NUP98-HOXD13 Fusion Gene Research Articles

Abstract 624: Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia

Abstract Background: Acute lymphoid leukemia is characterized by inherited or acquired mutations that effect critical differentiation and proliferation pathways. We have shown that Mcm2 deficient mice developed T cell acute lymphoblastic leukemia, due to copy number variations, most commonly interstitial deletions throughout the genome. When crossed with mice that expressed a NUP98::HOXD13 (NHD13) fusion gene, Mcm2−/−NHD13 mice developed B cell precursor ALL (BCP-ALL). A majority of these BCP-ALL had acquired homozygous deletions of Ptpn1, a protein tyrosine phosphatase, leading to the hypothesis that Ptpn1 deficiency combined with NHD13 expression leads to BCP ALL. Objective: To investigate the role of Ptpn1 deletion in BCP-ALL development using mouse models. Methods: Mice expressing an NHD13 fusion gene were crossed with Ptpn1 knockout mice, generating 6 possible genotypes. Mice were followed for 18 months. Mice with signs of leukemia were characterized by clinical evaluation, CBC, flow cytometry, and IHC. Primary BCP-ALL and derived BCP-ALL cell lines were also evaluated with RNA-seq and molecular pathway analysis. Results: NHD13+Ptpn1-/- mice developed BCP-ALL with 65% penetrance, characterized by hyperleukocytosis, anemia, thrombocytopenia, and invasion of non-hematopoietic tissues. Similar to human BCP-ALL, NHD13+Ptpn1-/- BCP-ALL had clonal IGH as well as clonal Tcrd gene rearrangements. Flow cytometry revealed CD19 and/or B220 expression. NHD13+Ptpn1+/- mice with BCP-ALL frequently lost the wild-type (WT) Ptpn1 allele in leukemic cells, reinforcing the hypothesis that Ptpn1 can function as a classic tumor suppressor gene in this context. Whole exome sequencing revealed acquired mutations in B-cell differentiation genes (Pax5 or Bcor) and activating mutations in tyrosine kinase genes (Jak1/3 and Flt3). Transcription signature analysis showed significant upregulation of Hoxa/b gene clusters, RNase12 and LncRNAs subsets. Conclusion: This study demonstrates that Ptpn1 loss combined with expression of NHD13 fusion gene leads to highly penetrant BCP-ALL in mice, suggesting a role for Ptpn1 in preventing malignant transformation. These findings present a collaborative model for BCP-ALL in which the NHD13 transgene leads to increased stem cell self-renewal, somatic Bcor or Pax5 mutations block normal B cell differentiation, and somatic signaling mutations (Jak1/3,Flt3) lead to hyperproliferation, which is potentiated by Ptpn1 deficiency. Citation Format: Nupur Nigam, Toshihiro Matsukawa, Ryan Bertoli, Michel L. Tremblay, Mianmian Yin, Peter D. Aplan. Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 624.

Abstract 5026: Protein tyrosine phosphatase Ptpn1 knockout in mouse models drives B-cell hematological malignancies

Abstract Background: Ptpn1 is a member of Protein Tyrosine Phosphatase family that dephosphorylates tyrosine residues. We previously reported that an Mcm2 hypomorphic mouse (designated Mcm Cre/Cre) develops T cell acute lymphoblastic leukemia (T-ALL), due to acquisition of 100-1000 kb interstitial deletions involving tumor suppressor genes. When crossed to mice that express a NUP98-HOXD13 (NHD13) fusion gene, NHD13+;Mcm2Cre/Cre mice develop B-cell precursor ALL (BCP-ALL). Sparse whole genome sequencing revealed somatic copy number variants (CNV) involving Pax5 and Ptpn1. The role of Pax5 in B-cell development, differentiation, and leukemia has been widely studied. However, little is known about deletions of Ptpn1 and its role in BCP-ALL. Objective: The principal objective is to investigate how deletion of Ptpn1 impacts BCP-ALL development in mouse models. Methods: Mice expressing NHD13 fusion (NHD13+) were crossed with Ptpn1 knockout mice. The F2 cross generated 6 genotypes that were positive or negative for NHD13 and wild type/heterozygous/homozygous for Ptpn1. Mice were studied for incidence of BCP-ALL, which was characterized by whole-exome sequencing, molecular pathway analysis and transcriptome-sequencing. Results: NHD13+;Ptpn1-/- mice developed BCP-ALL, characterized by hyperleukocytosis, variable anemia and thrombocytopenia, expression of B220 and/or CD19, and invasion of non-hematopoietic tissues (liver, kidney, lung). In addition, NHD13+;Ptpn1+/- mice that developed BCP-ALL lost the wild-type (WT) Ptpn1 allele in the leukemic cells. Similar to human BCP-ALL, NHD13+;Ptpn1-/- mice that developed BCP-ALL showed presence of clonal TCR-delta rearrangements as well as IGH rearrangements in the leukemic cells. Whole exome sequencing showed presence of acquired mutations in genes known to be involved in B-cell differentiation, such as Pax5 or Bcor, as well as genes involved in B cell signaling pathways, such as Jak3, Jak1 and Flt3 and Western blots demonstrated accumulation of phosphorylated STAT3 protein. Conclusion: This study demonstrates that Ptpn1 loss along with expression of an NHD13 fusion gene leads to a highly penetrant BCP ALL in mice, suggesting a role for Ptpn1 in preventing malignant transformation. Taken together, these findings are consistent with a collaborative model for BCP ALL in which the NHD13 transgene leads to increased stem cell self-renewal, somatic Bcor or Pax5 mutations block normal B cell differentiation, and somatic signaling mutations (Jak1/3, Flt3) lead to hyperproliferation, which is potentiated by Ptpn1 deficiency. Citation Format: Toshihiro Matsukawa, Nupur Nigam, Ryan Bertoli, Michel L. Tremblay, Mianmian Yin, Peter D. Aplan. Protein tyrosine phosphatase Ptpn1 knockout in mouse models drives B-cell hematological malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5026.

