein with X function through heterodimerization may have dramatic consequences in leukemia pathogenesis, as recently confirmed genetically by intercrossing X-‘mice with X-RARcz TM (Table 1 and see below). On the other hand, X-RARa can also interfere with RARcx function at multiple levels: (1) through its ability to bind DNA via the RARa DNA-binding domain52; (2) through its ability to bind RA with the same affinity as the wild-type receptor78; and (3) through its ability to form heterodimeric complexes with RXR85 (Fig 1). The concomitant interference with both the X and the RARa pathways could cooperate toward APL leukemogenesis.

APL Transgenic Mouse Models The generation of TM harboring a mutated gene in their genome introduced by microinjetting a fertilized egg is the most straightfor-

58

Rego and Pan&&

Figure 1. Hypothetical model for the mechanisms of action of X-RARo oncoproteins in APL pathogenesis. Schematic representation of the modular organization of RARol, subdivided into its conserved AF domains, whose attributed functions are indicated. PML, PLZF, NPM, NuMA, and Stat5B are designated as X proteins. The X-RARu fusion proteins have the ability to homo/heterodimerize with the X proteins and with RXR, thus potentially interfering with both X and RXR/ RARol pathways. In addition, because PML is part of a TGS RA-dependent RXRol/RARcu nuclear complex (TGS-RANC), the PML-RARol fusion proteins can also interfere with the transcriptional activity of this complex, thus further affecting the retinoic acid pathway. The exact function of the various RARa-X proteins is unclear, but RARo-PLZF retains 7 of the 9 zinc finger domains of PLZF and can bind DNA, thus possibly interfering with the function of PLZF. The dominant negative action of the X-RARa oncoproteins results in leukemogenesis.

ward approach to testing the pathogenic potential of any aberrant gene product. To study the oncogenic potential of the APL associated fusion proteins, we and others have generated a human cathepsin G (hCG) minigene expression vector to direct the expression of the X-RARa and RARwX transgenes to the promyelocytic cellular compartment in mice.18,38,39~47 An expression vector generated using the regulatory elements of the hMRP8 gene was also used for the same purpose. l1 Both cathepsin G and MRPS genes are expressed at the promyelocytic

stage of differentiation. However, whereas cathepsin G gene expression peaks at the promyelocytic stage of myeloid differentiation, the MRPS gene is expressed at high levels in early myeloid progenitors but also in more mature myeloid cells. l l hCG-PML-RARa TM develop a lethal form of leukemia that closely resembles the human ApL.39~~’ He et al*’ reported that overt leukemia in these mice occurred after a long preleukemic phase (> 12 months) and affected between 10% and 15% of the TM. The leukemic cellular population in bone marrow and spleen consisted of myeloid blasts, promyelocytes, and myelocytes that partially retained the ability to terminally differentiate toward mature granulocytes (Table 2). In the PB anemia, thrombocytopenia and leukocytosis with increased number of myeloblasts and promyelocytes was detected.*’ Grisolano et al found that 100% of hCG-PML-RARa TM displayed altered myeloid development, manifested by myeloid expansion in their bone marrow and spleen, but normal PB counts.39 Over the course of 6 to 13 months, 30% of the transgenic mice from three different founder lines developed AML. These leukemias showed similar hematologic features to the hCG-PML-RARa leukemia described by He et a139,*7 (Table 2). The hMRPS-PML-RARa mice generated by Brown et al developed APL between 3 to 9 months of age. i1 The leukemic features in these mice were similar to those in hCG-PML-RARa mice in some aspects, especially the extensive infiltration of leukemic cells. However, leukocytosis was not observed in hMRPS-PMLRARa! TM. Once again, before developing overt leukemia, the hMRPS-PML-RARa mice showed a preleukemic state characterized by the expansion of myeloid cells in the bone marrow and spleenli In addition, all of the hMRPSPML-RARa mice developed epidermal papillomas before or at the same time as leukemia onset (Table 2). This further demonstrated the PML-RARa oncogenic potential activity in other cellular compartments but complicated the analysis of the natural history of the disease and rendered even more difficult the maintenance and propagation of these transgenic lines. hCG-PLZF-RARa TM also develop a lethal

Modular

Table TM Model hCG-PML/RARa

of APL

2. Summary

in Mouse

hMRP&PML/RARa

hCG-PLZF-RARa

hCG-NPM-RARa

knock-in

of the APL TM Models Frequency/Age of Onset of Leukemia

Phenotype

Leukemia is characterized by the infiltration of bone marrow, spleen, and liver by immature myeloid cells. In the PB, anemia and thrombocytopenia without leukocytosis are detected. Leukemia is RA sensitive. TM also develop epidermal papillomas. Leukemia is characterized by extensive leukocytosis, anemia, and thrombocytopenia. Both PB and organ infiltration are by mature myeloid cells (CML-like leukemia). Leukemia is resistant to RA. Some founders developed leukemia with a heterogeneity in cytologic/pathologic features from typical APL to CML-like leukemia. Leukemia is RA sensitive. Only founder analysis is reported. The knock-in of PLM-RARa into the murine cathepsin G locus resulted in increased leukemia incidence, but the disease had similar features as in hCGPML/RARol TM.

leukemia with onset between 6 and 18 months of age. *> This leukemia displayed a dramatic leukocytosis accompanied by modest anemia and thrombocytopenia in the PB but was characterized by the infiltration of all organs by leukemic cells that fully retained the capacity to terminally differentiate (Table 2). Thus, the leukemia in hCG-PLZF-RARa mice lacks the distinctive block of myeloid differentiation that characterizes APL and more closely resembles a chronic myeloid leukemia (CML)-like syndrome. In agreement with our observation, Cheng et al also recently confirmed that the leukemia in their hCG-PLZF-RARa TM were characterized by CML like morphological features.r8 Leukemia in hCG-PLZF-RARa TM was once again preceded by a myeloproliferative disorder affecting the bone marrow and spleen.45,*7 In this phase, proliferation of myeloid precursors increased, but again the cells retained the ability to terminally differentiate into granulocytes.45 Transplantation of BM or spleen cells from both hCG-PML-RARa and hCG-PLZF-RARa mice with leukemia or myeloproliferative disorder caused leukemia in both nude and sublethally irradiated recipient mice.11,18,j9,45,47 Cheng et al reported that

59

Mod.&

Leukemia is characterized by leukocytosis associated with anemia and thrombocytopenia, extensive organ infiltration by leukemic myeloblasts and promyelocytes. Leukemia is RA sensitive. Same as above.

