Modeling Acute Promyelocytic Leukemia in the Mouse: New Insights in the Pathogenesis of Human Leukemias

Merghoub et al.

Blood Cells, Molecules, and Diseases (2001) 27(1) Jan/Feb: 231–248 doi:10.1006/bcmd.2001.0385, available online at http://www.idealibrary.com on

Modeling Acute Promyelocytic Leukemia in the Mouse: New Insights in the Pathogenesis of Human Leukemias Submitted 12/30/00 (Communicated by M. Lichtman, M.D., 01/03/01)

Taha Merghoub,1 Carmela Gurrieri,1 Francesco Piazza,1 and Pier Paolo Pandolfi1 ABSTRACT: Acute promyelocytic leukemia (APL) is characterized by the expansion of malignant myeloid cells blocked at the promyelocytic stage of differentiation and is associated with reciprocal chromosomal translocations always involving the retinoic acid receptor ␣ (RAR␣) gene on chromosome 17. As a consequence of the translocation, RAR␣ variably fuses to the PML, PLZF, NPM, NuMA, and Stat5b genes (X genes), respectively, leading to the generation of RAR␣-X and X-RAR␣ fusion genes. The aberrant chimeric proteins encoded by these genes, as well as the inactivation of the X and RAR␣ functions, may exert a crucial role in leukemogenesis. To define the molecular genetics of APL and the contribution of each molecular event in APL pathogenesis, we have generated transgenic mice harboring X-RAR␣ and/or RAR␣-X genes as well as mice where the various X genes have been inactivated by homologous recombination. Here we show that while the X-RAR␣ fusion gene is crucial for leukemogenesis, the presence of RAR␣-X and the inactivation of X function are critical in modulating the onset as well as the phenotype of the leukemia. © 2001 Academic Press Key Words: cancer; chromosomal translocation; cytogenetics; leukemia.

INTRODUCTION

therapy. Moreover, in the majority of cases relapse is accompanied with RA resistance (2–7). At the molecular level APL, is associated with reciprocal chromosomal translocations, invariably involving the Retinoic-Acid Receptor ␣ (RAR␣) locus on chromosome 17, that translocates and fuses to a distinct partner gene (for brevity, hereafter referred to as X genes). To date, five different fusion partners of RAR␣ have been identified (Fig. 1). In the vast majority of cases (⬎98%) the RAR-␣ gene is fused to the Promyelocytic Leukemia gene, PML (originally named myl), located on chromosome 15q22 (8 –10). In a small subset of APL, RAR-␣ fuses to the promyelocytic leukemia zinc-finger (PLZF) on 11q23, the nucleophosmin (NPM) gene on 5q35, the nuclear mitotic apparatus (NuMA) gene on 11q13 or to the signal transducer and activator of transcription 5b (STAT5b) gene on 17q11 (11–15). The invariable involvement of RAR␣ in APL

Acute promyelocytic leukemia (APL), the M3 subtype of the French–American–British classification of acute myeloid leukemias (AMLs), accounts for more than 10% of all AMLs. This leukemia is characterized by the clonal expansion of malignant cells blocked at the promyelocytic stage of myeloid maturation (1–3). Because of the unique sensitivity of the APL blasts to the differentiating effects of all-trans retinoic acid (ATRA), this malignancy has become the paradigm for a new therapeutic approach utilizing agents capable of reprogramming the neoplastic cells to differentiate normally. However, treatment with retinoic acid (RA) alone in APL patients induces only transient complete remission of the disease. The relapse is inevitable if remission is not consolidated with conventional chemo-

Correspondence and reprint requests to: Pier Paolo Pandolfi, Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Fax: (212) 717-3374. E-mail: [email protected]. 1 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, New York 10021. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved

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FIG. 1. Schematic representation of the X-RAR␣ and RAR␣-X fusion proteins generated as a consequences of the various APL specific translocations. The modular organization of RAR␣ and its various partners is shown: P, proline rich; R, ring finger domain; B1 and B2, B-boxes 1 and 2; S/P, serine/proline rich; POZ, BTB/POZ domain (Bric a brac, tram-track, broad complex/Pox virus and Zinc finger) Z, zinc-finger; GD, globular domain; MBD, metal binding domain; 1,2, acidic amino acid clusters; SH3, SH2, src-homology domains 3 and 2; TAD, transactivation domain. The arrows indicate junctional points between X and RAR␣ proteins.

made this disease the first convincing example of aberrant transcriptional regulation in cancer pathogenesis (3, 5, 6, 16). The RARs belong in fact to the super family of nuclear hormone receptors, which act as transcription factors. These molecules are involved in fundamental biological processes, such as development and differentiation (17). RARs form heterodimers with a second class of retinoid receptor, the retinoid-X-receptors (RXRs) (17). In the absence of their physiological ligand, 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 (HDAC1 or 2), thereby resulting in nucleosome assembly and transcriptional repression (18). RARs in their heterodimeric form act as ligand-inducible transcriptional activators. RA leads to the dissociation of the corepressors complex, determining the re-

cruitment of transcriptional coactivators to the RAR-RXR complex, thus resulting in the activation of gene expression. This, in turn, can induce terminal differentiation and growth arrest of cells of various histological origins including normal myeloid cells (19). However, cells derived from RAR␣ knock out mice undergo normal myeloid differentiation (20). Thus, the lack of activation of RAR␣ target genes is not sufficient to cause a block in myeloid differentiation and it is instead the disruption of the RAR␣ pathway, through the active repression of RAR␣ target genes that may lead to the block of myeloid differentiation observed in APL. PML is the most frequent partner of RAR␣ in APL. PML has been implicated in several cellular processes including cell proliferation, apoptosis, senescence and tumorigenesis (21). PML belongs to a family of proteins characterized by the presence of a RING-B-box-coiled-coil (RBCC) motif 232

