Myelodysplastic Syndrome In Mice Research Articles

Oncogenic RAS promotes leukemic transformation of CUX1-deficient cells.

-7/del(7q) is prevalent across subtypes of myeloid neoplasms. CUX1, located on 7q22, encodes a homeodomain-containing transcription factor, and, like -7/del(7q), CUX1 inactivating mutations independently carry a poor prognosis. As with loss of 7q, CUX1 mutations often occur early in disease pathogenesis. We reported that CUX1 deficiency causes myelodysplastic syndrome in mice but was insufficient to drive acute myeloid leukemia (AML). Given the known association between -7/del(7q) and RAS pathway mutations, we mined cancer genome databases and explicitly linked CUX1 mutations with oncogenic RAS mutations. To determine if activated RAS and CUX1 deficiency promote leukemogenesis, we generated mice bearing NrasG12D and CUX1-knockdown which developed AML, not seen in mice with either mutation alone. Oncogenic RAS imparts increased self-renewal on CUX1-deficient hematopoietic stem/progenitor cells (HSPCs). Reciprocally, CUX1 knockdown amplifies RAS signaling through reduction of negative regulators of RAS/PI3K signaling. Double mutant HSPCs were responsive to PIK3 or MEK inhibition. Similarly, low expression of CUX1 in primary AML samples correlates with sensitivity to the same inhibitors, suggesting a potential therapy for malignancies with CUX1 inactivation. This work demonstrates an unexpected convergence of an oncogene and tumor suppressor gene on the same pathway.

Activation Levels of b-Catenin Signaling in Osteoblasts Define a Threshold for the Development of Myeloid Neoplasia of Variable Severity

A growing body of evidence indicates that osteoblasts have a direct pathogenetic role in the development of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and transformation of MDS to AML. Models of osteoblast-induced MDS or AML (OIAML) include b-catenin activation in osteoblasts inducing AML in mice and seen in more than 30% of MDS and AML patients; deletion of Dicer1 in osteoblast progenitors leading to MDS in mice; Noonan syndrome mutation (PTPN11) in stromal cells driving MPN in mice; and Schwachman-Diamond syndrome mutation in stromal cells driving MDS in mice and predicting AML progression in patients. To further understand the mechanism of OIAML we examined whether a defined threshold of AML-inducing signal by osteoblasts is required for disease development. For this purpose we constitutively activated b-catenin in osteoblasts using three different lines of 2.3Col1 a 1-Cre transgenic mice characterized by different recombination efficiencies in bone. Line 1, in which recombination efficiency was 80%. Line 2 was the same original line in which after several inter-breedings we detected a reduction in recombination efficiency to 20%. Line3, which has 75% recombination efficiency. Activation of b-catenin in osteoblasts using Line 1, led to the development of MDS which rapidly transformed to full-blown AML characterized by multi-organ infiltration with myeloid blasts, hematopoietic deregulation and lethality by 6 weeks of age. In contrast, activation of b-catenin in osteoblasts using Line 2 did not lead to AML development. Mice remained healthy and survived until at least 10 months of age, the entire time they were observed. Activation of b-catenin in osteoblasts using Line 3 led to anemia, with the appearance of myeloid blasts in the blood, marrow and spleen and segmented neutrophils in blood. In addition to what was observed with Line 1, Pelger Huet neutrophils and Howell-Jolly bodies were detected in the blood. The latter are RBCs with inclusions of nuclear chromatin remnants (damaged chromosome fragments) indicating genomic instability and a dysfunctional spleen, unable to clear them from the circulation, due to dense infiltration with blasts and megakaryocytes. A spectrum of atypical immature monocytes with abundant pale blue agranular cytoplasm with vacuoles were observed in the blood. Taken together these features resemble characteristics of human MDS/CMML. Mice die between 4 and 14 weeks of age, when they undergo “blast crisis” indicating transformation to AML. To examine whether and how these observations relate to the human disease, we compared the levels of b-catenin activation in osteoblasts (calculated as a function % osteoblasts (Lin-/CD34-/RUNX2+ cells) expressing activated b-catenin) with the different MDS diseases subtypes as those are defined by the 2008 World Health Organization (WHO) classification system. We found that increased levels of b-catenin activation in osteoblasts correlate with the severity of the MDS type and with conversion to AML. Collectively, these observations suggest that a minimal activation level of a key leukemogenic signal from the stroma, is required to initiate cancer development, and provides mechanistic insight into the formation and progression of preleukemic stem cells in AML and the transformation of MDS to AML. DisclosuresRaza:Onconova Therapeutics: Research Funding, Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Genoptix: Speakers Bureau; Janssen R&D: Research Funding; Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding.