hCG-PML/RARa

PLM-RARo

Genetics

References

lO%-15% >12 mo

He et al, 199 747

30% 6 to 13 mo 30% 3-9 mo

Grisolano et al, 199739 Brown et al,

100% 6-18 mo

He et al, 199845

3 of 7 founders XL2 mo

Cheng et al, 199918

77% 71 wk

Westervelt 199w9

199711

et al,

hCG-NPM-RARa mice develop leukemia after a period of 1atency exceeding 1 year. The leukemia in hCG-NPM-RARa mice were characterized by heterogeneity in cytologyipathology, with a spectrum of manifestations from typical APL to a CML-like syndromeI (Table 2). However, the analysis was performed on founder mice only. We have also generated hCG-NPM-RARo! TM and performed the analysis of their progeny. Preliminary observations show a phenotype distinct from that reported by Cheng et al in that these mice developed a solid myeloid neoplasm that closely resembled human histiocytic sarcomas (E. Rego, D. Ruggero, and P. P. Pandolfi, unpublished observations). Although the conventional transgeni.c approach constitutes a very powerful tool to study leukemogenesis in vivo, it may have several limitations, such as expression of the transgene in the inappropriate cell compartment and its variable level of expression. These aspects are heavily influenced by the number of copies of the transgene integrated into the mouse genome and by the variable and unpredictable position of insertion into the genome (positiondependent effects). To circumvent these limita-

60

Rego and Pando&

tions, a so-called knock-in approach has been used recently to introduce an aberrant gene into a predefined locus in the mouse genome by homologous recombination in mouse embryonic stem (ES) cells. These ES cells are subsequently used to generate a mutant mouse/ embryo and/or chimeras.81 Westervelt and Ley have targeted by knock-in the PML-RAR~Y, oncogene into the murine cathepsin G 10cus.~‘s PML-RAR~Y knock-in mice had a 77% cumulative probability of leukemia by 71 weeks of age, compared with a 15 % leukemia incidence by the same age observed in a concurrent cohort of hCG-PML-RARa TM.lla Thus, the expression of the knocked-in PML-RARa under the control of murine cathepsin G promoter caused leukemia with higher incidence than previously reported in standard transgenic experiments. Phenotypic characteristics of leukemia in the knock-in animals were similar to those of hCG-PML-RARa transgenic animals, including leukocytosis, anemia, thrombocytopenia, massive hepatosplenomegaly with extensive leukemic cell infiltration, and accumulation of promyelocytes in the bone marrow and spleen (Table 2). The difference in the leukemia onset between transgenic and knock-in mice might be explained by the relatively higher level of expression driven by the endogenous murine cathepsin G gene promoter. However, the knock-in approach has also been used to replace a normal gene with an aberrant version of the same gene, thus closely mimicking what occurs at the somatic level in the altered cells in the human disease. Based on the analysis of hCGPLZF-RARa TM, PLZF-RARar seems critical but not sufficient for leukemogenesis and for induction of a blockade in myeloid differentiation. However, these conclusions may be hampered by the use of the hCG promoter, which may not entirely mimic the PLZF promoter in driving appropriate PLZF-RARa expression. To circumvent this disadvantage, to define the role of PLZF-RARa in leukemogenesis, and to unravel its possible dominant negative action on PLZF and/or RARa, we used a knock-in approach and generated knock-in ES cells, in which the PLZF-RARa! fusion gene was targeted into the PLZF locus by homologous recombination. If PLZF-RARcx indeed acts as a

dominant negative inhibitor of PLZF function, a phenotype similar to the one observed in PLZF-‘mutants would be expected in knock-in chimeric or heterozygous mice. Furthermore, these knock-in mice should develop leukemia. The characterization of these mutants is currently ongoing. Comparative analysis of RA response in APL TM models showed the same differential response observed in APL patients harboring different X-RARa proteins. As in human APL, the administration of RA to leukemic hCGPML-RARcx TM in a dose equivalent to that used for the treatment of APL patients induced complete albeit transient disease remission.lr,39,*7 The cells from hCG-PML-RARa leukemia differentiated upon RA treatment both in vitro and in vivo.11,39,47 Bone marrow cells of hCG-NPM-RARa were also sensitive to RA treatment in vitro, as reported for human patients harboring t(5;17).18 On the contrary, although RA could prolong the survival of the hCG-PLZF-RARa leukemic mice, complete remissions were never achieved, precisely as observed in human t( 11;17) APL upon RA treatment.45 Collectively, these data show that (1) the X-RARa! fusion proteins are necessary but not sufficient to cause leukemia; (2) they act as biologically distinct RAR~Y mutants; and (3) the X-RARa directly mediates differential responses to RA. The fact that in all these APL transgenic models leukemia is preceded by a long preleukemic phase suggests several possibilities that are not mutually exclusive (see also following paragraphs): (1) the reciprocal translocation (RARa-X) is also required for fullblown leukemogenesis; (2) other mutations (or secondary hits) are required for full-blown neoplastic transformation; and (3) the relative level of expression of the X-RARa transgene with respect to the wild-type X and RARct genes may not be appropriate.

In Vivo Analysis of the Mechanisms of Leukemogenesis by X-RARcl! The chromosomal translocations associated with APL result in fusion proteins in which the

Mdecuh

Genetics

of APL

B through F domains of RARa (including the DNA-binding and ligand-binding domains) are linked C-terminal to the five different proteins containing self-association domains (Fig 1). Because the various translocations of APL are always balanced and reciprocal, they result in the formation of X-RARa and RARa-X fusion genes and the coexpression of their products in the leukemic blasts in most APL patients.35,4xT71 As mentioned before, the X-RARa fusion products invariably have the potential ability to interfere with both X and RARiRXR pathways (Fig 1). This picture is further complicated by the fact that PML may function in mediating RA responses by participating in a tumor growth-suppressive (TGS) RA-dependent RXRIRARa nuclear complex (TGS-RANC).125 PML-RARct, through its het-

in Mome

61

MO&

erodimerization with PML, interferes with the function of the TGS-RANC, therefore exerting its effects on the functional interaction between PML and RXR/RARa.12j A model has been proposed by which the X-RARa proteins act as biologically distinct RARa mutants, leading to active transcrip(Fig 2). In the abtional repression 32,42,45,66,94 sence of RA, RAR-RXR heterodimers can repress transcription through histone deacetylation by recruiting nuclear receptor corepressors (N-CoR or SMRT), Sin3A, or Sin3B, which in turn form complexes with histone deacetylases (HDACl or 2), resulting in nucleosome assembly and transcriptional repression (Fig 2). Physiologic concentrations of RA cause the dissociation of the corepressor complex and the recruitment of transcriptional

Growth Suppression and Differentiation

APL

RA

resistant leukemia

Mechanisms of transcriptional silencing in APL leukemogenesis. In the absence of RA, the RXR-RARa complex is Figure 2. bound to HDAC, via nuclear corepressors (N-CoR), and is transcriptionally inactive (upper panel). Physiologic doses of RA induce the release of HDAC from the RXR-RARa complex, and the expression of the target genes is initiated. The complexes formed of RXR/PML-RARol/N-CoR/HDAC or of RXR/PLZF-RARo/N-CoR/HDAC cannot be dissociated by physiologic doses of RA; therefore, these oncoproteins act as transcriptional repressors (middle panel). Pharmacologic doses of RA induce the dissociation of the HDAC from the PML-RARol complex, allowing expression of RA-dependent genes, which in turn leads to differentiation of the APL blasts and disease remission. In contrast, PLZF-RARa is still able to repress transcription, even at pharmacologic doses of RA, providing a rationale for the unresponsiveness of t(11;17) APL to treatment as well as for the use of HDACls in APL therapy.