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(22) which consists of a C3HC4 zinc finger (Ring Finger) and one or two additional Cys-rich regions (B boxes) followed by a predicted coiledcoil region (Fig. 1). PML has been shown to act as a tumor suppressor gene. The PML protein is typically found concentrated in discrete nuclear speckles along with other proteins. These discrete structures are named PML nuclear bodies (NB) or PODs (for PML oncogenic domains) (23–28). In APL, PML, together with the other nuclear body components, is delocalized from the NB into aberrant nuclear structures as a result of its heterodimerization with PML-RAR␣ through the coiled-coil domains (25–28). Moreover, treatment of APL patients with RA induces degradation of the PML-RAR␣ fusion protein and subsequent relocalization of the various NB components into nuclear bodies (29, 30). These observations have led to the hypothesis that the function of PML is deregulated in the presence of PML-RAR␣ (2, 3, 30). PLZF is a DNA binding transcriptional repressor belonging to the POK (POZ and Kru¨ppel) family of proteins which shares an N-terminal POZ motif and a C terminal DNA binding domain made by Kru¨ppel-like C2-H2 zinc-fingers (Fig. 1) (11, 31, 32). The POZ domain of PLZF allows PLZF to self-associate and to form heterodimeric complexes with other proteins such as corepressor molecules or other members of the POK family of proteins. In PLZF-RAR␣, the POZ domain recruits corepressors and HDAC to RAR␣ target genes, inhibiting the expression of key genes required for normal myeloid differentiation. In overexpression studies, PLZF has growth suppressive activity in myeloid cell lines (33). In addition, its expression is accompanied by accumulation of cells in the G1/S compartment of cell cycle and increased apoptosis (33, 34), suggesting an important role of PLZF in hemopoietic development. NPM is implicated in APL pathogenesis and involved in the t(2;5) and t(3;5) translocations associated with CD30⫹ anaplastic large cell-lymphoma (35, 36) and myelodysplasia/AML-M6 (40) respectively, suggesting that this protein may play an important role in tumorigenesis. NPM is a major nucleolar phosphoprotein that is more

abundant in tumor cells than in normal resting cells (Fig. 1) (37, 38). NPM has a role in ribosome biogenesis in the shuttle of proteins between the cytoplasm and nucleolus and in the modulation of transcriptional factors such as YY-1 (39, 40). Furthermore, high levels of NPM expression have been associated with increased cell proliferation. NPM has also been shown to be a target of CdK2/ cyclin E phosphorylation, which is essential for effective centrosome duplication during cell division (41). However, the physiological function of NPM and how it relates to leukemogenesis remain still unclear also in view of the lack of in vivo studies. NuMA is a nuclear matrix-associated protein critical for coordination of mitosis (Fig. 1) (42, 43). NuMA interacts with DNA-matrix attachment regions (MAR), which are important for chromatin compaction and isolation of transcriptionally active loops of DNA (44, 45). Moreover, NuMA may also act as a proapoptotic factor. Nevertheless, as for NPM the in vivo role of NuMA is still unclear. Stat5b is a transcription factor belonging to the Janus kinase (JAK)-STAT signaling pathway. Phosphorylation of Stat5b by JAK induces its homodimerization as well as its heterodimerization with Stat5a, leading to gene transcription regulation (46, 47). This transcription factor has been involved in hematopoiesis and several evidences have shown Stat5 to be part of the signaling pathways for a number of cytokines relevant for hematopoiesis such as G-CSF, GM- CSF, IL-2, IL3, IL7, and erythropoietin (48 –51). APL translocations result in the generation of X-RAR␣ and RAR␣-X fusion genes and the coexpression in the leukemic blasts of their products, which are identical in their RAR␣ moieties (Fig. 1) (2, 3). It may therefore appear that the disruption of the RAR␣ function is the major and only cause of APL. Indeed, the RAR␣ portion is able to mediate heterodimerization with RXR, as well as ligand and DNA binding through the RAR␣ RA and DNA binding domains, respectively (2, 3, 29). However, despite their diversity, the various X-RAR␣ fusion molecules are able to form heterodimeric complexes with the respective X protein (2, 3). In addition, the normal gene 233

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a more aggressive clinical behavior, worse prognosis and little or no response to the treatment with ATRA and/or chemotherapy (52). This highlights the fundamental role of the RA pathway, but also suggests that the RAR␣ partner genes could also play important roles in APL pathogenesis. The initial information accumulated on the function of the genes involved in APL and their fusion products was mainly based on studies performed in various leukemic cell lines, which, obviously, do not constitute an ideal system for the study of oncogenic transformation. Recently mouse modeling based both on transgenic and Knock out approaches has been instrumental in unraveling the molecular genetics of APL. Here we will present the different animal models generated and various approaches utilized in order to answer fundamental questions concerning the molecular mechanism underlying APL pathogenesis. FIG. 2. Hypothetical model for the role of the various X-RAR␣ and RAR␣-X fusion proteins in APL pathogenesis. Each X-RAR␣ protein has the ability to heterodimerize with PML, PLZF, NPM, NuMA or Stat5b (X proteins) as well as with RXR. X-RAR␣ can also interfere with RAR␣ transcriptional function on DNA, since it invariably retains the RAR␣ DNA binding domain, as well as through its ability to bind the ligand (RA). The functional inactivation/ interference with these pathways would result in growth advantage, tumor susceptibility and the differentiation block at the promyelocytic stage. The biochemical functions of the various RAR␣-X proteins are currently largely unknown although data obtained in transgenic mice reveal that their contribution to APL leukemogenesis could be critical. By contrast, a discrete biochemical function has been attributed to RAR␣-PLZF. This protein can bind DNA through the PLZF zinc finger DNA binding domain, but loses the PLZF transcriptional repressive ability which is mediated, at least in part, by the N terminal PLZF POZ domain. Thus, RAR␣PLZF can interfere with the biological functions of PLZF through deregulation of its target genes.