Oncogenic KrasG12D Activation in the Nonhematopoietic Bone Marrow Microenvironment Causes Myelodysplastic Syndrome in Mice

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic RAS mutations, causing constitutive activation of multiple tumor-promoting pathways, are frequently found in human cancer. So far in hematologic malignancies, RAS mutations have only been reported to occur in hematopoietic cells. In this study, we investigated the effect of oncogenic Kras expression in the BMME in a chimeric mouse model. We observed that an activating mutation of Kras in the non-hematopoietic system leads to a phenotype resembling myelodysplastic syndrome (MDS) characterized by peripheral cytopenia, marked dysplasia within the myeloid lineage as well as impaired proliferation and differentiation capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotypical changes could be reverted when the BM was re-isolated and transferred into healthy recipients, indicating that the KrasG12D-activation in the non-hematopoietic BMME was essential for the MDS phenotype. Gene expression analysis of sorted non-hematopoietic bone marrow (BM) niche cells from KrasG12D mice revealed upregulation of multiple inflammation-related genes including IL-1-superfamily members (Il1α, Il1β, Il1f9) and the NLPR3 inflammasome. Thus, pro-inflammatory IL-1-signaling in the BMME may contribute to MDS development. Our findings show that a single genetic change in the non-hematopoietic BMME can cause an MDS phenotype. Oncogenic Kras activation leads to pro-inflammatory signaling in the BMME which impairs HSPCs function. Implications: These findings may help to identify new therapeutic targets for MDS.

ZRSR1 co-operates with ZRSR2 in regulating splicing of U12-type introns in murine hematopoietic cells.

Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. In order to further investigate the role of this splice factor in RNA splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not in human cells contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to accurately model ZRSR2-mutant MDS in mice.