62

Rego and Pandolfi

coactivators to the RAR-RXR complex, thus resulting in the activation of gene expression, which can in turn induce terminal differentiation and growth arrest (Fig 2).*0,1°1 The various X-RARa fusion proteins homodimerize and/or heterodimerize with RXRa and form corepressor complexes with NcoR/SMRT-SinsAHDAC, which are less sensitive to RA (Fig 2).32,42,45,66,94 Therefore, X-RARcz fusion proteins of APL can exert their leukemogenic potential through an aberrant HDACdependent chromatin remodeling and transcriptional repressive ability. The importance of the transcriptional repressive activity of the X-RARa oncogenes on the normal RARa function was underscored by the recent reports by Lin and Evans65 and by Minucci et a1.72 These studies suggest that the transforming ability of the X-RARct fusion proteins may depend on their oligodimerization capabilities. Lin and Evans65 demonstrated that PML-RARQ competes with RARct for binding of RXR and recruits corepressors-HDAC with higher affinity than RARa-RXR. Moreover, these authors showed that the fusion of any dimerization motif to RARa is sufficient for oligodimerization, recruitment of corepressors, and RA unresponsiveness. In addition, Minucci et a172 showed that PML-RARct! can oligodimerize to form trimer-trimer complexes that can bind more than two nuclear corepressor molecules. In complete agreement with this model, PML-RARa and PLZF-RARa, which are both RARar-dominant negative transcriptional repressors, cause RA-sensitive and -resistant leukemia, respectively, in TM.** Furthermore, a PML/RARa mutant (PMLIRARcx-M4) that cannot activate transcription in the presence of RA is fully capable of triggering leukemia in TM, and the corresponding RARct mutant is not,53 showing that activation of RA-dependent transcription by PMLIRARa is dispensable for leukemogenesis. However, PMLRARa, PLZF-RARa, and PML/RARa-M4 are all capable of affecting the X pathway as well. Thus, additional studies in vivo in TM are needed to address whether the sole dominant negative interference with the RARa pathway is crucial for leukemogenesis. Furthermore, at

this stage this simple model does not account for the distinct phenotypes observed in PMLRARa and PLZF-RARa TM because both molecules are effective transcriptional repressors, but only the former can cause a block in myeloid differentiation.

Relevance of the X Functional Inactivation for APL Pathogenesis In APL blasts, the normal gene dosage of X and RARa is reduced to heterozygosity as a consequence of the chromosomal translocation. In contrast, in X-RARct TM, the two normal copies of the gene X and RARa are still intact and may antagonize the dominant negative action of the X-RARa! oncoproteins. To determine whether X inactivation would accelerate leukemia onset and/or penetrance caused by PML-RARa, we first crossed hCG-PML-RARa TM*’ with PMLLmice.llO Compared with hCG-PML-RARa+‘-/PML+‘+ mice, hCGPML-RARct+‘-/PML+‘or PML-‘mutants presented a dramatic decrease in leukemia-free survival (LFS) and increase in the incidence of leukemia (Rego et al, unpublished observations; Table 1). Thus, we showed that PML inactivation contributes to APL leukemogenesis and the inactivation of one PML allele already has a striking effect on the incidence and latency of the disease (Table 1). Similarly, we studied the effects of the PLZF inactivation on the hemopoiesis of PLZFRARa TM. To this end, we intercrossed PLZFRARa TM with PLZF-‘mice* and characterized the leukemogenesis and hemopoiesis in littermates with different PLZF backgrounds. PLZFFmice do not have overt defects in myeloid hemopoiesis. 5 Leukemia onset and incidence were not increased by the inactivation of PLZF (Table 1). However, whereas the PLZF-RARa PLZF+‘+ mice developed a CMLlike leukemia indistinguishable from those observed in our parental PLZF-RARa TM,*5 leukemia in PLZF-RAR&PLZF-‘mice was characterized by a dramatic accumulation of blast/promyelocytic cells in the bone marrow and spleen and the absence of leukocytosis, thus displaying classic features of APL (Table 1). Leukemia in PLZF-RARaIIPLZF heterozygous

Moledar

Genetics of APL in Mouse Models

mice already had a more severe block in differentiation than leukemia in PLZF+‘+ mice. Therefore, similar to what was observed in the RARa-PLZF TM, the reduction in PLZF dose metamorphoses the CML phenotype (typical of PLZF-RARa TM) into a leukemia with classical APL features. These results underscore the relevance of the X pathway in APL leukemogenesis and support the notion that the RARaPLZF fusion protein may act as a dominant negative inhibitor of PLZF function (see next paragraph).

Role of the RARa-X Fusion Proteins RARa-PML transcripts are detected in 70% to 80% of t(15;17) APL cases.rJ7 The reciprocal RAR~Y-PML fusion protein consists of RARa transactivation domain A,r3 fused to the COOH terminus of PML, to which no discrete biochemical functions have been attributed to datelJOJ1 (Fig 1). No apparent difference has been observed in RA sensitivity or clinical outcomes in APL patients who do or do not harbor the RARa-PML transcript.37,63 APL patients with prolonged remissions do not appear to express the PML-RARa transcript, as evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR), but are in some cases still positive for the expression of the RARaPML fusion transcript which can nevertheless be attributed to a differential sensitivity of the RT-PCR assay.lo5 However, an APL patient whose blasts harbored the RARsPML but not the PML-RARa fusion gene was also identified, underscoring the potential relevance of this molecule in leukemogenesis.58 RARa-PML TM do not develop leukemia, but the coexpression of RARa-PML and PMLRARa increased the penetrance and accelerated the onset of leukemia in a PML-RARa TM model (Table 1).86 Thus, RARa-PML acts as a tumor modifier, a molecule that does not cause leukemia per se but whose expression affects tumor onset, burden, or multiplicity. The reciprocal translocation RARsPLZF was detected in 9 of 11 reported cases of APL associated with PLZF-RARa rearrangement.35 RARa-PLZF transcripts were detected in all 6 cases in which the analysis was performed.35 In