RESULTS AND DISCUSSION Transgenic Mouse Models of APL Although several approaches have been utilized to test the potential oncogenic role of an aberrant gene product, the generation of transgenic animals remains a straightforward and effective approach. The key step to this end is the ability to direct the expression of the aberrant gene to a specific target tissue and/or to a restricted cellular compartment, thus mimicking the human malignancy of interest in the experimental animal. The genetic alterations associated with cancer, such as chromosome translocations in the case of leukemia, are acquired somatic lesions, which occur in a specific cell type. The ubiquitous or unrestricted expression of the transgene might result in embryonic lethality or in a defect that is not related to the human disease. In the case of APL the search of an appropriate myeloid specific promoter in order to model APL in the mouse proved not to be an easy task. We and others, have tried a variety of expression vectors which targeted transgene expression ubiquitously and/or to differentiated or totipotent hemopoietic cells un-

function of X is reduced to heterozygosity. As a consequence these fusion products have the potential ability to interfere with both X and RAR/ RXR pathways (Fig. 2). It should also be pointed out that APL cases associated with the translocation t(11;17) involving the PLZF gene, are typically characterized by 234

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hCG-PML-RAR␣ transgenic mice generated by our group (53). Brown et al. have generated transgenic mice expressing PML-RAR␣ under the control of the hMRP8 promoter (57). The resulting hMRP8-PML-RAR␣ mice developed APL at 3–9 months of age (57). The features of leukemia in these mice were similar to those in hCG-PMLRAR␣ mice on some aspects, especially the extensive infiltration of leukemic cells, in several organs. However, no leukocytosis was observed in the PB. Once more, prior to overt leukemia, the hMRP8-PML-RAR␣ went through a preleukemic phase, characterized by the expansion of myeloid cells in the BM and spleen (57). In addition, all the hMRP8-PML-RAR␣ mice developed epidermal papillomas before or simultaneously to the leukemia onset. This may be due to the fact that MRP8 drives expression in the skin as well and further demonstrates the neoplastic activity of PML-RAR␣. However, it further complicated the analysis of the natural history of the disease leading to difficulties in the maintenance and propagation of these transgenic lines. Taken together, the analysis of PML-RAR␣ transgenic mice suggests that PML-RAR␣ is necessary but not sufficient to induce leukemia, and that the leukemia onset may be triggered by additional genetic events which, accumulate throughout time. To test the potential role, and the importance of the X moiety in modulating the function of the X-RAR␣ fusion gene, and to investigate the leukemic potential of other X-RAR␣ fusion gene, we have generated hCG-PLZF-RAR␣ transgenic mice. These mice developed leukemia at a higher frequency and with an earlier onset. All mice succumbed to the disease between 6 and 18 months of age. This leukemia displayed a dramatic leukocytosis accompanied with modest anemia and thrombocytopenia in the PB, but was characterized by the infiltration of leukemic cells, that fully retained the capacity to terminally mature, in all the organs examined. Thus, leukemia in hCG-PLZF-RAR␣ mice resembled more human chronic myeloid leukemia (CML) rather than classical APL, which is on the contrary characterized by a distinctive block in myeloid differentiation at the promyelocytic stage (59, 60). In

successfully. Embryonic lethality or unperturbed hemopoiesis was the invariable outcome. These problems were solved by the generation of promyelocytic specific and pan-myeloid expression vectors such as (i) a human cathepsin-G (hCG) minigene expression vector that restricts the expression of the transgene to the promyelocytic cellular compartment (53–56); (ii) the expression cassette (hMRP8), which drives expression in transgenic mice at high levels in early myeloid progenitors, but also in more mature myeloid cells (57, 58). Role of the X-RAR␣ in leukemogenesis. Several groups including ours have generated mice expressing X-RAR␣ and/or RAR␣-X, in the promyelocytic compartment, in order to elucidate, in vivo, the possible leukemogenic role of these fusion genes (Table 1). We have generated hCG-PML-RAR␣ transgenic mice. These mice developed a form of leukemia that closely resembles human APL. After a long latency period (12 months), 10 to 15% of the mice developed leukemia (55). The full-blown leukemia was preceded by a myeloproliferative stage of variable durations characterized by the accumulation of immature myeloid cells in the spleen and bone marrow (55). The leukemia was characterized by a profound leukocytosis, anemia, thrombocytopenia, and extensive organ infiltration by leukemic cells, including bone marrow, spleen, liver, lymph nodes, kidneys, reproductive organs and lungs. The leukemic cells in bone marrow and spleen consisted of myeloid blasts, promyelocytes and myelocytes that partially retained the ability to terminally differentiate toward mature granulocytes (55). Two other groups have generated PMLRAR␣ transgenic mice. Grisolano et al. have shown that 100% of their hCG-PML-RAR␣ transgenic mice developed a preleukemic syndrome characterized by myeloid expansion in bone marrow and a splenomegaly due to extramedullary hematopoiesis. About 10 to 30% of their animals developed leukemia between 6 to 13 months of age. Although they presented full myeloid maturation in peripheral blood (PB) and bone marrow, these leukemia showed similar features to the 235