Abstract 2641: Examining the potential therapeutic efficacy of the CDK inhibitor alvocidib in a pre-clinical model of myelodysplastic syndrome

Abstract Myelodysplastic Syndrome (MDS) represents a group of hematologic malignancies characterized by pancytopenia and dysplastic morphology. High risk MDS is invariably fatal due to either severe cytopenia, or transformation to acute myeloid leukemia (AML). Inhibitors of transcription offer promising potential for new therapies. Modulation of CDK9 activity, which plays a critical role in controlling gene transcription through phosphorylation of RNA polymerase II, has been proposed as a novel therapeutic approach. In this study, we examined the efficacy of alvocidib, a broad spectrum CDK inhibitor with highest potency against CDK9, which is hypothesized to slow transcription via inhibition of CDK9, leading to stalled RNA Polymerase II and cell death. We employed a previously characterized pre-clinical model of MDS based on the NUP98-HOXD13 (NHD13) fusion gene, which induces MDS that progresses to AML in mice. In addition to alvocidib, we evaluated the in vitro efficacy of 5-Azacytidine (5-Aza) and Decitabine (DAC), which are hypomethylating agents used in treatment of MDS. The in vitro assays demonstrated a potent effect of alvocidib on cell lines that expressed the NHD13 fusion gene. Alvocidib induced a clear cytostatic effect and showed increasing cytotoxic effects with time, peaking at 70% cell death after 72 hours. Annexin-V staining further supported these results, revealing that most cells were apoptotic by 72 hours. Neither 5-Aza nor DAC treatment matched these effects in vitro. The in vivo assays were based on treatment of chimeric mice with both wild type (WT) and MDS hematopoiesis, generated by allogeneic hematopoietic stem cell transplant with bone marrow from WT and NHD13 mice. Four treatment groups (saline, 5-Aza, Alvocidib, and 5-Aza+Alvocidib) were assessed by serial flow cytometry and complete blood counts. In contrast to in vitro studies, in vivo results did not show a clear survival benefit from either group receiving alvocidib, whereas 5-Aza treatment demonstrated statistically significant survival. However, this study revealed several “exceptional responder” mice, defined as mice that demonstrated a ≥75% reduction in WBC count and a ≥50% reduction in NHD13 engraftment levels following transformation to AML; these exceptional responses were observed only in mice receiving alvocidib. This type of dramatic response has not previously been observed in over 20 prior chimeric transplant experiments employing the NHD13 mouse model. Ongoing efforts include follow-up studies to evaluate the frequency of exceptional responders, as well as identification of acquired mutations which collaborate with the initiating mutation (NHD13) to generate AML. Citation Format: Ryan M. Bertoli, Yang Jo Chung, Jason M. Foulks, Anthony S. Wokasch, Peter D. Aplan. Examining the potential therapeutic efficacy of the CDK inhibitor alvocidib in a pre-clinical model of myelodysplastic syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2641.

Role of p21 in a Mouse Model of NUP98-HOXD13 Fusion Driven Myelodysplastic Syndromes (MDS)

The myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective hematopoiesis leading to cytopenias and a high rate of progression to acute myeloid leukemia (AML). The NUP98-HOXD13 (NHD13) fusion has been found in patients with MDS and AML. A transgenic (Tg) mouse model, generated by Peter Aplan’s group, which utilizes Vav 1 regulatory elements to direct expression of the NHD13 transgene in hematopoietic tissues, displays the phenotypic features of MDS including a chronic phase of cytopenias followed by transformation to AML (Lin et al., 2005). We previously reported that loss of one or both alleles of p53 did not rescue the MDS phenotype in NHD13+ Tg mice, but rather exacerbated the MDS phenotype and accelerated the development of AML (Xu et al., 2012). Expression of p21WAF1/CIP1(p21) was increased in the Lin−Sca-1+c-Kit+ (LSK) cells isolated from NHD13+ Tg mice, so we generated and analyzed NHD13+p21+/– and NHD13+p21–/– mice to further investigate whether the accelerated MDS and AML that occurs in the absence of p53 relates to the defective expression of the p53 target gene. Deletion of p21 significantly altered the fate of the NHD13+ Tg mice. All of the NHD13+p21–/– mice died of AML, rather than MDS. Only 18% (4 out of total 22 mice) of the NHD13+p21+/– mice developed MDS with a median survival of 289 d; in contrast 31% (9 out of total 29 mice) of NHD13+ Tg mice died from MDS, with a median survival of 230 d (p<0.05). We examined the peripheral blood counts of the “clinically healthy” NHD13+p21–/– mice at 3 to 5 months, and found increased white blood cell (WBC) and neutrophil (NE) counts, compared to the age matched NHD13+ Tg mice. Clearly the deletion of one or two alleles of p21 increases the median survival of NHD13+ Tg mice with MDS, and the complete loss of p21 rescues the fatal MDS induced by NHD13 fusion gene. However the deletion of one or two p21 alleles does not significantly affect the development of AML, which in 82% of NHD13+p21+/– mice resulting in a median survival of 291 d, AML in the NHD13+p21–/– mice was with a median survival of 320 d, and 69% of NHD13+ Tg mice showed AML with a median survival of 315 d. p21 is important for maintaining a normal-sized HSPC pool (Cheng et al., 2000), and both p21 and p53 have been shown to be involved in the determination of asymmetric vs symmetric cell divisions of epithelial cells (O’Brien et al., 2012; Cicalese et al., 2009). To determine whether the symmetric division of NHD13+ HSCs is affected by the loss of p21 or p53 in vitro, we performed paired daughter cell assays. Single LSKCD34–Flt3–CD150+ cells isolated from wild type (WT), NHD13+, NHD13+p21–/– and NHD13+p53–/– mice bone marrow were sorted into 96-well plates one cell/well. After the first cell division, the two daughter cells were split into two wells for 12 days in culture. We examined the ability of sorted single LSKCD34–Flt3–CD150+ cells to generate daughter cells that retain multipotent lineage differentiation potential and found that NHD13+p53–/– CD150+CD34–Flt3–LSK cells underwent symmetric self-renewal divisions 85% of the time (both daughter cells are multipotent), with 15% asymmetric divisions (only one daughter cell is multipotent); the NHD13+p21–/– CD150+CD34–Flt3–LSK cells produced 13% symmetric self-renewal divisions, 50% asymmetric divisions and 37% symmetric commitment divisions (both daughter cells are not multipotent); the NHD13+ CD150+CD34–Flt3–LSK cells produced 50% symmetric self-renewal divisions, 40% asymmetric divisions and 10% symmetric commitment divisions; and the WT CD150+CD34–Flt3–LSK cells produced 27% symmetric self-renewal divisions, 42% asymmetric divisions and 31% symmetric commitment divisions. These data indicate that loss of p53 increases symmetric self-renewal divisions of NHD13+ HSCs, and loss of p21 increases asymmetric self-renewal divisions of NHD13+ HSC in vitro. Collectively, our data indicate that loss of p21 maintains the survival of MDS driven by NUP98-HOXD13 fusion, which is independent of the function of p53; and the increased asymmetric self-renewal divisions of NHD13+p21–/– HSCs may contribute to the increased survival observed in NHD13+p21–/– MDS mice. DisclosuresArmstrong:Epizyme : Consultancy.