63

the RARar-PLZF fusion protein, the RARa transactivation domain A is fused to the last seven zinc fingers of PLZF.36,64 RARa-PLZF can bind DNA through 7 of the 9 Kriippel-type zinc fingers that constitute the PLZF DNAbinding domain.48a71 However, RARa-PLZF does not retain the transcriptional repression BTB/POZ domain of PLZF, capable of negatively regulating transcription through recruitment of histone deacetylases, which is replaced by one of the transacting domains from RARa (A domain).13 Thus, RARa-PLZF can interfere with the transcriptional repressive ability of PLZF.44,45 RARa-PLZF TM displayed a slow and progressive accumulation of myeloid cells in the bone marrow and spleen.** The infiltrating cells retained the ability to terminally differentiate toward mature granulocytes, and the progressive hyperplasia of the myeloidlgranulocytic compartment occurred in the absence of a block of differentiation. After the first year of life, approximately 20% of the mice developed overt splenomegaly, but leukemia was never observed. When coexpressed with PLZF-RARa, RARa-PLZF did not affect leukemia onset or penetrance, unlike a classical tumor modifier. In contrast, comparative analysis of the morphologic and hematologic features of the leukemia presented by PLZF-RARa TM harboring or not harboring the reciprocal RARa-PLZF showed striking differences: although leukemia in PLZF-RAR~u TM without the reciprocal was characterized by leukocytosis in the PB and infiltration in all organs by myeloid leukemic cells that fully retained the capacity to terminally mature, leukemia in TM harboring both PLZF-RARa and RARa-PML (double TM) was characterized by a dramatic accumulation of immature blasts and of cells blocked at the promyelocytic stage of differentiation (Table 1). Furthermore, as in human APL, leukemia was characterized by low platelet counts, anemia, and mildly increased white blood cell counts in the PB.** Hematologic and pathologic follow-up of the double TM showed that as for the PLZF-RARa and PML-RARa TM, leukemia was preceded by a myeloproliferative disorder characterized by accumulation in the bone marrow and the spleen of immature myeloid cells and promyelocytes.

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Thus, RARcx-PLZF does not function as a classical tumor modifier, but rather metamorphoses the CML-like leukemic phenotype observed in single PLZF-RARa TM into an APL-like phenotype. Altogether, the analysis in TM points out the critical importance of both products of the APL-associated translocations. These data also suggest that in APL patients who do not express RARa-X or, more rarely, X-RARo!, the oncogenic function normally exerted by these molecules is probably exerted by additional genetic events yet to be identified.

Therapeutic Trials in APL Transgenic Mouse Models RAand ArsenicTrioxide As previously mentioned, APL harboring t(15; 17) is uniquely sensitive to the differentiating action of RA, becoming the paradigm for therapeutic approaches using differentiating agents.33s71J1*,r15 However, although effective, treatment with RA alone in APL patients induces disease remission transiently, and relapse is inevitable if remission is not consolidated with chemotherapy. In addition, in the majority of cases, relapse is accompanied by RA resistance.28J02J14 Unlike t(15;17) APL, t(l1; 17) leukemias show a distinctly worse prognosis with poor response to chemotherapy and little or no response to treatment with RA, thus defining a new APL syndrome.35s64,92 Analyses of RA response in X-RARa TM are in complete agreement with the results obtained in the treatment of APL patients.11,39,45,88 PMLRARa mice attained transient complete remission with RA treatment, whereas PLZF-RARa mice at the same dose of RA never achieved remission, although RA treatment prolonged survival.45 However, in both PML-RARcz and PLZF-RARcx mice, leukemia relapsed, becoming insensitive to RA. 45 Thus, X-RARa mice represent an invaluable model system to study the mechanisms underlying constitutive and acquired resistance to RA in APL, as well as to develop novel therapeutic approaches to overcome RA resistance and potentiate the therapeutic effects of RA. At the molecular level, in addition to its

critical transcriptional effects, RA acts by inducing the degradation of PML-RARa through caspase activation and direct proteasome targeting. 27,123,129 Arsenic trioxide (As,03), a chemical used in traditional Chinese medicine, is extremely effective in the treatment of APL. Approximately 90% of t(15;17) APL patients treated with As,03 alone achieved complete remission.99plo2 In vitro studies on APL cell lines or fresh APL cells showed that As,03 could act as a growthinhibitory and proapoptotic agent.14,97,111 Lowdose As,Os may trigger partial differentiation of the leukemic blasts.l* As,O,-induced apoptosis might be mediated by down-regulation of BCL-2 gene expression, up-regulation of the expression of the proenzymes of caspase 2 and 3, and activation of both caspase 1 and 3.15J02,111 In addition, As,03 may induce the degradation of the PML-RARa protein through ubiquitination of the PMT. moiety.14,76,97J03 This event could be critical in mediating the biological effects of As,03 in APL. However, NB4-306 cells, an RA-resistant cell line derived from NB4 that no longer expresses the PML-RARa fusion protein, responded to As,03 as the parental NB4 cells. 97,98~111In addition, in vitro, As,O, shows antitumoral and proapoptotic activity in cancer cells that do not harbor the t(15;17).6,9iJ1’ Recently, we and others have analyzed the effects in vivo of the treatment combining RA and As,O, in leukemic PML-RARa and PLZFRARa TM model and in transplantation models using leukemic cells obtained from these mice.59~ss In PML-RAR~J TM, RA + As,03 was more effective than either drug alone in prolonging disease-free and overall survival in both models. In contrast, RA + As,Os did not induce complete disease remission either in PLZF-RARa TM or in nude mice transplanted with PLZF-RARo! cellsSs8 Unexpectedly, therapeutic doses of RA and RA + As,O, could induce the complete degradation of either PML-RAR~Y or PLZF-RARa proteins, suggesting that the maintenance of the leukemic phenotype depends on the continuous presence of the former, but not of the latter.88 These data suggest that As,O, and/or the As,03 + RA combination might be beneficial for the treat-

Molecular Genetics of APL in Mouse Models

ment of t(15;17) but not for t(11;17) APL and prompt an evaluation of the efficacy of this drug combination in t( 15; 17) APL patients at disease presentation.

RA and Cytokines IFN, granulocyte colony-stimulating factor (GCSF), IL-la, IL-4, and TNF were found to increase RA-induced differentiation in HL60 and U937 cells, although these cytokines had no cytodifferentiating effect per se.77,84 In agreement with this notion, the lack of TNF-a expression or the expression of IL-3, G-CSF, or granulocyte-macrophage colony-stimulating factor (GM-CSF) in blasts from APL patients significantly reduced the differentiating potential of RA cells.26 In a relapsed APL patient, IFN could restore RA sensitivity, and complete remission was achieved after combination therapy.55 However, this result could not be confirmed in other patients.ii3 IFN may synergize with RA by stimulating PML, as well as PML-RARa expression.60,77 Among the cytokines that may have a beneficial role in APL treatment, the most promising is without a doubt G-CSF. Cassinat et al reported that G-CSF in combination with RA enhanced the terminal differentiation of fresh t(l5;17)-positive APL blasts.12 Striking granulocytic maturation was also observed in vitro when APL blasts from a t( 11;17) patient were cultured with RA and G-CSF, although treatment with a single agent did not induce differentiation of the leukemic cells.5O When RA and G-CSF were administered to the patient at leukemia relapse, in vivo, granulocytic maturation and consequent complete remission at hematologic, cytogenetic, and RT-PCR levels was achieved.>O Moreover, bone marrow cells isolated from hCG-PLZF-RARa! leukemic mice responded to RA when cultured in a 2% murine spleen cell-conditioned medium (MSCCM) containing IL-3 and GM-CSF (unpublished observation). Therapeutic trials with RA and G-CSF combinations in hCG-PLZFRARa leukemic mice will further assess the efficacy of this promising therapeutic strategy.