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TABLE 1 Phenotype of the X-RAR␣ and RAR␣-X Transgenic Mice

Genetic modification hCG-PML-RAR␣

hCG-PML-RAR␣ hMRP8-PML-RAR␣

hMRP8-PML-RAR␣m4

hMRP8-RAR␣m4 hCG-PLZF-RAR␣

hCG-NPM-RAR␣

hCG-RAR␣-PML hCG-RAR␣-PLZF

Phenotype of the transgenic mutant (TM)

Frequency age of onset

Onset of a RA-sensitive leukemia characterized by leukocytosis, anemia, trombocytopenia, and extensive organ infiltration by leukemic myeloblasts and promyelocytes Same as above

10–15% ⬎12 months

Presence of BM, spleen, and liver infiltration by immature myeloid cells together with anemia and trombocytopenia. RA-sensitive leukemia Onset of acute leukemias with promyelocytic features; anemia, trombocytopenia, and blast/ promyelocytes are present in the PB along with extensive infiltration of the spleen and the liver; RA-resistant leukemia TM mice are healthy; no perturbation in myeloid development RA-resistant CML-like leukemia characterized by conspicuous leukocytosis in the PB and BM failure; the leukemic myeloid infiltrating cells are mature Analysis of founders evidences in some cases development of RA-sensitive leukemia with heterogeneous (from typical APL to CML-like leukemia) features Normal hemopoiesis Slow and progressive accumulation of myeloid cells in BM and spleen; mutants do not develop leukemia in a 2.5year follow-up

Mutual influence of X-RAR␣ and RAR␣-X molecules on the leukemic phenotype hCG PML-RAR␣/RAR␣-PML double transgeneic mice develop APL-like leukemia with higher penetrance

References He et al. (55), Pollock et al. (77)

30% 6–13 months 30% 3–9 months

Grisolano et al. (53)

30% 3–11 months

Kogan et al. (61)

Brown et al. (57)

Kogan et al. (61)

100% 6–18 months

He et al. (59)

3 of 7 founders ⬎12 months

Cheng et al. (56)

100%

agreement with our observation, Cheng et al. confirmed that the leukemia in their hCG-PLZFRAR␣ transgenic mice was characterized by

See hCG-PML-RAR␣ hCG-PLZF-RAR␣/RAR␣-PLZF double transgeneic mice develop leukemia characterized by dramatic accumulation of immature blasts and promyelocytes; anemia and trombocytopenia are present, as well as slight increase in WBC counts in PB; RA-induced differentiation is further reduced compared with single PLZF-RAR␣ TM

He et al. (60)

CML-like morphological features (56). The leukemia was once more preceded by a myeloproliferative disorder (59, 60). In this phase, prolifera236

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tion of myeloid precursors increased, but the cells retained once again the ability to terminally differentiate into granulocytes. Thus, also in the case of PLZF-RAR␣ other genetic events are required in order to trigger full-blown leukemia. However, in this case the latency period is shorter and the penetrance of the disease is complete suggesting that the PLZF moiety confers a higher oncogenic potential to the X-RAR␣ fusion molecule. Preliminary data indicate that hCG-NPMRAR␣ mice, after a latency period exceeding one year, develop a solid myeloid neoplasm that closely resembles human histiocytic sarcomas (E. Rego, D. Ruggero, and P. P. Pandolfi, unpublished observation). In contrast, the hCG-NPMRAR␣ mice generated by Cheng et al. (56) reveal a distinct phenotype: after a long latency (once again exceeding one year) these mice developed leukemia with a phenotype characterized by heterogeneity in cytology/pathology, with a spectrum of manifestations from typical APL to CMLlike syndrome (56). In conclusion, this in vivo analysis demonstrates that the various X-RAR␣ mutants represent biologically distinct RAR␣ mutants in spite of their identity in the RAR␣ moiety. This difference is mostly prominent in comparing hemopoiesis of hCG-PLZF-RAR␣ and hCG-PML-RAR␣ transgenic mice. This is demonstrated by the fact that the hCG-PLZF-RAR␣ fusion protein fails to cause the block at the promyelocytic stage which, is distinctive of APL. Nevertheless, in human t(11;17) blasts, the differentiation block at the promyelocytic stage is observed. In human APL associated with t(11;17), this block might be conferred by the coexistence of the RAR␣-PLZF fusion protein (see below). The specificity of the X-RAR␣ activity and the differentiation block at the promyelocytic stage might therefore be conferred by the X-moiety. Moreover, it has recently been shown that in the PML-RAR␣ transgenic mice, the PML domain plays a critical role while retinoic acid mediated transactivation is dispensable (61). Several PML-RAR␣ mutations, such as the one that impair the ability of the fusion protein to bind RA and to activate transcription both in patients with