Non-homologous end joining mediated DNA repair is impaired in the NUP98-HOXD13 mouse model for myelodysplastic syndrome

Chromosomal translocations typically impair cell differentiation and often require secondary mutations for malignant transformation. However, the role of a primary translocation in the development of collaborating mutations is debatable. To delineate the role of leukemic translocation NUP98-HOXD13 (NHD13) in secondary mutagenesis, DNA break and repair mechanisms in stimulated mouse B lymphocytes expressing NHD13 were analyzed. Our results showed significantly reduced expression of non-homologous end joining (NHEJ)-mediated DNA repair genes, DNA Pkcs, DNA ligase4, and Xrcc4 leading to cell cycle arrest at G2/M phase. Our results showed that expression of NHD13 fusion gene resulted in impaired NHEJ-mediated DNA break repair.

A NUP98-HOXD13 leukemic fusion gene leads to impaired class switch recombination and antibody production

Myelodysplastic syndrome is a clonal process characterized by ineffective hematopoiesis and progression to acute leukemia. Although many myelodysplastic syndrome and leukemic patients have compromised immunity, the role of underlying mutations in regulating immune function is poorly understood. Recent studies show that NUP98-HOXD13 (NHD13) fusion gene results in myelodysplastic syndrome and impairs lymphocyte differentiation in transgenic mice. In our studies, we sought to elucidate the mechanism by which NHD13 affects B-lymphocyte development and function. Based on our preliminary findings that transgenic mice had increased levels of IgM and reduced IgG1 and IgE, we hypothesized that the fusion gene might impair class switch recombination (CSR). Mice were immunologically challenged with dinitrophenol. NHD13 mice showed a marked reduction in B-lymphocyte differentiation in their bone marrow and spleen following dinitrophenol stimulation and had reduced production of dinitrophenol-specific antibodies. Spleen follicles from these mice were small and hypocellular, indicating failure of clonal expansion. When isolated NHD13 B lymphocytes were stimulated in vitro using Escherichia coli lipopolysaccharide or lipopolysaccharide + interleukin-4, they failed to undergo sufficient CSR and proliferation. Taken together, our findings show that expression of NUP98-HOXD13 impairs CSR and reduces the antibody-mediated immune response, in addition to its role in leukemia. Further delineation of the NUP98-HOXD13 transgene may reveal novel pathways involved in CSR.

Loss of p53 Accelerates the Complications of Myelodysplastic Syndromes (MDS) In the NUP98-HOXD13 Transgenic Mouse Model

Abstract 2804The myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis leading to blood cytopenias and a high rate of progression to acute myelogenous leukemia (AML) (Nimer, 2008). Recent studies implicated an important role for p53 in 5q−MDS, a subtype where haploinsufficiency of the RPS14 ribosomal protein drives the anemia that accompanies this disease (Ebert et al., 2008; Barlow et al., 2010 and Dutt et al., 2011). The elevated level of p53 activity apparently triggers the excessive apoptosis and the dysplastic morphology seen in the erythroid cells of the “5q-mice” that lack one copy of the chromosomal region syntenic to the human 5q region that contains the RPS14 gene (Barlow et al., 2010). Thus, the cytopenias in some patients with MDS may be the result of defective ribosomal biosynthesis, leading to activation of p53 and excessive apoptosis. The nucleoporin gene NUP98 is fused to a number of different genes including HOXD13 by chromosomal translocations that are found in patients with MDS, AML and CML, blast crisis. Genetically engineered mice that express a NUP98-HOXD13 (NHD13) transgene (Tg) display the phenotypic features of MDS, including cytopenias, bone marrow dysplasia, and transformation to acute leukemia (Lin et al., 2005). We obtained these mice and analyzed the hematopoietic stem cells (HSC) and the erythroid compartment and found significantly decreased rps14 mRNA expression in the NHD13+ CD71+Ter119+ cells compared to the WT controls. Furthermore, flow cytometry analysis revealed increased intracellular p53 levels in the NHD13+ Lin−Sca-1+ c-Kit+ (LSK) and CD71+Ter119+ cells. These data suggest that defective rps14 ribosomal protein production and an increased p53 level may contribute to the anemic phenotype in NHD13+ Tg mice. To examine whether inhibition of p53 function can improve the cytopenias of NHD13+ Tg mice, mice were injected with Pifithrin-α (a reversible inhibitor of p53-mediated apoptosis and p53-dependent gene transcription, 2 mg/kg body weight) daily for five weeks. We observed partial rescue of the myeloid and lymphoid lineage differentiation defects, with no improvement in the hemoglobin level. To further investigate whether the presence or absence of p53 affects the MDS or AML phase of NHD13 driven disease, we generated NHD13+p53+/− and NHD13+p53−/− mice. Deletion of one allele of p53 rescues the myeloid progenitor cell compartment, while deletion of both alleles reversed the deficit in both the LSK and the MPP populations that was observed in the NHD13+ Tg mice. The clinically healthy NHD13+p53−/− mice (aged 3 to 5 months) and the NHD13+p53+/− mice (aged from 3 to 7 months) both had more severe leukopenia and anemia than the NHD13+ Tg mice or the WT controls (aged 4 to 7 months). 60% of the NHD13+p53−/− mice and the NHD13+p53+/− mice developed MDS with a median survival of 138 d and 190 d respectively; in contrast 30% of the NHD13+ Tg mice had MDS with a median survival of 236 d. These data indicate that the relative or absolute lack of p53 hastens the development of MDS, and shortens the median survival. Lack of one or two p53 alleles significantly accelerated the development of AML, which in NHD13+p53+/− mice resulting in a median survival of 278 d. However, AML in the NHD13+p53−/− mice is more undifferentiated with a median survival of 133 d, and the NHD13+ Tg mice showed AML with a median survival of 324 d. Taken together, these data demonstrated that the chronic loss one allele or two alleles of p53 does not rescue the MDS phenotype induced by NHD13 fusion gene. Rather it accelerates the development of NHD13 driven MDS and leukemia. Our studies suggest that targeting p53 transiently may temporally improve hematopoiesis in MDS, but over the long term has detrimental effects on hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