FoxM1 Haploinsufficiency Drives Clonal Hematopoiesis

Hematopoiesis is an orchestrated process in which hematopoietic stem cells (HSCs) can self-renew and produce all lineages of blood cells. Majority of HSCs are in a quiescent state with a low growth rate. However, some genetic mutations that occur in HSCs impel HSCs to exit the quiescent state and to proliferate excessively, which enables mutant HSCs to outcompete normal HSCs and leads to clonal expansion of mutant HSCs. Myelodysplastic syndromes (MDSs) as a clonal disease, arise from the expansion of mutant HSCs and are characterized by morphologic dysplasia, ineffective hematopoiesis and an increased risk of transformation to acute myeloid leukemia. FoxM1 is one of transcription factors in the family of Fox ('Forkhead box') proteins. Analysis of public database revealed that the expression level of FOXM1 was decreased significantly in CD34 + cells from a subset of patients with MDS as compared to healthy individuals. Thus, we sought to determine whether haploinsufficiency of FOXM1 contributes to the development of MDS in mice. Our study showed that haploinsufficiency of Foxm1 led to an expansion of hematopoietic stem/progenitor cells in mice. Since FoxM1 has previously been implicated in regulation of cell cycle, we determined the cell cycle status of Foxm1 heterozygous HSCs. By BrdU incorporation assay, we showed that Foxm1 heterozygous HSCs have an increased S phase and G2/M phase as compared to control HSCs from wildtype mice. Additional analysis with Hochest33342/Pyronin-Y staining and Ki67/DAPI staining showed a significant decrease in the number of quiescent (G0) cells in Foxm1 heterozygous HSCs as compared to control HSCs. These results suggest that FoxM1 haploinsufficiency promotes HSCs to exit quiescence and to enter cell cycle, thereby leading to exhaustion of HSCs. To further assess the function of Foxm1 heterozygous HSCs in vivo, we performed competitive repopulation assay. We found that Foxm1 haploinsufficiency HSCs exhibited competitive repopulation advantage in the first and secondary recipient mice, but displayed significantly decreased capacity of repopulation in tertiary recipient mice as compared to control HSCs, suggesting that Foxm1 haploinsufficiency promoted clonal expansion of HSCs, which leads to an exhaustion of HSCs eventually. HSC proliferation can be induced by some specific immune effectors such as Toll-like receptor 4 (TLR4). Lipopolysaccharide (LPS) stimulates HSC proliferation by activating TLR4 signaling pathway. Low dose of LPS treatment over time accelerated the development of MDS in mice. Interestingly, low dose of LPS injection chronically induced defects in hematopoiesis in Foxm1 haploinsufficient mice but not the control wildtype mice. Recipient mice transplanted with Foxm1 heterozygous BM cells but not the control BM cells developed MDS-like disease with cytopenia and a decreased number of hematopoietic stem/progenitor cells after LPS stimulation. Moreover, we found that nearly half of aged Foxm1 haploinsufficient mice (20 months) developed splenomegaly. Analysis of histologic sections in Foxm1 haploinsufficient mice showed that the mice developed hematopoietic dysplasia including dysplastic megakaryocytes with bizarre-shaped nuclei in bone marrow and extramedullary hematopoiesis with accumulation of myeloid cells in spleen. RNA-seq analysis indicated that haploinsufficiency of Foxm1 perturbed multiple stem cell-maintenance mechanisms especially in metabolic processes. Taken together, our studies suggest that Foxm1 haploinsufficiency in mice causes clonal expansion of HSCs and promotes MDS-like disease, which underscores the significant role of FOXM1 downregulation in the initiation and development of human MDS. Disclosures No relevant conflicts of interest to declare.

Inhibition of HIF1α Signaling: A Grand Slam for MDS Therapy?

The recent focus on genomics in myelodysplastic syndromes (MDS) has led to important insights and revealed a daunting genetic heterogeneity, which is presenting great challenges for clinical treatment and precision oncology approaches in MDS. Hayashi and colleagues show that multiple mutations frequently found in MDS activate HIF1α signaling, which they also found to be sufficient to induce overt MDS in mice. Furthermore, both genetic and pharmacologic inhibition of HIF1α suppressed MDS development with only mild effects on normal hematopoiesis, implicating HIF1α signaling as a promising therapeutic target to tackle the heterogeneity of MDS. Cancer Discov; 8(11); 1355-7. ©2018 AACR See related article by Hayashi et al., p. 1438.

Abstract B42: Modeling MDS in mice through precise molecular lesions in APC and mDia1.

Abstract While the murine equivalent to “5q– syndrome” should map to the distal arms of murine chromosomes 11 and 18, molecular attempts have been unsuccessful in generating a reliable mouse model of myelodysplasia leading to exacerbated myeloid leukemia. Genetic studies suggest interactions between individual proteins in this locus suppress tumor initiation. More specifically, cellular and biochemical studies demonstrated collaboration between two proteins in the commonly deleted regions of 5q– syndrome: mDia1 (mammalian Diaphanous-related Rho GTPase effector formin) and APC (adenomatous polypolis coli). Therefore, we generated mice compounding these two putative disease alleles. Because both mDia1 knockout [Drf1(–/–)] mice and Apcmin (min = multiple intestinal neoplasia; haploinsufficient) mice develop age-dependent myelodysplastic syndrome (MDS), we hypothesized that a mouse strain lacking both proteins will exhibit a more severe MDS phenotype than either mutant allele alone. To define the disease caused by mutation in Drf1 and Apcmin, we monitored erythropoiesis, myelopoiesis, and overall survival. At all ages, mice with both mutant alleles exhibit granulocytosis. Anemia occurs in aged mice (>270 days), exhibiting drastic differences in blood cell morphology that corresponded to differences in long-term survival in comparison to either Drf1–/– or Apcmin animals. Furthermore, immunophenotyping of all subpopulations indicate an accumulation of CD71+/TER-119+ cells in mice with both mutant alleles, however no significant differences in CD11b expression appeared among CD45+ bone marrow subpopulations. Our studies indicate that combined loss of mDia1 and APC expression and function direct disease progression. We are currently defining the hematopoietic precursor cell responsible for anemia and bone marrow failure. This enhanced mouse model also serves as molecular laboratory to study disease progression and to test potential therapeutics under development in our laboratory. Citation Format: Julie Davis Turner, Susan Kitchen-Goosen, Andrew M. Howard, Heather Schumacher, Art S. Alberts. Modeling MDS in mice through precise molecular lesions in APC and mDia1. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B42.