RA resistant APL as well as in some RA resistant subclones of the NB4 APL cell line, have been described (62– 65). Kogan et al. have generated transgenic mice with such a PML-RAR␣ mutant (M4) as well as with its RAR␣ counterpart (RAR␣m4) both unable to activate transcription in response to retinoic acid (61) (Table 1). PMLRAR␣m4 transgenic mice developed leukemia indicating that transcriptional activation by PMLRAR␣ is not required for leukemic transformation. However, the characteristics of the leukemia in these mice were different from the one observed in PML-RAR␣ transgenic mice indicating that the ligand responsiveness might influence the leukemic phenotype. The RAR␣m4 transgenic mice did not develop leukemia suggesting that PML-RAR␣ plays a specific and critical role in leukemogenesis, and that the X moiety in the X-RAR␣ is not only important to determine the phenotypic specificity, but it is necessary for leukemogenesis. The comparative analysis of RA treatment in APL transgenic mice, demonstrates that the XRAR␣ fusion proteins directly mediate differential response to RA. Administration of RA to hCG-PML-RAR␣ leukemia mice at a dose equivalent to the one utilized for the treatment of APL patients induced, as in human APL, complete albeit transient disease remission (53, 55, 57). Cells from hCG-PML-RAR␣ leukemias differentiated upon RA treatment in vitro and in vivo (53, 55, 57). Bone marrow cells from hCG-NPMRAR␣ leukemic mice, were also sensitive to RA treatment in vitro, as reported for human patients harboring t(5;17) (56). On the contrary, while RA could prolong the survival of the hCG-PLZFRAR␣ leukemic mice complete remissions were never achieved, precisely as observed in human t(11;17) APL upon RA treatment (59). These data provide conclusive in vivo evidence that X-RAR␣ directly mediates differential response to RA. NuMA-RAR␣ transgenic mice and mice harboring various RAR␣ mutants have been also generated and are currently being characterized. These data taken together demonstrate that: (1) The various X-RAR␣ fusion proteins are necessary but not sufficient to cause leukemia. 237

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This has several possible implications such as that (i) The reciprocal translocation product may be required to trigger full blown leukemia. (ii) Other mutations (or secondary hits) may be required for full blown leukemogenesis, (iii) The relative level of expression of the X-RAR␣ transgene with respect to the wild-type X and RAR␣ genes may not be appropriate. (2) The various X-RAR␣ fusion proteins act as biologically distinct RAR␣ mutants. (3) The X moiety of the X-RAR␣ oncoprotein directly mediates differential response to RA.

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PLZF through binding to its responsive elements (32, 34, 73). To date, PLZF responsive elements have been identified in regulatory elements of several genes such as Cyclin A, Hox B2 and HOXD11 genes (34, 74, 75). The deregulation of these genes by RAR␣-PLZF might confer growth and survival advantage to the leukemic APL blasts. We and others have generated RAR␣-X transgenic mice in order to unravel the possible oncogenic effect of RAR-X in vivo and to study their possible role in modulating X-RAR␣ oncogenic function (60, 77). The hCG-RAR␣-PML transgenic mice did not develop leukemia, but coexpression of PMLRAR␣ with RAR␣-PML significantly increased the penetrance of leukemia while the features of these leukemias were unchanged (Fig. 3, Table 1) (77). Thus, RAR␣-PML does not cause leukemia, but affects tumor burden. Hence RAR␣-PML can be considered as a classic tumor modifier, whereas PML-RAR␣ causes and determines the APL phenotype observed in these transgenic models (Table 1). We have also generated hCG-RAR␣-PLZF transgenic mice (Table 1). These mice displayed a slow progressive accumulation of myeloid cells in the BM and spleen (60). The infiltrating cells retained the ability to terminally differentiate toward mature granulocytes and the progressive hyperplasia of the myeloid/granulocytic compartment occurred in the absence of a distinct block of differentiation. Thus, RAR␣-PLZF although not sufficient for leukemogenesis, can affect myelopoiesis. The characterization of hCG-PLZFRAR␣/RAR␣-PLZF double transgenic mice showed that the coexpression of both fusion genes did not accelerate the leukemia onset in hCGPLZF-RAR␣ mice (Fig. 3, Table 1). However, the comparative analysis of the leukemic features in hCG-PLZF-RAR␣ transgenic mice with hCGPLZF-RAR␣/RAR␣-PLZF double transgenic mice revealed a striking difference. While leukemia in hCG-PLZF-RAR␣ transgenic mice lacked the classical block of differentiation at the promyelocytic stage, which defines APL, leukemia in PLZF-RAR␣/RAR␣-PLZF double transgenic mice was characterized by a dramatic accumula-

Role of RAR␣-X proteins in leukemogenesis. RAR␣-PML transcripts are found in 70 to 80% of APL patients with t(15;17) (66 – 68). No apparent difference has been observed in RA sensitivity or clinical outcomes between APL patients who express both PML-RAR␣ and RAR␣-PML translocation products, and those expressing only the PML-RAR␣ transcript (69, 70). In addition, RAR␣-PML fusion protein consists of the RAR␣ transactivation domain A (17) which, is fused to the COOH-terminus of PML (Fig. 1). Given the fact that no biochemical functions have been attributed to the COOH-terminus of PML, it is difficult to predict its potential role in APL pathogenesis (9, 66, 67, 71). However, APL patients whose blasts harbored the RAR␣-PML but not the PML-RAR␣ fusion gene were also identified (72). In the case of APL associated with t(11;17) the majority of patients coexpress the reciprocal RAR␣-PLZF transcript (52, 68). Contrasting with the difficulty of attributing a biochemical function to RAR␣-PML, the role of the RAR␣-PLZF molecule can be predicted according to its structure. RAR␣-PLZF fusion protein is composed of the RAR␣ transactivation domain A fused to the last seven zinc fingers of PLZF (52, 68) (Fig. 1). Thus, RAR␣-PLZF retains the PLZF binding domain, but not the protein–protein interaction domain (POZ domain), which is responsible for the recruitment of corepressor complexes by PLZF. Consequently, unlike the wild-type PLZF, RAR␣PLZF can bind DNA, but can no longer repress transcription. Thus, RAR␣-PLZF might interfere with the transcriptional repressive activity of 238