A NUP98-HOXD13 Fusion Gene Impairs B Lymphocyte Development and Function In a Mouse Model for Myelodysplastic Syndrome

AbstractAbstract 279Transgenic mice which express the fusion gene NUP98-HOXD13 (NHD13) have been shown to develop characteristic features of Myelodysplastic syndrome (MDS) including impaired hematopoietic differentiation and peripheral blood cytopenias in the presence of normocellular or hypercellular bone marrow (BM). It is evident that B-cells play a role in the progression of MDS by immune modulation or as direct targets of mutations resulting in ALL, or as cells that influence the BM microenvironment in which a neoplastic myeloid clone evolves. Choi and colleagues suggested a block in differentiation during the early development of B lymphocytes in the BM of NHD13 mice leading to lymphopenia consistent with the observation in some MDS patients. In this study, we sought to further delineate the role of NHD13 on B lymphocytes which escaped the initial differentiation block in the BM. We hypothesized that NHD13 impairs maturation and function of IgM+ B lymphocytes contributing to immunodeficiency. To study this, we performed blood smear examination, Complete Blood Counts (CBC), quantitative ELISA for antibody concentrations, and flow cytometric analysis of B cell fractions from the BM and spleen in 8–12 week-old transgenic and wild type (WT) mice. CBCs revealed significant lymphopenia and ELISA showed higher IgM concentrations (n=10, p<0.001), reduced levels of IgG1 (n=10, p<0.05) and IgE (n=10, p<0.01). The IgG2a, IgG2b, and IgG3 antibody levels were comparable to WT counterparts. Flow cytometric analysis of BM and splenic B cell fractions revealed reduced numbers of B cells in Hardy fractions D and F (n=10, p<0.01) indicative of impaired differentiation prior to these stages; splenic fractions in NHD13 mice were comparable to WT controls. Next, to assess the peripheral maturation and functional efficiency of B lymphocytes in the context of a comprehensive immune stimulation, a cohort of five WT and five preclinical transgenic mice were injected with 100 μ g dinitrophenol (DNP) followed by a booster dose on day 21. Mice were euthanized on day 28 and whole blood, spleen, lymphnodes and BM were harvested. CBC evaluation revealed significant lymphopenia in NHD13 mice (n=5, p<0.001). Quantitative ELISA for DNP specific antibodies showed comparable levels of serum IgM and significantly reduced levels of serum IgG1 (n=5, p<0.001), IgG2a (n=5, p<0.001), IgG2b (n=5, p<0.01), IgG3 (n=5, p<0.001) and IgE (n=5, p<0.01). Flow cytometric analysis of peripheral blood showed reduced numbers of B220+ IgM+ B cells (n=5, p<0.01), but comparable percentages of CD4+ and CD8+ T-cells. Detailed flow cytometric analysis of B-cell fractions in the BM and spleen of DNP-stimulated mice revealed a reduction in subpopulations of B lymphocytes. The earliest B cell lineage population, Pre-Pro B, was comparable to the WT controls. Hardy Pro B fraction B (n=5, p<0.001) and Pre B fractions E (n=5, p<0.01) and F (n=5, p<0.01) from BM of stimulated mice were significantly reduced in contrast to fractions C and C', which were higher (n=5, p<0.05 and p<0.001 respectively), indicative of cell growth arrest at these stages. Flow cytometry of splenic B-cell fractions from the DNP-stimulated mice showed significantly lower Transitional 1 (n=5, p<0.01), Follicular (n=5, p<0.05) and Marginal Zone (n=5, p<0.001) populations upon antigenic stimulation suggestive of defective clonal expansion of IgM+ cells even after escaping the block in the BM. Histopathology of the spleen revealed smaller lymphoid follicles with poorly developed mantle and marginal zone regions in the transgenic mice when compared to WT controls, consistent with the flow cytometric data. This study indicates that when NHD13 mice are immunologically challenged, B lymphocytes undergo impaired differentiation in the BM and maturation in the spleen, as well as reduced antibody class switching and subsequently lower antibody production. Analysis of B cell subsets during development and specific IgG/IgE antibody production, suggest that the NHD13 transgene might impair VDJ gene recombination and class switch recombination that are critical during these phases of B cell development. Disclosures:No relevant conflicts of interest to declare.