RA and HDAC Inhibitors As mentioned above, the X-RARa! fusion proteins can exert their leukemogenic potential

65

through an aberrant HDAC-dependent chromatin remodeling and transcriptional repressive ability. Several classes of HDAC inhibitors (HDACIs) have been identified, including short-chain fatty acids (for example, butyrates) and organic hydroxamic acids (for example, trichostatin A {TSA} and hybrid polar compounds {HPCS)).~~ HDACIs such as TSA, suberanilohydroxamic acid (SAHA), and sodium phenylbutyrate (SPB) invariably exerted a dramatic growth-inhibitory and proapoptotic activity in leukemia cell lines such as NB4 (APL cell line) or in HL60 and U937 cell lines, which do not harbor the APL fusion genes.*” Inhibition of proliferation and induction of apoptosis by HDACIs were potentiated by RA. RAinduced differentiation was potentiated by these HDACIs, although no differentiation could be induced by HDACIs. These results suggest that the association between RA and HDACI might be useful in the treatment of RA-sensitive APL.** Sodium phenylbutyrate (SPB) has been previously used as a single agent in the treatment of /3-thalassemia because of its ability to induce the expression of the y-globin gene.3,25 Recently, we have successfully used SPB in combination with RA for the treatment of one case of APL refractory to multiple chemotherapeutic regimens as well as to RA.i16 In addition, studies in PLZF-RARa TM model showed that TSA and RA in combination exerted a synergistic growth-inhibitory and differentiating activity on PLZF-RARa leukemic cells.*> Strikdespite their marked antitumoral ingly, activity, HDACIs show negligible toxicity in mice at doses that were able to induce accumulation of acetylated histones in PB and bone marrow cells.*(j These promising preliminary results prompted us to initiate preclinical trials in our RA-sensitive and RA-resistant APL leukemia models with HDACIs or HDACIiRA combinations. Once again, these transgenic models of leukemia will be instrumental in testing the efficacy of these novel therapeutic modalities.

Conclusions In summary, the analysis of the molecular pathogenesis of APL is one of the most com-

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pelling examples of the straightforward connection between clinical and basic research. The data generated so far not only illuminate the molecular mechanisms underlying APL pathogenesis, but also provide the molecular basis for understanding the clinical peculiarities of leukemias associated with distinct translocations. Mouse models of APL have been invaluable tools not only to unravel these mechanisms, but also to validate in vivo the efficacy of the new therapeutic strategies that are developed on the basis of this new understanding. Furthermore, what has been learned by studying APL in vivo in the mouse provides strong support for the more general tenet that cancer treatment must be tailored on the basis of the specific genetics and molecular mechanisms of each tumor/leukemia subtype.

10.

11.

12.

13. 14.

15.

References 1. Alcalay M, Zangrilli D, Fagioli M, et al: Expression pattern of the RARaIPML fusion gene in acute promyelocytic leukemia. Proc Nat1 Acad Sci USA 89:4840-4844, 1992 2. Arnould C, Philippe C, Bourdon V, et al: The signal transducer and activator of transcription STAT5b gene is a new partner of retinoic acid receptor alpha in acute promyelocytic-like leukaemia. Hum Mol Genet 8:1741-1749, 1999 3. Atweh GF, Sutton M, Nassif I, et al: Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease. Blood 93:1790-1797, 1999 4. Bardwell VJ, Treisman R: The POZ domain: A conserved protein-protein interaction motif. Genes Dev 8:1664-1677, 1994 5. Barna M, Hawe N, Niswander L, et al: Plzf regulates limb and axial skeletal patterning. Nat Genet 25:166-172, 2000 6. Bazarbachi A, El-Sabban ME, Nasr R, et al: Arsenic trioxide and interferon-alpha synergize to induce cell cycle arrest and apoptosis in human T-cell lymphotropic virus type I-transformed cells. Blood 93:278-283, 1999 7. Bennett JM, Catovsky D, Daniel D, et al: Proposal for the classification of the acute leukemias. Br J Haematol 33:451-458, 1976 8. Bhatia M, Bonnet D, Wu D, et al: Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells. J Exp Med 18931139-1148, 1999 9. Borden KLB, Boddy MN, Lally J, et al: The solution structure of the RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML. EMBO J 14:1532-1541, 1995

16.

17.

18.

19.

20.

21.

22.

Borer RA, Lehner CF, Eppenberger HM, et al: Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56:379-390, 1989 Brown D, Kogan S, Lagasse E, et al: A PMLRARalpha transgene initiates murine acute promyelocytic leukemia. Proc Nat1 Acad Sci USA 94:2551-2556, 1997 Cassinat B, Balitrand N, Zassadowski F, et al: Combination of ATRA with G-CSF or RARa agonists enhances differentiation induction of fresh APL cells. Blood 92:S596a, 1998 (suppl 1, abstr) Chambon P: A decade of molecular biology of retinoic acid receptors. FASEB J 10:940-954, 1996 Chen GQ, Shi XG, Tang W, et al: Use of arsenic trioxide (As203) in the treatment of acute promyelocytic leukemia (APL): I. As203 exerts dosedependent dual effects on APL cells. Blood 89: 3345-3353, 1997 Chen GQ, Zhu J, Shi XG, et al: In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As203) in the treatment of acute promyelocytic leukemia: As203 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood 88:1052-1061, 1996 Chen SJ, Zelent A, Tong JH, et al: Rearrangements of the retinoic acid receptor alpha and promyelocytic leukemia zinc finger genes resulting from t(11;17)(q23;q21) in a patient with acute promyelocytic leukemia. J Clin Invest 91:2260-2267, 1993 Chen Z, Brand NJ, Chen A, et al: Fusion between a novel Kruppel-like zinc finger gene and the retinoic acid receptor-alpha locus due to a variant t(l1;17) translocation associated with acute promyelocytic luekaemia. EMBO J 12:1161-1167, 1993 Cheng G, Zhu X, Men X, et al: Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF-RARalpha and NPM-RARalpha. Proc Nat1 Acad Sci USA 96: 6318-6323, 1999 Collins SJ, Robertson KA, Mueller L: Retinoic acid-induced granulocytic differentiation of HL-60 myeloid leukemia cells is mediated directly through the retinoic acid receptor (RAR-alpha). Mol Cell Biol 10:2154-2163, 1990 Compton DA, Cleveland DW: NuMA is required for the proper completion of mitosis. J Cell Biol 120:947-957, 1993 Compton DA, Szilak I, Cleveland DW: Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis. J Cell Biol 116:1395-1408, 1992 de The H, Chomienne C, Lanotte M, et al: The t( 15 ; 17) translocation of acute promyelocytic leukemia fuses the retinoic acid receptor (Y gene to a novel transcribed locus. Nature 347:558-561, 1990

Moleczllar

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

de The H, Marchio A, Tiollais expression and ligand regulation receptor alpha and beta genes.