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FIG. 3. Effect of the reciprocal translocation product and the X inactivation on leukemogenesis in transgenic mice. (A) Crosses of PML-RAR␣ transgenic mice, with PML⫺/⫺ mice or RAR␣-PML transgenic mice, induce the appearance of the leukemia at an earlier age. (B) Crosses of PLZF-RAR␣ transgenic mice, with PLZF⫺/⫺ mice or RAR␣-PLZF transgenic mice, induces the transformation of the CML like phenotype observed in the PLZF-RAR␣ transgenic mice, into an APL like phenotype.

tion of immature blasts and of cells blocked at the promyelocytic stage of differentiation. Moreover, it was characterized by low platelet number, anemia and a mild increase of the white blood cell count in the PB, features that are also frequently observed in APL (60). Thus, RAR␣-PLZF does not function as a classical tumor modifier, but rather metamorphoses a CML-like phenotype into an APL like phenotype. In sum, the overall analysis of the various transgenic models of APL demonstrates the critical importance of both products issued from the balanced reciprocal translocation which are associated with APL. While this points at the relevance of the various RAR␣-X fusion proteins in APL pathogenesis, it also suggests that, in their absence, additional genetic events yet to be identified have to replace RAR␣-X functions.

very effective tool to study leukemogenesis in vivo. However, this approach has several limitations: (i) the transgene is under the control of a heterologous promoter which does not necessarily reflect the expression of the aberrant gene in vivo in the leukemic cells; (ii) the expression of the transgene may be inappropriate and vary due to the unpredictable transgene copy number and the effect of the transgene integration site; (iii) in the APL leukemic blast the dosage of X and RAR␣ is reduced to heterozygosity as consequence of the chromosomal translocation, while in the transgenic mice the two normal copies of the wild-type alleles are still intact. This aspect is of particular relevance in modeling APL in the mouse because the fusion gene (X-RAR␣) is believed to interfere with the normal function of its parental genes (X or RAR␣) (Fig. 2). Therefore the two normal copies of X and RAR␣ may oppose the dominant negative action of the X-RAR␣ oncoproteins. An alternative strategy referred to as “knock in” approach has been recently utilized to overcome the disadvantages encountered employing a

Modeling APL Using a “Knock in” Approach The analysis of the various X-RAR␣ and RAR␣-X transgenic mice, further confirms that the conventional transgenic approach constitutes a 239

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FIG. 4. Modeling APL using a “knock in” approach. (A) Schematic representation of a classical transgenic approach. In this case, the transgene copy number is variable, and the integration site of the transgene is random. Thus, the expression of the transgene may vary depending on the surrounding regulatory elements and copy number. Moreover, two copies of the wild-type allele are still present in the transgenic animal, contrasting with the situation observed in APL cells where the wild-type allele is reduced to heterozygosity. (B) Schematic representation of the “knock in” approach. In this case, the fusion gene (X-RAR␣) is targeted by homologous recombination into the genomic locus of one of the genes involved in the translocation (X gene). As a result, the expression of the fusion gene is now under the control of the promoter of the X gene mimicking the situation observed in the APL blast at the somatic level. In addition, the gene dosage (at least for one of the two genes involved in the chromosomal translocation) is comparable to the one observed in the APL blasts. However, the expression of the fusion gene is not restricted to the hemopoietic compartment. It is on the contrary expressed throughout development leading to a possible embryonic lethality of the “knocked-in” heterozygous embryos, or to unexpected phenotypes in the adult mutant.

classical transgenic approach (Fig. 4). This methodology consists in targeting the aberrant fusion gene directly into a predefined locus in mouse Embryonic Stem (ES) cells by homologous recombination. These cells are subsequently utilized to generate mice expressing the fusion gene (78 – 80). As an example, the AML1-ETO fusion cDNA resulting from the t(8;21) observed in the AML-M2 subtype of myeloid leukemia was targeted to replace the normal AML1 gene in order to model t(8;21) leukemias in mice. In this case, the aberrant AML1-ETO fusion gene, which replaces the normal AML1 gene, is now under the control of its natural promoter, thus closely mimicking what is occurring, at the somatic level, in the leukemic blast (Fig. 4). The relative dosage of the fusion gene and its wild-type counterpart are

comparable and their expression should be concordant. The variegation effect due to the random insertion of the aberrant gene at different location of the genome in transgenic mice is also eliminated. However, this replacement resulted in the embryonic lethality of the heterozygous mutants due to the block of fetal hemopoiesis, with a phenotype similar to the one observed in the AML-1 “knock out” homozygous mice (79, 80). Despite the impossibility of characterizing the leukemogenic role of AML1-ETO, the “knock in” experiment was extremely informative since it supported the notion that the fusion protein could act in a dominant negative manner thus blocking AML1 function (80). In the case of APL, the results obtained utilizing a classical transgenic approach suggest that 240