Increased Reactive Oxygen Species Induced by NUP98-HOXD13 Fusion Gene in MDS Mouse Model

The myelodysplastic syndromes (MDSs) comprise a group of pre-malignant hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). The causative agents for de novo or therapy-related MDS (t-MDS) include exposure to known genotoxins, such as benzene and other solvents, anti-cancer chemotherapy, and ionizing radiation. It is known that MDS is closely related to chromosomal aberrations including deletions, amplifications, inversions, and translocations, however, the molecular mechanism of disease progression from MDS to AML not been completely elucidated. Recently, several studies have reported that oxidative stress contributes to the disease progression mechanism in MDS. Reactive oxygen species (ROS) play a role in regulating several biologic phenomena involving activation of signaling pathways in response to cytokines, and the gene expression induced by this signaling. ROS is also known to induce oxidative DNA damage which generates DNA base modifications and DNA helix alterations. Therefore, increased intracellular levels of ROS can induce mutations which lead to transformation of MDS to AML. A mouse model for MDS, generated by expressing a NUP98-HOXD13 (NHD13) fusion gene was exploited to investigate ROS levels in this study. Lineage negative (Linneg) bone marrow mononuclear cell (BMNC) from NHD13 mice with MDS had a 7.8±5.6 fold increased level of ROS compared with wild-type (WT) Linneg BMNC, while ROS levels of unfractionated BMNC were similar in NHD13 and WT mice. The increase ROS level in NHD13 Linneg BMNC was associated with an increase in G2/M phase (2 fold). Interleukin 3 (IL-3) dependent NHD13 ‘knock-in’ cell lines also showed an increased level of ROS (3 to 5 fold), in comparison with other IL-3 dependent cell lines, BaF3 and 32D. To determine if the increase in ROS could be generated by an NHD13 fusion, BaF3 and 32D cell were transfected with an NHD13 expression vector. The BaF3 and 32D transfectant showed 2 fold (2.1±0.3 and 1.9±0.6 fold) increase ROS level compared to controls transfected with an empty vector. These findings suggest that expression of an NHD13 fusion gene can induce ROS in hematopoietic progenitor cells, which may contribute to the development of MDS and subsequent disease progression to AML through DNA damages.

Heterogeneity of Acute Myeloid Leukemia at the Stem Cell Level.

Leukemias are considered hierarchically organized being maintained by a leukemia stem cell (LSC). Whereas LSCs become the primary focus for targeted therapies, little is known about the pathways regulating LSC fidelity. Using retroviral gene transfer of MN1, NUP98HOXD13 (ND13), or HOXA9 oncogenes and limiting-dilution transplantation we modelled leukemias with different LSC frequencies, and characterized critical signaling pathways by loss-of-function analysis. Here we establish the concept that LSCs are heterogeneous based on the number of activated transcriptional networks, and functionally characterize downstream targets that are critical for LSC activity. Constitutive expression of the very potent myeloid oncogene MN1 with the ND13 fusion gene in murine bone marrow cells results in acute myeloid leukemia (AML) that is phenotypically very similar to MN1-induced AML. However, limiting dilution analysis showed that the LSC frequency was 33 fold higher in MN1+ND13 cells compared to MN1 cells, and disease latency at the limiting dilution was significantly shorter in the combination model (p=.009). Whereas MN1-LSCs expanded 68-fold over a period of 6 days, MN1+ND13-LSCs expanded 131-fold more than MN1-LSCs as determined by the competitive repopulation unit (CRU) assay. To screen for functional differences of the two models we screened for differential cytokine responses in vitro. Interestingly, MN1+ND13 expressing cells proliferated in response to GM-CSF, whereas MN1 cells or ND13 cells did not. This was confirmed as well for MN1+HOXA9 expressing cells and their MN1+CTL or HOXA9+CTL expressing counterparts. We found that Stat1, Stat3, Stat5, and Erk1/2 were selectively phosphorylated upon cytokine stimulation in MN1+ND13 and MN1+HOXA9 cells compared to single-oncogene transduced cells. To test the role of Stat1 and Stat5b for LSC fidelity Stat1 −/− and Stat5b −/− cells were co-transduced with MN1 and HOXA9 and compared to wildtype cells in vitro and in vivo. Stat1 −/− cells transduced with MN1+HOXA9 proliferated slower than wildtype cells in response to GM-CSF but not with IL3/IL6/SCF. Proliferation of Stat5b −/− cells transduced with MN1+HOXA9 proliferated slower than wildtype cells in response to both GM-CSF and IL3/IL6/SCF (p<.05). CRU assays with MN1+HOXA9-transduced Stat1 −/− and Stat5b −/− cells demonstrated that the day 6 CRU was 6 and 77 fold reduced, respectively, compared to wildtype cells. As MN1 and HOXA9 are upregulated in distinct subsets of normal karyotype AML we speculated that their combined overexpression may model subsets of complex karyotype AML. We performed gene set enrichment analysis on cytogenetic subsets of previously published gene expression data from 285 AML patients. 12 of 13 Stat-related pathways were enriched in complex karyotype patients compared to 4 and 8 of 13 Stat-related pathways in inv(16) and normal karyotype AML, respectively, thus supporting a critical role of Stat activation in LSCs of AML with multiple active pathways like complex karyotype AML. In conclusion we demonstrate considerable heterogeneity of LSC fidelity depending on the number of activated oncogenes and establish a critical role of Stat1 and Stat5b in mediating this LSC fidelity. Stat1 and Stat5b may become important therapeutic targets in complex karyotype AML.

A NUP98-HOXD13 Fusion Gene Induces a Transplantable Myelodysplastic Syndrome in Mice.

The myelodysplastic syndromes (MDSs) are a group of hematologic stem cell disorders characterized by ineffective hematopoiesis and dysplasia. A large number of chromosomal aberrations including deletions, amplifications, inversions, and translocations, some of which involve the NUP98 gene, have been associated with MDS. Recently an MDS mouse model expressing a NUP98-HOXD13 (NHD13) fusion gene was developed, which faithfully recapitulates all of the key features of MDS. Although it is well-established that acute myeloid leukemia (AML) is transplantable, there is no evidence that MDS is a transplantable condition. Therefore, in order to develop evidence for MDS as a hematopoietic stem cell (HSC) disease, we attempted to transfer MDS to normal recipients through bone marrow transplantation (BMT). All the recipients transplanted with bone marrow (BM) cells from NHD13 mice with MDS showed anemia, leukopenia, lymphopenia, and neutropenia when compared to recipients of wild-type (WT) littermates. The homing efficiency of the NHD13 primitive progenitor cells (Lineage negative [Lin−], Sca-1+) was about 2 fold higher than WT, and there was no significant difference in BM cellularity between the recipients of NHD13 and WT BM, indicating that the NHD13 recipients had ineffective hematopoiesis. These phenomena were reproduced in secondary recipients using primary recipients of NHD13 BM as donor mice. In secondary transplantation assays, 3 out of 5 recipients developed acute myeloid leukemia (AML) at 16 weeks post-transplantation. Morphological features of MDS, including nuclear-cytoplasmic asynchrony, binucleate cells, hypersegmented neutrophils, and giant platelets were detected in BM and peripheral blood of NHD13 donor, primary and secondary recipients by cytospin preparations. In competitive repopulation assays, mice transplanted with equal numbers of WT and NHD13 BM cells showed a decreased percentage of NHD13 cells in the peripheral blood, but an increased percentage of NHD13 cells in the BM, again providing evidence of ineffective hematopoiesis of the NHD13 cells. The transplantation of lineage depleted cells from BM has shown that the transplantable cells for MDS reside in the Lin− population of NHD13 BM. These findings demonstrate that MDS can be transferred to healthy recipients by BMT, supporting the concept that MDS originates in a transplantable multilineage hematopoietic stem cell.