67

Genetics of APL in Mome Moakh

P, et al: Differential of the retinoic acid EMBO J 8:429-433,

1989 Dong F, Liu X, de Koning JP, et al: Stimulation of Stat5 by granulocyte colony-stimulating factor (GCSF) is modulated by two distinct cytoplasmic regions of the G-CSF receptor. J Immunol 161: 6503-6509, 1998 Dover GJ, Smith KD, Chang YC, et al: Fetal hemoglobin levels in sickle cell disease and normal individuals are partially controlled by an X-linked gene located at Xp22.2. Blood 80:816-824, 1992 Dubois C, Schlageter MH, de Gentile A, et al: Hematopoietic growth factor expression and ATRA sensitivity in acute promyelocytic blast cells. Blood 83~3264-3270, 1994 Fanelli M, Minucci S, Gelmetti V, et al: Constitutive degradation of PMLiRARalpha through the proteasome pathway mediates retinoic acid resistance. Blood 93:1477-1481, 1999 Fenaux P, Degos L: Differentiation therapy for acute promyelocytic leukemia. N Engl J Med 337:1076-1077, 1997 Feuerstein N, Spiegel S, Mond J: The nuclear matrix protein, numatrin (B23), is associated with growth factor-induced mitogenesis in Swiss 3T3 fibroblasts and with T lymphocyte proliferation stimulated by lectins and anti-T cell antigen receptor antibody. J Cell Biol 107:1629-1642, 1988 Goddard AD, Borrow J, Freemont I, et al: Characterization of a zinc finger gene disrupted by the t( 15; 17) in acute promyelocytic leukemia. Science 254:1371-1374, 1991 Goddard AD, Borrow J, Freemont PS, ec al: Characterization of a zinc finger gene disrupted by the t( I5 ; 17) in acute promyelocytic leukemia. Science 254:1371-1374, 1991 Grignani F, De Matteis S, Nervi C, et al: Fusion proteins of the recinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia. Nature 391:815-818, 1998 Grignani F, Fagioli M, Alcalay M, et al: Acute promyelocytic leukemia: From genetics to treatment. Blood 83:10-25, 1994 Grimley PM, Dong F, Rui H: StatSa and Stat5b: Fraternal twins of signal transduction and transcriptional activation. Cytokine Growth Factor Rev 10:131-157, 1999 Grimwade D, Biondi A, Mozziconacci MJ, et al: Characterization of acute promyelocytic leukemia cases lacking the classic t(15;17): Results of the European Working Party. Groupe Frangais de CytogCnttique Hematologique, Groupe de Fransais d’Hematologie Cellulaire, UK Cancer Cytogenerics Group and BIOMED 1 European CommuniryConcerted Action “Molecular Cytogenetic Diagno-

sis 36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

in

1297-1308, Grimwade terization

Haematological 2000 D, Gorman of cryptic

Malignancies.” P, Duprez rearrangements

Blood

96:

E, et al: Characand variant

translocations in acute promyelocytic leukemia. Blood 90:4876-4885, 1997 Grimwade D, Howe K, Langabeer S, et al: Escablishing the presence of the t(15;17) in suspected acute promyetocytic leukaemia: Cytogenetic, molecular and PML immunofluorescence assessment of patients entered into the M.R.C. ATRA trial. M.R.C. Adult Leukaemia Working Party. Br J Haematol 94:557-573, 1996 Grisolano JL, Sclar GM, Ley TJ: Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice. Proc Nat1 Acad Sci USA 91:89&9-8993, 1994 Grisolano JL, Wesselschmidt Altered myeloid development

RL, Pelicci and acute

PG, et al: leukemia

in transgenic mice expressing PML-RAR alpha under control of cathepsin G regulatory sequences. Blood 891376-387, 1997 Gudas L, Sporn M, Roberts A: The Retinoids: Cellular Biology and Biochemistry of the Retinoids. New York, Raven, 1994 Gueth-Hallonet C, Weber K, Osborn M: Cleavage of the nuclear matrix protein NuMA during apopcosis. Exp Cell Res 233:21-24, 1997 Guidez F, Ivins S, Zhu J, et al: Reduced retinoic acid-sensitivities of nuclear receptor corepressor binding to PMLand PLZF-RARalpha underlie molecular pathogenesis and treatment of acute promyelocytic leukemia. Blood 91:2634-2642, 1938 Guo A, Salomoni P, Luo J, et al: The function of PML in p53-dependent apoptosis. Nat Cell Biol 21730-736, 2000 He LZ, Bhaumik M, Tribioli C, et al: Two hits for promyelocytic leukemia. Mol Cell 6: 113 1-l 14 1, 2000 He LZ, Guidez F, Tribioli C, et al: Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL. Nat Genet I&126-135, 1998 He LZ, Toletino T, Warrell RP Jr, et al: Transcription therapy for cancer effects of histone deacetylase (HDAC) inhibitors on proliferation, apoptosis and differentiation of leukemic cell lines. Blood 92: 510a, 1998 (suppl, abstr) He LZ, Tribioli C, Rivi R, et al: Acute leukemia with promyelocytic features in PMLiRARalpha transgenic mice. Proc Nat1 Acad Sci USA 94:53025307, 1997 He L-Z, Merghoub T, Pandolfi PP: In vivo analysis of the molecular pathogenesis of Acute Promyelocycic Leukemia in the mouse and its therapeutic implications. Oncogene 18:5278-5292, 1999

Rego and Pando&

49.

50.

Hodges M, organization, mia protein 63:297-304,

Tissot C, Howe K, et al: Structure, and dynamics of promyelocytic leukenuclear bodies. Am J Hum Genet 1998

62.

63.

51.

Jansen JH, de Ridder MC, Geertsma WMC, et al: Complete remission of t(l1;17) positive acute promyelocytic leukemia by all-trans retinoic acid and G-CSF. Blood 92, 1998 Kakizuka A, Miller WH Jr, Umesono K, et al:

64.

52.

Chromosomal translocation t( 15 ; 17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell 66663-674, 1991 Kastner P, Perez A, Lutz Y, et al: Structure, localization and classes of retinoic

65.

53.

54.

55.

56.

57.

58.

59.

60.

61.

transcriptional acid receptor

properties alpha fusion

of two proteins

in acute promyelocytic leukemia (APL): Structural similarities with a new family of oncoproteins. EMBO J 11:629-642, 1992 Kogan SC, Hong SH, Shultz DB, et al: Leukemia initiated by PML-RARalpha: The PML domain plays a critical role while retinoic acid-mediated transactivation is dispensable. Blood 95:1541-

66.

1550,2000 Koken MHM, Reid A, Quignon F, et al: Leukemiaassociated retinoic acid receptor alpha fusion partners, PML and PLZF, heterodimerize and colocalize

68.

to nuclear bodies. Proc Nat1 Acad Sci USA 94: 10255-10260, 1997 Koller E, Krieger 0, Kasparu H, et al: Restoration of all-trans retinoic acid sensitivity by interferon in

69.

acute promyelocytic 1155, 1991 Kondo T, Minamino Identification and minlB23lnumatrin

leukaemia.

Lancet

67.