BRCA1-Down-Regulation In Chronic Myeloid Leukemia (CML) Occurs Via The Deubiquitinase / Tumor Suppressor BRCA1-Associated Protein 1 (BAP1)

BCR-ABL has been shown to lead to a genetic instability in leukemic cells either directly by inducing oxidative stress or indirectly by compromising DNA repair mechanisms. BRCA1 is a major DNA repair gene as it promotes homologous recombination and plays thereby a critical role for preserving genomic integrity. We have previously reported that BCR-ABL down-regulates BRCA1 protein using a post-transcriptional mechanism (Deutsch et al, Blood 2003). The precise mechanism of this down regulation had not been established so far. BAP1 (BRCA1 associated protein-1) is a tumor suppressor gene that encodes a nuclear ubiquitin carboxy-terminal hydrolase, which interacts with BRCA1 protein and with many other cell cycle regulators. BAP1 is mutated in hereditary cancers and the overexpression of a deleted form of BAP1 has been shown to lead to a myelodysplastic syndrome in mice (Dey et al, Science 2012). In a gene profiling analysis of the human UT7 cells expressing BCR-ABL, we have discovered that the expression of BAP-1 is down-regulated as compared to parental UT7 cells. Using qRT-PCR and Western blotting analyses, we have confirmed the reduction of BAP1 transcript and protein levels in UT7 cells expressing BCR-ABL. This occurs in a tyrosine kinase dependent manner as exposure to Imatinib reverted BCR-ABL-associated BAP1 down-regulation. To determine the effects of BAP1 complementation in leukemic cells, we have transfected UT7-BCR-ABL cells with a BAP1 expression vector. The enforced expression of BAP1 in BCR-ABL expressing cells restored BRCA1 protein levels without affecting its mRNA level. As BAP1 is a deubiquitinase, we wondered whether there was an increased ubiquitination of BRCA1 in BCR-ABL expressing cells, due to a BAP1 deficiency. In the UT7-BCR-ABL model, we have performed immunoprecipitation of BRCA1 followed by Western blotting using anti-ubiquitin antibodies. These experiments revealed that BRCA1 was highly ubiquitinylated in BCR-ABL-expressing cells as compared to parental UT7 cells, explaining potentially its down-regulation in CML at the protein level via a proteasome-related mechanism. We next wished to validate these findings in primary human CML samples using qRT-PCR. In a cohort of newly diagnosed chronic phase CML patients before any therapy (n= 21) blood mRNA levels of BAP1 were significantly reduced ( p=0.0032, Mann Whitney Test ) as compared to normal controls (n= 8). Thus, our report reveals for the first time loss of BAP1 expression as a mechanism of the down-regulation of the DNA repair protein BRCA1 in CML. In addition to its contribution to genetic instability, BAP1 could be directly involved in the pathophysiology of CML due to its interactions with epigenetic factors such as polycomb proteins. The molecular mechanisms of BAP1 downregulation in BCR-ABL-expressing leukemic cells is under investigation. Disclosures:Guilhot:Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:BMS, Novartis: Honoraria, Research Funding.