A NUP98-HOXD13 Fusion Gene Impairs Differentiation of T and B Lymphocytes.

Chromosomal translocations leading to NUP98 gene fusions are associated with a wide range of hematologic malignancies, including acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), and myelodysplastic syndrome (MDS). The NUP98-HOXD13 (NHD13) gene fusion was first identified in a patient with a MDS that progressed to AML. Recently, we used an NHD13 fusion gene to develop a mouse model of MDS that recapitulates all of the key findings of the human disease, including ineffective hematopoiesis leading to peripheral blood cytopenias, dysplasia, and progression to AML. In addition to the features noted above, we observed that the NHD13 mice were lymphopenic, and 10–30% of these mice, depending on the strain background, progressed to T-ALL. These findings prompted us to define the lymphocyte development of NHD13 mice. CBCs obtained from clinically healthy NHD13 mice showed lymphopenia (2.21 vs. 8.72 K/μL, p<0.01) compared with wild type (WT) littermate controls. This lymphopenia was due to a decrease in both T and B cells, as FACS analysis of peripheral blood (PB) from NHD13 mice revealed a marked decrease in CD4 single positive (SP) and B220+/IgM+ cells (p<0.05) compared to WT controls; similar findings were observed in the spleen (p<0.01). The percentage of CD8 SP cells was not different between the NHD13 and WT mice in either the PB or spleen. To investigate the cause for the B-cell lymphopenia, we determined the Hardy fractions of bone marrow B cells. Although the pro-B cell (B220+/CD43+, Hardy fractions A–C) populations showed no difference between NHD13 and WT BM, the NHD13 BM showed decreased pre-B and B cell (B220+/CD43−, Hardy fractions D–F) populations, suggesting impaired differentiation at the pro-B to pre-B stage. Thymi from NHD13 mice (n=7, median age=7 months) showed grossly decreased size and decreased total number of thymocytes (1.75×107 vs. 9.21×107, p<0.01). The CD4/CD8 DN population was markedly increased (p<0.001) and the CD4/CD8 DP population markedly decreased (p<0.001) in NHD13 compared to WT mice. In addition, there was a variable increase in the DN1 and DN2 population, as well as a decrease in the DN3 and DN4 population in thymi from the NHD13 thymus, suggesting a partial block at the DN2 to DN3 transition. To determine clonality of the NHD13 thymocyte population, we used degenerate RT-PCR to identify clonal TCRβ gene rearrangements. As expected, the WT thymi showed polyclonal TCRβ gene rearrangements. However, 5 of 6 NHD13 thymus samples showed clonal DJ rearrangements, with over half of the TCRβ rearrangements in the thymus showing an identical D-J junction, but distinct V-D junctions. This finding suggested that there was a massive clonal expansion of DN2 cells that had undergone a DJ rearrangement, but not completed a VDJ rearrangement, further supporting the contention of a partial block at the DN2 to DN3 transition. Interestingly, analysis of TCRβ gene rearrangements in NHD13 spleens showed no evidence of cells with clonal DJ rearrangements, suggesting that the thymocytes with clonal DJ rearrangements did not mature and migrate from the thymus. Finally, we noted that although there is a marked increase in DN thymocytes from the NHD13 mice, the T-ALL that developed in these mice were typically DP or CD4 SP, suggesting that a rare cell that “escapes” the T-cell differentiation block is susceptible to leukemic transformation. Taken together, these findings demonstrate that the NHD13 transgene inhibits lymphoid as well as myeloid and erythroid differentiation, and is oncogenic in lymphoid as well as myeloid cells.

A complex karyotype, including a three-way translocation generating a NUP98-HOXD13 transcript, in an infant with acute myeloid leukemia

We report the case of an infant with acute myeloblastic leukemia who had the abnormal karyotype 46,XX,t(2; 11;9)(q31;p15;q22),t(6; 11;15)(q21;q23;q22),t(8;10)(q13;q22). At relapse, a different three-way translocation emerged. Fluorescence in situ hybridization and a reverse transcription-polymerase chain reaction assay detected the NUP98-HOXD13 fusion gene in bone marrow cells of the patient at diagnosis and at relapse. Sequence analysis showed that exon 12 of NUP98 was fused in-frame with exon 2 of HOXD13. The patient had neither a rearrangement of the MLL gene nor aberrations for FLT3, KIT, NRAS, KRAS, or PTPN11. The NUP98-HOXD13 fusion transcript created by t(2;11;9)(q31;p15;q22) may play an important role in the leukemogenesis in this case.