3 38: 1154-

N, Nagamura-Inoue T, et al: characterization of nucleophoswhich binds the anti-oncogenic

70.

transcription factor IRF-1 and manifests oncogenic activity. Oncogene 15:1275-1281, 1997 Labrecque J, Allan D, Chambon P, et al: Impaired granulocytic differentiation in vitro in hematopoi-

71.

etic cells lacking retinoic acid receptors alpha1 and gamma. Blood 92:607-615, 1998 Lafage-Pochitaloff M, Alcalay M, Brunnel V, et al: Acute promyelocytic leukemia cases with nonreciprocal PMLIRARo or RARaiPML fusion genes. Blood 85:1169-1174, 1995

72.

Lallemand-Breitenbach V, Guillemin MC, Janin A, et al: Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J Exp Med 189:10431052, 1999 Lavau C, Marchio A, Fagioli M, et al: The acute promyelocytic leukaemia-associated PML gene is induced by interferon. Oncogene 11:871-876, 1995 Levy DE, Gilliland DG: Divergent roles of STAT1 and STAT5 in malignancy as revealed by gene

73.

74.

75.

76.

disruptions in mice. Oncogene 19:2505-2510, 2000 Li JY, English MA, Ball HJ, et al: Sequence-specific DNA binding and transcriptional regulation by the promyelocytic leukemia zinc finger protein. J Biol Chem 272~22447-22455, 1997 Li YP, Andersen J, Zelent A, et al: RAR alphali RAR alpha2-PML mRNA expression in acute promyelocytic leukemia cells: A molecular and laboratory-clinical correlative study. Blood 90:306-312, 1997 Licht JD, Chomienne C, Goy A, et al: Clinical and molecular characterization of a rare syndrome of acute promyelocytic leukemia associated with translocation (11;17). Blood 85:1083-1094, 1995 Lin RJ, Evans RM: Acquisition of oncogenic potential by RAR chimeras in acute promyelocytic leukemia through formation of homodimers. Mol Cell 5:821-830, 2000 Lin RJ, Nagy L, Inoue S, et al: Role of the histone deacetylase complex in acute promyelocytic leukaemia. Nature 391:811-814, 1998 Liu JH, Mu ZM, Chang KS: PML suppresses oncogenic transformation of NIH/ST3 cells by activated neu. J Exp Med 181:1965-1973, 1995 Liu QR, Chan PK: Formation of nucleophosmini B23 oligomers requires both the aminoand the carboxyl-terminal domains of the protein. Eur J Biochem 200:715-721, 1991 Luderus ME, den Blaauwen JL, de Smit OJ, et al: Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove. Mol Cell Biol 14:6297-6305, 1994 L&in T, Lohnes D, Mark M, et al: High postnatal lethality and testis degeneration in retinoic acid receptor (Y mutant mice. Proc Nat1 Acad Sci USA 90:7225-7229, 1993 Melnick A, Licht JD: Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 93:3167-3215, 1999 Minucci S, Maccarana M, Cioce M, et al: Oligomerization of RAR and AMLl transcription factors as a novel mechanism of oncogenic activation. Mol Cell 5:811-820, 2000 Morris SW, Kirstein MN, Valentine MB, et al: Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science 263:1281-1284, 1994 Mu ZM, Chin KV, Liu JH, et al: PML, a growth suppressor disrupted in acute promyelocytic leukemia. Mol Cell Biol 14:6858-6867, 1994 Mu ZM, Le XF, Glassman AB, et al: The biologic function of PML and its role in acute promyelocytic leukemia. Leuk Lymphoma 23:277-285, 1996 Muller S, Miller WH Jr, Dejean A: Trivalent antimonials induce degradation of the PML-RAR

Molecular

Genetics

of APL

oncoprotein and reorganization of the promyelocytic leukemia nuclear bodies in acute promyelocytic leukemia NB4 cells. Blood 92:4308-4316, 1998 Nason-Burchenal K, Gandini D, Botto M, et al: Interferon augments PML and PMLiRARa expression in normal meyloid and acute promyelocytic cells and cooperates with all-trans-retinoic acid to induce maturation of a retinoid resistant promyelocytic ceil line. Blood 889:3926-3936, 1996 78. Nervi C, Poindexter EC, Grignani F, et al: Characterization of the PML/RARa! chimeric product of the acute promyelocytic leukemia specific t( 15 ; 17) translocation. Cancer Res 52:3687-3692, 1992 79. Okuda M, Horn HF, Tarapore P, et al: Nucleophosmin/B23 is a target of CDK2iCyclin E in centrosome duplication. Cell 103:127-140, 2000 80. Onodera M, Kunisada T, Nishikawa S, et al: Overexpression of retinoic acid receptor alpha suppresses myeloid cell differentiation at the promyelocyte stage. Oncogene 11:1291-1298, 1995 8 1. Pandolfi PP: Knocking in and out genes and tram genes: The use of the engineered mouse to study normal and aberrant hemopoiesis. Semin Hematol 35:136-148, 1998 82. Pandolfi PP, Grignani F, Alcalay M, et al: Structure and origin of the acute promyelocytic leukemia myl/RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties. 77.

83.

84.

85.

86.

87.

58.

Oncogene 6:1285-1292, 1991 Pearson M, Carbone R, Sebastiani C, et al: PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Nature 406:207-210, 2000 Peck R, Bollag W: Potentiation of retinoid-induced differentiation of HL-60 and U937 cell lines by cytokines. Eur J Cancer 27:53-57, 1991 Perez A, Kastner P, Sethi S, et al: PML/RAR homodimers: Distinct DNA binding properties and heteromeric interactions with RXR. EMBO J 12: 3171-3182, 1993 Pollock J, Grisolano J, Goda P, et al: Development of acute promyelocytic leukemia (APML) in transgenie mice expressing both the PML-RAR and the reciprocal fusion protein, RAR-PML. Blood 90: 32Oa, 1997 (suppl, abstr) Redner RL, Rush EA, Faas S, et al: The t(15;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion. Blood 3:882-886, 1996 Rego EM, He LZ, Warrell RP Jr, et al: Retinoic acid (RA) and As203 treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature of the leukemogenic process induced by the PML-RARalpha and PLZFRARalpha oncoproteins. Proc Nat1 Acad Sci USA 97:10173-10178, 2000

in Mouse

89.

90.

91.

92.

93. 94.

95.

96.

97.

98.

99.

100.

101.