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PLZF-RAR␣ is necessary but not sufficient for leukemogenesis and does not induce block of differentiation at the promyelocytic stage. However, in this mouse model the expression of PLZF-RAR␣ is not driven by the PLZF promoter, as in the human APL blast. Furthermore, the wild-type PLZF gene dosage is not reduced to heterozygosity as in human APL. Thus, the absence of APL features in these mice could be due to an inappropriate level and/or pattern of expression of the oncogene. To circumvent these disadvantages and to define the possible dominant negative effect of the PLZF-RAR␣ fusion gene on PLZF and/or RAR␣ function, we have utilized a “knock in” approach to generate mice harboring the fusion gene PLZF-RAR␣ targeted to the PLZF locus (T. Merghoub, J. Costoya, and P. P. Pandolfi, unpublished observation). If PLZFRAR␣ acts as dominant negative inhibitor of PLZF function, we should expect in PLZF-RAR␣ knocked-in heterozygous mutants a phenotype similar to the one observed in PLZF KO mice. Chimeric mice obtained injecting ES cells are presently being characterized. Other groups have used the “knock in” approach to target the aberrant gene into a desired locus other than the ones involved in the translocation. In this case, the gene to be targeted is selected on the basis of its expression pattern, which should be restricted to the cell type where the aberrant gene is expressed in the human disease. In this approach the only advantage compared to a classic transgenic strategy is the possibility to better control the copy number and the expression level of the fusion gene, however the gene dosage of the wild-type gene involved in the translocation is not corrected. A successful example of this “Knock in” strategy, by targeting the PML-RAR␣ to the murine Cathepsin G locus has been reported (81). The “knocked in” mice had a 77% cumulative probability of leukemia by 71 weeks of age, compared with 15% leukemia incidence by the same age observed in a concurrent cohort of hCG-PML-RAR␣ transgenic mice (81). Thus, the expression of the knocked-in PMLRAR␣ under the control of murine Cathepsin G promoter caused leukemia with higher incidence than previously reported in standard transgenic

experiments. The leukemia features in the “knocked in” animals were similar to those of hCG-PML-RAR␣ transgenic animals, including leukocytosis, anemia, thrombocytopenia, massive hepatosplenomegaly with extensive leukemic cell infiltration, and accumulation of promyelocytes in the marrow and the spleen. The difference between the leukemia onset in transgenic and “knocked in” mice might be explained by the relatively higher level of expression driven by the promoter of the murine Cathepsin G gene versus the human Cathepsin G promoter utilized in the transgenic approach. Although the “knock in” approach presents several advantages it has also several fundamental limitations. Unlike somatic translocations, which results in the expression of an aberrant gene in a specific subset of cells, as a consequence of a “knock in” event, the altered gene is expressed in all tissues and throughout the developmental stages of the embryo where the targeted gene is normally expressed. Indeed, many of the genes involved in cancer-associated translocations play an important role not only in hemopoiesis, but also during embryonic development. Hence, a major disadvantage of “knock in” approach is the unrestricted expression pattern of the mutated gene, that may result in unexpected phenotypes or even cause early embryonic lethality. X Functional Inactivation and APL Pathogenesis As mentioned before the fusion proteins in APL may have the potential ability to interfere with both X and RAR/RXR pathways. To understand the function of the various X genes, knock out (KO) mice have been generated in which the X genes have been disrupted by homologous recombination. Inactivation of PML function. As mentioned before, PML-RAR␣ delocalizes PML from the NB suggesting that the PML function might be impaired in APL. PML KO (PML⫺/⫺) mice have defined a crucial role for this protein in critical physiological functions. PML null mice display a reduced number of circulating monocytes and 241

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granulocytes, although their hematopoietic myeloid precursors retain the ability to terminally differentiate (82). In addition, PML⫺/⫺ mutants show an increased susceptibility to spontaneous Botryomycotic infections (82). PML⫺/⫺ mice are protected from multiple caspase-dependent apoptotic signals such as Fas, tumor necrosis factor (TNF), ceramide, interferons (INF) and ionizing radiations (83), demonstrating a role of PML as a proapoptotic factor which is essential for multiple apoptotic pathways (83). PML⫺/⫺ mice are more sensitive to the tumor promoting activity of 12-O-tertradecanoyphorbol-13-acetate (TPA) giving rise to a high frequency of papiloma and carcinomas. Moreover, when injected with carcinogens such as dimethybenzanthracene (DMBA), PML null mice develop B, T lymphomas and histiocytomas (82) at a much higher frequency than in wild-type controls. Therefore, PML acts as a putative tumor suppressor in vivo. Furthermore, as reported in cells from Bloom syndrome patients, an increased rate of sister chromatid exchange is observed in PML null cells implicating PML and the PML-NB in the maintenance of genomic stability (84). In this respect it is important to note that PML and BLM colocalize in the NB. All together, these data indicate that PML might exert its tumor suppressive activity at multiple levels, by controlling cell growth cell survival and genomic stability. Consequently, the dominant negative effect of PML-RAR␣ on PML might promote tumorigenesis.

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might lend a selective growth and survival advantage to the leukemic cells through deregulated expression of PLZF target genes. In this respect, Hox genes and BMPs have been implicated in proliferation and differentiation of primitive hemopoietic cells (86, 87). Aberrant regulation of Hox genes in APL may be exacerbated by the fact that PLZF-RAR␣ concomitantly interfere with the transcriptional function of PLZF and RAR␣ which has also been implicated in the regulation of Hox gene expression. Inactivation of Stat5b function. STAT5a and STAT5b KO mice present deficiencies in prolactin and growth hormone functions. However, although these transcription factors have been involved in hematopoiesis, the PB counts and in vitro colony formation from bone marrow hemopoietic progenitors are not affected in these mutants (88). By contrast, the STAT5a⫺/⫺b⫺/⫺ double mutants have a profound deficiency in peripheral T cell proliferation accompanied by reduced number of IL-3, IL-5, and G-CSF induced colonyforming units in bone marrow cells (88). STATs appear to play an important role in various myeloid malignancies, which are characterized by arrested maturation and cytokine-independent proliferation of myeloid progenitors (89, 90). Moreover, leukemic transformation by tyrosine kinase fusion genes such as BCR/ABL and TELJAK2 has been consistently linked to Stat5 activation (89, 90). In agreement with this notion, Stat5a/b deficient mice do not develop the myeloand lymphoproliferative disorder that wild-type mice would develop when their bone marrow cells are transduced with a retrovirus expressing TEL-JAK2 (91). All these data point out the important role of Stat5 in leukemogenesis. However, it is important to note that Stat5b acts as an oncogenic factor contrasting with the tumor suppressor role played with PML and the anti-proliferative effect of PLZF. Consequently, in this case the Stat5b-RAR␣ fusion protein may not act as a dominant negative on Stat5 function, but rather activate this pathway in a constitutive manner. In this respect it is important to point out that Stat5bRAR␣ can directly bind DNA through the RAR␣ as well as the Stat5b DNA binding domain. This