Modeling Myelodysplastic Syndromes In Mice By Altered Hoxa1 Spliceform Expression

Myelodysplastic syndromes (MDS) are a heterogeneous group of blood cell diseases with 30% of the patients developing acute myeloid leukemia (AML). The etiology of MDS is largely unknown and as a result there are no curative therapies. Mouse models that faithfully represent MDS are based on rare genetic abnormalities and currently represent only a very small subset of MDS patients.Using in silico analysis we have identified that homeobox A1 (HOXA1) mRNA is significantly upregulated in 50% of MDS patients (1.8-fold increase, n=183 MDS, 17 controls, P<0.05). Importantly, the upregulation was observed across all subsets of patients, including those with a normal karyotype. Both human HOXA1 and mouse Hoxa1 are expressed as two different spliceforms generated by alternative splicing within exon 1 of the wildtype Hoxa1 (WT-Hoxa1). These spliceforms encode a full-length (Hoxa1-FL) and a truncated form (Hoxa1-T), the latter lacking the homeobox domain. Given that mutations in splicing machinery have been identified in up to 85% of MDS patients we hypothesised that deregulated HOXA1 spliceforms may contribute to MDS.We identified that the Hoxa1 isoforms were differentially expressed in murine hematopoietic stem cells (HSCs) and progenitors. Retroviral overexpression of either WT-Hoxa1 (which generates both Hoxa1-FL and Hoxa1-T) or Hoxa1-T in murine bone marrow (BM) cells showed opposing effects on in vitro cell proliferation and colony forming cell (CFC) production respectively, suggesting that Hoxa1-T may negatively regulate Hoxa1-FL. We therefore generated a mutant Hoxa1 (MUT-Hoxa1), which expresses normal Hoxa1-FL but not Hoxa1-T, by oligomutagenesis at the splice site of Hoxa1-T. We transplanted recipient mice (n>28 per group) with bone marrow (BM) overexpressing either MUT-Hoxa1, WT-Hoxa1 or empty vector (MXIE) control.All recipients of MUT-Hoxa1 BM developed peripheral blood (PB) macrocytic anemia (mean ± SEM: Hb (g/L): MUT-Hoxa1: 126±2.9**; WT-Hoxa1: 134±2.7; MXIE: 135±1.4; **P<0.01 vs MXIE), without leukocytosis. Furthermore, thrombocytopenia was also observed in MUT-Hoxa1 and WT-Hoxa1 recipients (PB platelets (x 106/ml): MUT-Hoxa1: 738±88*; WT-Hoxa1: 777±57*; MXIE: 922 ± 37, *P<0.05 vs MXIE). Overexpression of MUT-Hoxa1 or WT-Hoxa1 was associated with significant apoptosis in BM erythroid cells (% apoptosis in GFP+ Ter119+ cells: MUT-Hoxa1: 20.7±4.9*; WT-Hoxa1: 25.3±5.2** MXIE: 13.6±2.5, *P<0.05, **P<0.01 vs MXIE). We also observed numerous dysplastic erythroid cells and micromegakaryocytes in the MUT-Hoxa1-overexpressing cells.Strikingly, 88% of recipients of MUT-Hoxa1 BM, but no WT-Hoxa1 recipients, developed AML with a median time of 10.5 months post-transplant, independent of retroviral integration site (Kaplan-Meier survival curve, P<0.05 vs MXIE). Therefore, overexpression of MUT-Hoxa1 or WT-Hoxa1 in BM cells results in clinical features of MDS, with MUT-Hoxa1 overexpression resulting in a more aggressive MDS that spontaneously progresses to AML.Analysis of BM from non-leukemic mice identified that MUT-Hoxa1 and WT-Hoxa1 recipients had significantly decreased HSC-containing lineage -ve, c-kit+, Sca-1+ (LKS+) cells (% of lin- GFP+ BM: MUT-Hoxa1 1.72 ± 0.28#; WT-Hoxa1: 2.08 ± 0.37#; MXIE: 5.09 ± 0.56%; #P<0.0001 vs MXIE, n>12). This was accompanied by increased progenitor cell-containing LKS- (% of lin- GFP+ BM: MUT-Hoxa1: 53.32 ± 5.65*; WT-Hoxa1: 47.42 ± 4.50; MXIE: 38.37 ± 4.13; *P<0.05 vs MXIE, n>12). Strikingly, granulocyte/monocyte progenitors were significantly reduced in the MUT-Hoxa1 LKS- cells, accompanied by a marked accumulation of megakaryocyte/erythroid progenitors (MEPs). We have identified deregulated gene pathways in the MUT-Hoxa1 BM MEPs by microarray analyses. We are currently testing drug candidates identified from these screens for their effects on BM obtained from our MUT-Hoxa1 and WT-Hoxa1 mouse models and MDS patients.Taken together, these results suggest that overexpression of Hoxa1-FL results in MDS, and that concurrently inhibiting splicing of Hoxa1-T (MUT-Hoxa1) forms a more aggressive MDS phenotype. Preliminary analysis of CD34+ BM cells from MDS patients reveal significant numbers of patients with elevated HOXA1-FL mRNA compared to lower or absent levels of HOXA1-T. Our MUT-Hoxa1 and WT-Hoxa1 mouse models will therefore be highly valuable in identifying better therapies for a significant subset of MDS patients. Disclosures:No relevant conflicts of interest to declare.