Mod&

69

Richon VM, Emiliani S, Verdin E, et al: A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases. Proc Nat1 Acad Sci USA 95:3003-3007, 1998 Rimokh R, Magaud JP, Berger F, et al: A translocation involving a specific breakpoint (~135) on chromosome 5 is characteristic of anaplastic large cell lymphoma (‘Ki-1 lymphoma’). Br J Haematol 71:31-36, 1989 Rousselot P, Hardas B, Pate1 A, et al: The PMLRAR alpha gene product of the t( 15;17) translocation inhibits retinoic acid-induced granulocytic differentiation and mediated transactivation in human myeloid cells. Oncogene 9:545-551, 1994 Sainty D, Liso V, Cantu-Rajnoldi A, et al: A new morphologic classification system for acute promyelocytic leukemia distinguishes cases with underlying PLZFRARA gene rearrangements. Groupe Franfais de Cytogenetique Hematologique, UK Cancer Cytogenetics Group and BIOMED 1 European Community-Concerted Action “Molecular Cytogenetic Diagnosis in Haematological Malignancies.” Blood 96:1287-1296, 2000 Salomoni P, Guo A, Pandolfi PP: PML in tumor suppression. Nat Rev Mol Cell Biol 2000 (in press) Salomoni P, Pandolfi PP: Transcriptional regulation of cellular transformation. Nat Med 6:742-744, 2000 Schwaller J, Parganas E, Wang D, et al: Stat5 is essential for the myeloand lymphoproliferative disease induced by TELIJAKZ. Mol Cell 6:693-704, 2000 Shaknovich R, Yeyati PL, Ivins S, et al: The promyelocytic leukemia zinc finger protein affects myeloid cell growth, differentiation, and apoptosis. Mol Cell Biol 18:5533-5545, 1998 Shao W, Fanelli M, Ferrara FF, et al: Arsenic trioxide as an inducer of apoptosis and loss of PMLiRAR alpha protein in acute promyelocytic leukemia cells. J Nat1 Cancer Inst 90:124-133, 1998 Dermine S, Grignani F, Clerici M, et al: Occurrence of resistance to retinoic acid in the acute promyelocytic cell line NB4 is associated with altered expression of the PML/RARalpha protein. Blood 82:1573-1577, 1993 Shen 2X, Chen GQ, Ni JH, et al: Use of arsenic trioxide (As203) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood 89: 3354-3360, 1997 Shi Y, Lee JS, Galvin KM: Everything you have ever wanted to know about Yin Yang 1. Biochim Biophys Acta 1332:F49-F66, 1997 Smith MA, Parkinson DR, Cheson BD, et al: Retinoids in cancer therapy. J Clin Oncol 10:839864, 1992

70

102.

103.

104.

105.

106.

107.

108.

109.

Rego and Pandolfi

Soignet SL, Maslak P, Wang ZG, et al: Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med 339:1341-1348, 1998 Sternsdorf T, Puccetti E, Jensen K, et al: PIC-1I SUMO-l-modified PML-retinoic acid receptor alpha mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia. Mol Cell Biol 19: 5 170-5 178, 1999 Teglund S, McKay C, Schuetz E, et al: StatSa and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell 93:841850, 199s Tobal K, Saunders MJ, Grey MR, et al: Persistence of RAR alpha-PML fusion mRNA detected by reverse transcriptase polymerase chain reaction in patients in long-term remission of acute promyelocytic leukaemia. Br J Haematol 90:615-618, 1995 Tsai S, Bartelmez S, Heyman R, et al: A mutated retinoic acid receptor-a exhibiting dominant-negative activity alters the lineage development of a multipotent hematopoietic cell line. Genes Dev 6~2258-2269, 1992 Tsai S, Collins SJ: A dominant negative retinoic acid receptor blocks neutrophil differentiation at the promyelocyte stage. Proc Nat1 Acad Sci USA 90:7153-7157, 1993 Tsutsui K, Okada S, Watarai S, et al: Identification and characterization of a nuclear scaffold protein that binds the matrix attachment region DNA. J Biol Chem 268:12886-12894, 1993 van Oostveen J, Bijl J, Raaphorst F, et al: The role of homeobox hematological

110.

111.

112.

113. 114.

115.

116.

117.

118.

119.

120.

121. 122.

123.

genes in normal hematopoiesis and malignancies. Leukemia 13: 1675-

1690, 1999 Wang ZG, Delva L, Gaboli M, et al: Role of PML in cell growth and the retinoic acid pathway. Science 279:1547-1551, 1998 Wang ZG, Rivi R, Delva L, et al: Arsenic trioxide and melarsoprol induce programmed cell death in myeloid leukemia cell lines and function in a PML and PML-RARalpha independent manner. Blood 92:1497-1504, 1998 Wang ‘ZG, Ruggero D, Ronchetti S, et al: PML is essential for multiple apoptotic pathways. Nat Genet 20:266-272, 1998 Warrell RP Jr: Retinoid resistance. Lancet 341:126, 1993 (letter) Warrell RP Jr, de The H, Wang ZY, et al: Acute promyelocytic leukemia. N Engl J Med 329:177189, 1993 Warrell RP Jr, Frankel SR, Miller WH Jr, et al: Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med 324:1385-1393, 1991 Warrell RP Jr, He LZ, Richon V, et al: Therapeutic targeting of transcription in acute promyelocytic

124.

125.

126.

127.

128.

129.

leukemia by use of an inhibitor of histone deacetylase. J Nat1 Cancer Inst 90:1621-1625, 1998 Wells RA, Catzavelos C, Kamel-Reid S: Fusion of retinoic acid receptor alpha to N&IA, the nuclear mitotic apparatus protein, by a variant translocation in acute promyelocytic leukaemia. Nat Genet 17: 109-113, 1997 Wells RA, Hummel JL, De Koven A, et al: A new variant translocation in acute promyelocytic leukaemia: Molecular characterization and clinical correlation. Leukemia 10:735-740, 1996 Westervelt P, Ley TJ: Efficient generation of acute promyelocytic leukemia in mice using a PMLI RARalpha cDNA “knocked in” to the murine cathepsin gene G locus requires Cre-mediated excision of a linked selectable marker cassette. Blood 92:211a, 1998 (suppl 1, abstr) Wilbanks AM, Mahajan S, Frank DA, et al: TEL/ PDGFbetaR fusion protein activates STAT1 and STATS: A common mechanism for transformation by tyrosine kinase fusion proteins. Exp Hematol 28:584-593, 2000 Wilks AF, Oates AC: The JAKiSTAT pathway. Cancer Surv 27:139-163, 1996 Yoneda-Kato N, Look AT, Kirstein MN, et al: The t(3;5)(q25.l;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLFl. Oncogene 12:265-275, 1996 Yoshida H, Kitamura K, Tanaka K, et al: Accelerated degradation of PML-retinoic acid receptor (Y (PML-RARA) oncoprotein by all-truns-retinoic acid in acute promyelocytic leukemia: Possible role of the proteasome pathway. Cancer Res 56:29452948, 1996 Yung BY, Busch H, Chan PK: Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis. Biochim Biophys Acta 826:167-173, 1985 Zhong S, Delva L, Rachez C, et al: A RAdependent, tumour-growth suppressive transcription complex is the target of the PMLRARalpha and T18 oncoproteins. Nat Genet 23:287-295, 1999 Zhong S, Salomoni P, Pandolfi PP: The transcriptional role of PML and the nuclear body. Nat Cell Biol 2:E85-E90, 2000 Zhong S, Ye TZ, Stan R, et al: A role for PML and the nuclear body in genomic stability. Oncogene 18:7941-7947, 1999 Zhong S, Ronchetti S, Freemont PS, et al: Role of SUMO-l-modified PML in nuclear body formation. Blood 95~2748-2752, 2000 Zhu J, Gianni M, Kopf E, et al: Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor alpha (RARalpha) and oncogenic RARalpha fusion proteins. Proc Nat1 Acad Sci USA 96:14807-14812, 1999