Inactivation of PLZF function. PLZF⫺/⫺ mice display patterning defects affecting axial and limb skeletal structures including homeotic transformation of anterior skeletal elements into posterior structures (85). In the limb, PLZF⫺/⫺ cells show a greater proliferative capacity and a reduced rate of apoptosis (85). This suggests that PLZF may act, as PML, as a growth-inhibitory and proapoptotic factor. 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 KO mice (85). Thus, an aberrant PLZF function in APL, in view of PLZF-RAR␣ and RAR␣-PLZF activities, 242

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and PLZF-RAR␣ in double transgenic mice, the reduction of the PLZF dose metamorphoses the CML phenotype observed in PLZF-RAR␣ transgenic mice into an APL phenotype. These results underscore the relevance of the X pathway in APL leukemogenesis, and support the notion that the RAR␣-PLZF fusion protein may act as a dominant negative inhibitor of PLZF function (Fig. 3).

oncoprotein may therefore display two distinct aberrant transcriptional functions contributing to leukemogenesis through activation of Stat5 target genes and concomitant repression of RAR␣ target genes. Consequences of X functional inactivation in APL. As mentioned before, the leukemic phenotype in the various X-RAR␣ transgenic mice is preceded by a variable latency. One possible explanation is that in the mouse model the relative level of expression of the X-RAR␣ transgene with respect to the wild-type X and RAR␣ genes is not appropriate. In APL blasts the normal gene dosage of X and RAR␣ is reduced to heterozygosity as a consequence of the chromosomal translocation, while in X-RAR␣ transgenic mice the two normal copies of the gene X and RAR␣ are still intact and may antagonize the dominant negative action of the X-RAR␣ oncoproteins. To determine whether the X inactivation would accelerate leukemia onset and/or penetrance triggered by PML-RAR␣, we have crossed the hCG-PMLRAR␣ transgenic mice (55) with PML⫺/⫺ mice (82). Compared to PML⫹/⫹/hCG-PML-RAR␣ transgenic mice, PML⫹/⫺ or PML⫺/⫺/PMLRAR␣ transgenic mice mutants presented a dramatic decrease in the leukemia-free survival (LFS) and increased incidence of leukemia (92), thus demonstrating that PML inactivation contributes to APL leukemogenesis and the inactivation of only one PML allele is sufficient to affect the incidence and latency of the disease. Similarly, we have investigated the effects of the PLZF inactivation on leukemogenesis by PLZF-RAR␣. To this end, we have intercrossed PLZF-RAR␣ transgenic mice with PLZF⫺/⫺ mice (85). Whereas the PLZF-RAR␣/PLZF⫹/⫹ control cohort developed a “CML” like leukemia indistinguishable from those observed in the parental PLZF-RAR␣ transgenic mice (59), leukemia in PLZF-RAR␣/PLZF⫺/⫺ mice was characterized by a dramatic accumulation of blast/promyelocytic cells in the BM and spleen and the absence of leukocytosis, thus displaying classic APL features. However, leukemia onset and incidence were unaffected. Therefore, similarly to the effects caused by the coexpression of RAR␣-PLZF

CONCLUSION The systematic and comparative analysis of the genetics of APL and its genes and fusion genes in the mouse, has been invaluable in determining the relevance and role of these various players with important therapeutic implications that can now be tested in these genetically defined models of the disease. This analysis has served once again as a paradigm for many more studies to come in the future. There is no doubt, in fact, that in a post-genome era, the number of aberrant genes that will be associated with a specific form of cancer will grow exponentially. We have at end the technological tools to analyze the relative consequences of these genetic mutations in vivo providing the conceptual basis for a future pharmacogenetic approach to cancer therapy, whereby any therapeutic regiment will be tailored on the basis of the detailed knowledge of genetic make up of any individual tumor. ACKNOWLEDGMENTS We thank all the past and present members of the Molecular and Developmental Biology (MADB) lab at Memorial Sloan-Kettering Cancer Center, working on APL and related subjects: Austin Changou, Jose Costoya, Maria Barna, Mantu Bhaumik, Laurent Delva, Mirella Gaboli, Domenica Gandini, Marco Giorgio, Ailan Guo, Nicola Hawe, Sundeep Kalantry, Letizia Longo, Daniela Peruzzi, Eduardo Rego, Roberta Rivi, Simona Ronchetti, Davide Ruggero, Paolo Salomoni, Carla Tribioli, Zhu-Gang Wang, Hui Zhang, Sue Zhong, and Li-Zhen He. T.M. is a Scholar of the Lymphoma Leukemia Society. Our work is supported by the NCI, the De Witt Wallace Fund for Memorial Sloan-Kettering Cancer Center, the MMHCC (Mouse Models of Human Cancer Consortium), and NIH Grants to P.P.P. This 243

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paper was presented at a Focused Workshop on Animal Models of Leukemia and Lymphoma jointly sponsored by the Leukemia & Lymphoma Society and the MMHCC of the National Cancer Institute on September 19 –20, 2000.

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