Mouse embryonic stem cells that express a NUP98–HOXD13 fusion protein are impaired in their ability to differentiate and can be complemented by BCR-ABL

NUP98-HOXD13 (NHD13) fusions have been identified in patients with myelodysplastic syndrome, acute myelogenous leukemia and chronic myeloid leukemia blast crisis. We generated 'knock-in' mouse embryonic stem (ES) cells that express a NHD13 fusion gene from the endogenous murine NUP98 promoter, and used an in vitro differentiation system to differentiate the ES cells to hematopoietic colonies. Replating assays demonstrated that the partially differentiated NHD13 ES cells were immortal, and two of these cultures were transferred to liquid culture. These cell lines are partially differentiated immature hematopoietic cells, as determined by morphology, immunophenotype and gene expression profile. Despite these characteristics, they were unable to differentiate when exposed to high concentrations of erythropoietin (Epo), granulocyte colony-stimulating factor or macrophage colony-stimulating factor. The cell lines are incompletely transformed, as evidenced by their dependence on interleukin 3 (IL-3), and their failure to initiate tumors when injected into immunodeficient mice. We attempted genetic complementation of the NHD13 gene using IL-3 independence and tumorigenicity in immunodeficient mice as markers of transformation, and found that BCR-ABL successfully transformed the cell lines. These findings support the hypothesis that expression of a NHD13 fusion gene impairs hematopoietic differentiation, and that these cell lines present a model system to study the nature of this impaired differentiation.

Retroviral Insertion Tagging Identifies Mn1 as a Specific Collaborator of NUP98-HOXD13 in a Murine Model of Leukemogenesis.

The NUP98-HOXD13 (NHD13) fusion gene is formed by the t(2;11)(q31;p15) in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). This fusion gene encodes a protein that fuses the N-terminal portion of NUP98, a nucleoporin involved in mediating RNA and protein transport in and out of the nucleus, with the C-terminal portion of HOXD13, a homeodomain protein not expressed during normal haematopoietic development. We have previously demonstrated that expression of the NHD13 fusion gene in a transgenic mouse model results in an invariably fatal MDS. These mice either die of complications of severe pancytopenia or progress to a fatal acute leukemia; greater than 90% of mice die by 14 months of age. The prolonged latency suggests that additional genetic events are required for leukemic transformation. We used a MOL4070LTR retroviral mutagenesis screen to identify candidate genes that might collaborate with the NHD13 transgene. All transgenic mice infected with the MOL4070LTR virus developed an acute leukemia, and the median survival of these mice was only four months, with none surviving beyond seven months of age. These survival figures are significantly decreased compared to either the wild type infected group or the transgenic uninfected group, suggesting a true synergistic effect between the NUP98-HOXD13 transgene and the genes affected by retroviral insertion events. Cloning of the insertion sites allowed identification of candidate collaborating genes. The most frequently identified gene was Meis1, a gene known to collaborate with HOX genes during leukemic transformation. This result supports the contention that the retroviral infection screen identifies specific collaboration between retrovirus and transgene. The second most common insertion site was near Mn1. These insertions clustered in two regions: four occurred within the final intron of Mn1, and four occurred between 80 kb and 160 kb 3′ of the 3′ end of the gene. Of note, MN1 is involved in human leukemia by fusion to the TEL oncogene via the t(12;22)(p13;q11) in myeloid leukemia. This translocation occurs within the intron of the human MN1 gene equivalent to that in which the four retroviral insertions occur in the mouse Mn1 gene. Moreover, this fusion transcript has recently been shown to collaborate with HOXA9 overexpression in leukemogenesis (Kawagoe and Grosveld, Blood 106(13):4269–77). We identified a fusion transcript formed in these four mice by RNA splicing between the 5′ part of Mn1 and the retroviral env gene. In addition, all eight of the mice with Mn1 insertions (both intronic and 3′) showed elevated wild-type Mn1 expression, suggesting that overexpression of wild-type Mn1 as well as a retroviral Mn1-env fusion can collaborate with the NHD13 transgene during leukemic transformation. Further studies are underway to investigate the oncogenic potential of an Mn1-env fusion as well as overexpression of the wild-type Mn1.

Acceleration of Leukemogenesis in NUP98-HOXD13 Transgenic Mice by Infection with the MOL4070LTR Virus, and Identification of Collaborating Genes.

NUP98 is a promiscuous oncogene that is fused to at least sixteen different partner genes in a wide range of haematopoietic malignancies. The NUP98-HOXD13 (NHD13) fusion gene is formed by the t(2;11)(q31;p15), which has been observed in patients with MDS or AML. This fusion gene encodes a protein that fuses the N-terminal portion of NUP98, a nucleoporin involved in mediating RNA and protein transport in and out of the nucleus, with the C-terminal portion of HOXD13, a homeodomain protein not expressed during normal haematopoietic development. We have previously demonstrated that expression of the NHD13 fusion gene in a transgenic mouse model results in an invariably fatal myelodysplastic syndrome (MDS). The median survival for these transgenic mice is 10 months, with no mice surviving beyond 14 months of age. About one-third die due to complications of severe pancytopenia, and about two thirds progress to acute leukemia. To identify genes that might collaborate with the NHD13 transgene, a cohort of thirty mice (15 NHD13 transgenic, 15 normal) were infected with the MOL4070LTR virus at birth and the disease course monitored. All transgenic infected mice developed an acute leukemia, predominantly of the myeloid lineage. The median survival of the transgenic infected mice was only four months, with none surviving beyond 7 months of age. These survival figures are vastly decreased compared to either control group (the wild type infected group or the transgenic uninfected group), suggesting a true synergistic effect between the NUP98-HOXD13 transgene and the genes affected by retroviral insertion events. Cloning of these insertion sites has revealed proximity to numerous genes of interest, including several genes with a known role in haematopoiesis (EpoR, Ifnar2, Gata2, PU.1), development (HOXB8, HOXC9) and/or cancer (p53, Stat5b, Pim1, CyclinD1). In addition two recurrent integration sites were identified near uncharacterized ESTs. Since the NHD13 fusion has been shown to block differentiation, several of the genes we identified (p53, EpoR, Stat5b, Pim1, CyclinD1) fit the evolving AML paradigm that postulates one mutation blocks differentiation, and a second, complementary mutation leads to increased proliferation or decreased apoptosis. Ongoing analysis of these and additional collaborating genes should provide important insights into the process of the MDS to AML transition.

NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia

The myelodysplastic syndromes (MDSs) are a group of clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis and dysplasia. A wide spectrum of genetic aberrations has been associated with MDS, including chromosomal translocations involving the NUP98 gene. Using a NUP98-HOXD13 fusion gene, we have developed a mouse model that faithfully recapitulates all of the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia, and apoptosis, and transformation to acute leukemia. The MDS that develops in NUP98-HOXD13 transgenic mice is uniformly fatal. Within 14 months, all of the mice died of either leukemic transformation or severe anemia and leucopenia as a result of progressive MDS. The NUP98-HOXD13 fusion gene inhibits megakaryocytic differentiation and increases apoptosis in the bone marrow, suggesting a mechanism leading to ineffective hematopoiesis in the presence of a hypercellular bone marrow. These mice provide an accurate preclinical model that can be used for the evaluation of MDS therapy and biology.

NUP98-HOXD13 Transgenic Mice Develop Myelodysplastic Syndrome, Acute Myeloid Leukemia, and Pre-T Lymphoblastic Leukemia.

The NUP98 gene is located at chromosome 11p15 and encodes the 98 kd component of the nuclear pore complex; this protein normally functions as a docking protein involved in nucleocytoplasmic transport. NUP98 is fused to at least 15 different partner genes by chromosomal translocation in a wide spectrum of hematological malignancies including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and pre-T lymphoblastic leukemia (pre-T LBL). Over half of the known NUP98 gene fusions involve fusions to a HOX family member; these fusions invariably retain the amino terminal FG repeats of NUP98 and the homeodomain DNA-binding region of the HOX partner. The NUP98-HOXD13 fusion was initially identified in a patient with MDS that subsequently transformed to erythroleukemia, and has subsequently been identified in AML M1 and M2 patients as well. To model this disease in vivo, we generated transgenic mice which expressed the NUP98-HOXD13 (NHD13) fusion from vav regulatory elements.The NHD13 transgene is ubiquitously expressed in hematopoietic tissues such as thymus, spleen, and bone marrow, and is not expressed in other tissues. Serial CBCs from clinically healthy mice aged 4–7 months demonstrated a progressive neutropenia, lymphopenia, anemia, and macrocytosis. Peripheral blood smears showed signs of dysplasia including giant platelets and hypersegmented neutrophils; bone marrow exam showed an increase number of dysplastic binucleate erythroblasts and increased apoptosis, consistent with a diagnosis of MDS. 10/10 (100%) of the NHD13 mice died of hematologic disease by 14 months of age; in contrast, none of the non-transgenic control littermates developed evidence of hematologic disease. We classified the hematologic diseases according to the Bethesda proposals. Three mice died with MDS, two mice had pre-T LBL, two had acute undifferentiated leukemia, one had megakaryocytic leukemia, one had myeloid leukemia with maturation, and one had both pre-T LBL and erythroid leukemia.The malignant blasts from mice with pre-T LBL showed monoclonal T-cell receptor B gene rearrangements and were positive for CD3, 4, and 8. The mouse with megakaryocytic leukemia had serial CBCs documenting a platelet count of 3.2 million/uL, rising to >15million/uL at the time of death. This mouse had CD41+ megakaryocytes and megakaryoblasts invading the liver and spleen, and an osteosclerotic bone marrow reminiscent of chronic idiopathic myelofibrosis (CIMF). The mouse with concurrent pre-T LBL and erythroid leukemia had replacement of the thymus and infiltration of the lung with T-lymphoblasts which had a clonal TCRB gene rearrangement; interestingly, the spleen, liver, and bone marrow of this mouse were invaded with erythroblasts that were negative for CD3 and TCRB gene rearrangements.We conclude that the NHD13 transgene consistently induces an MDS, of variable severity, in these mice. Some mice die of severe anemia due to MDS, and MDS transforms into an acute non-lymphoid leukemia in other mice. Still other mice die of pre-T LBL which we believe evolves in the thymus separately from the MDS. These data demonstrate that the NHD13 fusion gene is transforming in both lymphoid and myeloid cells.

Induction of acute myeloid leukemia in mice by the human leukemia-specific fusion gene NUP98-HOXD13 in concert with Meis1

AbstractHOX genes, notably members of the HOXA cluster, and HOX cofactors have increasingly been linked to human leukemia. Intriguingly, HOXD13, a member of the HOXD cluster not normally expressed in hematopoietic cells, was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months), when some mice developed a myeloproliferative-like disease. In contrast, mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML), with a median disease onset of 75 days. In summary, this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation, and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways.

The NUP98-HOXD13 fusion gene implicated in human leukemia induces myeloproliferation and blocks early b cell development in murine hematopoiesis

The nucleoporin gene NUP98 (11p15) has been reported to be fused to a number of genes including the homeobox HOX genes A9 and D13 in therapy-related AML. To investigate the role and effects that the fusion gene may have on hematopoiesis and in the development of leukemia, we used the MSCV retrovirus (GFP as a selectable marker) to express the NUP98-HOXD13 cDNA in murine bone marrow (BM). NUP98-HOXD13 (GFP+) transduced BM cells had a 1.8 fold increase (n = 5) in clonogenic colony forming cell numbers in vitro, compared to control GFP+ BM cells. Moreover, they yielded a 20 fold increase in day 12 CFU-S following 1 week in liquid culture. To assess the effect on hematopoiesis in vivo, mice were transplanted (TX) with transduced BM cells (n = 9). BM biopsy and peripheral blood analysis (PB) 12 weeks post-TX revealed a reduced level of circulating B220+ B cells (0.72 vs 2.47 × 106/ml) and a 25 fold reduction in B cell clonogenic progenitors in the BM. Two NUP98-HOXD13 mice sacrificed 18 weeks post-TX for further analysis manifested varying degrees of increased myelopoiesis. One mouse had clear signs of early myeloproliferative syndrome (MPS), including a marked increase in splenic CFC-GM (65 fold) and Gr-1+ cells (4 fold). The myeloproliferation was transplantable and progressive with 2 of 5 secondary mice becoming moribund by 10 weeks post-TX and MPS, as shown by increased PB counts, splenomegaly and myeloid cell infiltration in multiple organs. These results provide direct evidence of the potential of NUP98-HOXD13 to grossly perturb hematopoiesis.