O-008 Modeling and targeting MLL-PTD/RUNX1 related myelodysplastic syndromes in mice

Results: WES identified 10 somatic mutations in our index patient. Among those, three previously reported genes in myeloid mailgnancies: BCOR, RUNX1 and STAG2. Direct sequencing of BCOR and the related BCORL1 genes in a cohort of 221 MDS patients identified additional 10 inactivating BCOR (4.5%) and five BCORL1 mutations (2.3%). Mutations were found in patients of all IPSS risk-groups and WHO subtypes. BCOR mutations were associated with RUNX1 (P=0.027). Similar frequencies for BCOR and BCORL1mutations were found in an extension cohort of 208 patients with various myeloid malignancies: 8 BCOR and 8 BCORL1 mutations (3.8% each). BCORL1 mutations were most often found in patients with AML-MRC disease (3/22=13.6%). Deep sequencing covered with a median of 6415 reads every mutation identified in 8 BCOR mutated patients. The quantified frequency of BCOR mutations affected BM cells with a range of 13.5% to 79%. With the exception of one patient, BCOR mutations never presented at the greatest frequency, which would be expected if they were involved in disease initiating events. BCOR mutations arise commonly after other mutations affecting the splicing machinery or epigenetic transcription. In univariate analysis, BCORmutationswere associated with a poor prognosis in MDS patients (Overall Survival: P=0.034, AML transformation: P=0.051). Multivariate analysis confirmed a significant inferior OS (HR: 3.3; 95%CI 1.4–8.1; P=0.008) for BCOR mutated patients. Conclusions: These data suggest that BCOR mutations are rather defining the clinical course than disease initiation and that despite their relatively low global frequency, BCOR might be considered as a key gene in risk stratification.

A NUP98-HOXD13 Fusion Gene Impairs Differentiation of B and T Lymphocytes and Leads to Expansion of Thymocytes with Partial TCRB Gene Rearrangement

Expression of a NUP98-HOXD13 (NHD13) fusion gene leads to myelodysplastic syndrome in mice. In addition to ineffective hematopoiesis, we observed that NHD13 mice were lymphopenic; the lymphopenia was due to a decrease in both T and B lymphocytes. Although the pro-B cell (B220(+)/CD43(+)) populations from the NHD13 and wild-type mice were similar, the NHD13 mice showed decreased pre-B cells (B220(+)/CD43(-)), indicating impaired differentiation at the pro-B to pre-B stage. Thymi from NHD13 mice were smaller and overexpressed Hoxa cluster genes, including Hoxa7, Hoxa9, and Hoxa10. In addition, the NHD13 thymi contained fewer thymocytes, with an increased percentage of CD4(-)/CD8(-) (double-negative (DN)) cells and a decreased percentage of CD4(+)/CD8(+) (double-positive) cells; the DN1/DN2 population was increased and the DN3/DN4 population was decreased, suggesting a partial block at the DN2 to DN3 transition. To determine clonality of the thymocytes, we used degenerate RT-PCR to identify clonal Tcrb gene rearrangements. Five of six NHD13 thymi showed an unusual Tcrb gene rearrangement pattern with common, clonal DJ rearrangements, but distinct V-D junctions, suggesting a marked clonal expansion of thymocytes that had undergone a DJ rearrangement, but not completed a VDJ rearrangement. Taken together, these findings demonstrate that expression of the NHD13 transgene inhibits lymphoid as well as myeloid and erythroid differentiation, results in overexpression of Hoxa cluster genes, and leads to a precursor T cell lymphoblastic leukemia/lymphoma.

Impaired differentiation and apoptosis of hematopoietic precursors in a mouse model of myelodysplastic syndrome

Expression of a NUP98-HOXD13 (NHD13) fusion gene, initially identified in a patient with myelodysplastic syndrome, leads to a highly penetrant myelodysplastic syndrome in mice that recapitulates all of the key features of the human disease. Expansion of undifferentiated lineage negative (lin(neg)) hematopoietic precursors that express NHD13 was markedly inhibited (30-fold) in vitro. Decreased expansion was accompanied by decreased production of terminally differentiated cells, indicating impaired differentiation of NHD13 precursors. Rather than differentiate, the majority (80%) of NHD13 lin(neg) precursors underwent apoptotic cell death when induced to differentiate. These findings demonstrate that NHD13 lin(neg) cells provide a tractable in vitro system for studies of myelodysplastic syndrome.

NUP98-HOX Translocations Lead to Myelodysplastic Syndrome in Mice and Men

The myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute myeloid leukemia (AML). A wide spectrum of genetic aberrations has been associated with MDS, including chromosomal translocations involving the NUP98 gene, most commonly leading to fusions of NUP98 with abd-b group HOX genes, including HOXD13. We used vav regulatory elements to direct expression of a NUP98-HOXD13 (NHD13) fusion gene in hematopoietic tissues. NHD13 transgenic mice faithfully recapitulate all the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia and apoptosis, and transformation to acute leukemia. The MDS that develops in NHD13 transgenic mice is highly lethal; within 14 months, 90% of the mice died of either leukemic transformation or severe anemia and leukopenia due to progressive MDS. These mice provide a preclinical model that can be used for the evaluation of MDS therapy and biology.

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.

Arsenic Trioxide Targets the MDS1 and EVI1- Containing Oncoproteins for Degradation by Proteasome Dependent and Independent Mechanisms.

The EVI1 gene was first identified as a common site of retroviral integration in murine myeloid tumors and expresses a zinc finger-containing transcriptional regulator. It also encodes for MDS1/EVI1, a longer, alternatively spliced form of EVI1. In blood development EVI1 is normally expressed in hematopoietic stem cells (HSCs) but not in committed cells. However, aberrant expression of EVI1 or MDS1/EVI1 has been frequently found in myelodysplastic syndrome (MDS), and in cases of chronic myeloid leukemia during blast phase (CML-BP) and acute myelogenous leukemia (AML). In addition, the MDS1/EVI1 gene fuses to the AML1 (Runx1) gene as a consequence of the t(3;21)(q26;q22) translocation found in patients with CML-BP and therapy related MDS and AML, producing the AML1/MDS1/EVI1 (AME) or AML1/MDS1 fusion genes. We have previously shown that expression of the AME fusion protein in mouse bone marrow cells by retroviral transduction impaired hematopoiesis and eventually induced an AML. EVI1 was also reported to induce MDS in mice. These results indicate that the EVI1 and AME oncoproteins play causal roles in t(3;21)- and EVI1-positive hematological malignancies. Therapies targeting oncoproteins that play causal roles in tumorigenesis have been shown to be effective in treating cancers. Arsenic trioxide (ATO) has been found as an effective treatment for patients suffering from acute promyelocytic leukemia (APL) that harbor the t(15;17) translocation. The success of ATO in treating APL patients has prompted many to consider expanding the drug's use to target other translocations and/or mutations found in hematological malignancies. In this study we found that ATO could target the EVI1, MDS1/EVI1, AML1/MDS1 and AME oncoproteins for degradation. We further found that ATO targeted EVI1 for degradation through the ubiquitin-proteasome pathway. Interestingly, ATO induced degradation of MDS1-containing oncoproteins via a proteasome-independent mechanism. Consistent with the downregulation of AME, the ATO treatment induced differentiation and apoptosis in AME leukemic cells in vitro and reduced splenomegally in mice transplanted with AME leukemic cells. Our results suggest that ATO could be used as a part of targeted therapy for AME-, AML1/MDS1-, MDS1/EVI1- and EVI1-positive hematological malignancies. We are currently studying the mechanism of increased ubiquitination of EVI1 by ATO, as well as the proteasome-independent mechanism by which ATO downregulates MDS1-containing proteins. Such research will help further the identification of new therapeutic targets and the development of new therapeutic strategies for human cancer.

EVI1 induces myelodysplastic syndrome in mice

EVI1 induces myelodysplastic syndrome in mice

EVI1 induces myelodysplastic syndrome in mice

EVI1 induces myelodysplastic syndrome in mice

EVI1 induces myelodysplastic syndrome in mice

EVI1 induces myelodysplastic syndrome in mice

EVI1 induces myelodysplastic syndrome in mice.

Myelodysplasia is a hematological disease in which genomic abnormalities accumulate in a hematopoietic stem cell leading to severe pancytopenia, multilineage differentiation impairment, and bone marrow (BM) apoptosis. Mortality in the disease results from pancytopenia or transformation to acute myeloid leukemia. There are frequent cytogenetic abnormalities, including deletions of chromosomes 5, 7, or both. Recurring chromosomal translocations in myelodysplasia are rare, but the most frequent are the t(3;3)(q21;q26) and the inv(3)(q21q26), which lead to the inappropriate activation of the EVI1 gene located at 3q26. To better understand the role of EVI1 in this disease, we have generated a murine model of EVI1-positive myelodysplasia by BM infection and transplantation. We find that EVI1 induces a fatal disease of several stages that is characterized by severe pancytopenia. The disease does not progress to acute myeloid leukemia. Comparison of in vitro and in vivo results suggests that EVI1 acts at two levels. The immediate effects of EVI1 are hyperproliferation of BM cells and downregulation of EpoR and c-Mpl, which are important for terminal erythroid differentiation and platelet formation. These defects are not fatal, and the mice survive for about 10 months with compensated hematopoiesis. Over this time, compensation fails, and the mice succumb to fatal peripheral cytopenia.

The transcription factor Evi-1

Evi-1 is a transcription factor with two sets of zinc finger domains. The temporally and spatially restricted pattern of Evi-1 expression in embryonic tissues suggests a role of Evi-1 in organogenesis and morphogenesis in mouse development. Mice lacking Evi-1, which die within the first few weeks of life with multiple defects in embryonic development, suggest that Evi-1 is essential for developmental cell proliferation, vascularization, and cell-specific signaling at midgestation. In hematopoietic cells, Evi-1 expression is restricted at a transient stage of myeloid cell differentiation. Constitutive expression of Evi-1 in hematopoietic cells, which is caused by retroviral insertions or chromosomal translocations and inversions, is closely associated with myelogenous leukemias and myelodysplastic syndromes in mice and humans. In the aspect of potential therapeutic approaches, some pharmaceutical drugs or antisense oligonucleotides that repress Evi-1 expression would be useful for the treatment of Evi-1-induced neoplastic tumors.