CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax.

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both in vitro and in vivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

Pathways, Processes, and Candidate Drugs Associated with a Hoxa Cluster-Dependency Model of Leukemia.

High expression of the HOXA cluster correlates with poor clinical outcome in acute myeloid leukemias, particularly those harboring rearrangements of the mixed-lineage-leukemia gene (MLLr). Whilst decreased HOXA expression acts as a readout for candidate experimental therapies, the necessity of the HOXA cluster for leukemia maintenance has not been fully explored. Primary leukemias were generated in hematopoietic stem/progenitor cells from Cre responsive transgenic mice for conditional deletion of the Hoxa locus. Hoxa deletion resulted in reduced proliferation and colony formation in which surviving leukemic cells retained at least one copy of the Hoxa cluster, indicating dependency. Comparative transcriptome analysis of Hoxa wild type and deleted leukemic cells identified a unique gene signature associated with key pathways including transcriptional mis-regulation in cancer, the Fanconi anemia pathway and cell cycle progression. Further bioinformatics analysis of the gene signature identified a number of candidate FDA-approved drugs for potential repurposing in high HOXA expressing cancers including MLLr leukemias. Together these findings support dependency for an MLLr leukemia on Hoxa expression and identified candidate drugs for further therapeutic evaluation.

Targeted deletion of the Hoxa cluster affects B lymphopoiesis through depletion of early lymphoid progenitors.

It is well established that Hoxa genes play a critical role in the proliferative capacity of adult hematopoietic stem and progenitor cells, but the importance of Hoxa genes in later stages of hematopoietic differentiation is less clear. Previously, we observed that B-cell numbers were reduced in adult mice in which Hoxa deletion was induced. In the current study, we investigated the requirement of Hoxa genes at different stages of B-cell development. Using an MxCre-inducible conditional knock-out mouse model, we showed that immature B-cell fractions and early lymphoid progenitors were markedly reduced in the absence of Hoxa, whereas mature B-cell populations were found at levels comparable to controls. Deletion of Hoxa genes in B-cell lineage-committed cells, however, did not affect B-cell development despite sustained Hoxa gene expression in immature CD19+ B-cell subsets. Together, these results suggest that the effect of Hoxa on B-cell development originates in early lymphoid progenitor cells.

Hoxa cluster genes determine the proliferative activity of adult mouse hematopoietic stem and progenitor cells.

Determination of defined roles for endogenous homeobox (Hox) genes in adult hematopoietic stem and progenitor cell (HSPC) activity has been hampered by a combination of embryonic defects and functional redundancy. Here we show that conditional homozygous deletion of the Hoxa cluster (Hoxa(-/-)) results in a marked reduction of adult HSPC activity, both in vitro and in vivo. Specifically, proliferation of Hoxa(-/-) HSPCs is reduced compared with wild-type (WT) cells in vitro and they are less competitive in vivo. Notably, the loss of Hoxa genes had little impact on HSPC differentiation. Comparative RNA sequencing analyses of Hoxa(-/-) and WT hematopoietic stem cells (CD150(+)/CD48(-)/Lineage(-)/c-kit(+)/Sca-1(+)) identified a large number of differentially expressed genes, three of which (Nr4a3, Col1a1, and Hnf4a) showed >10-fold reduced levels. Engineered overexpression of Hoxa9 in Hoxa(-/-) HSPCs resulted in partial phenotypic rescue in vivo with associated recovery in expression of a large proportion of deregulated genes. Together, these results provide definitive evidence linking Hoxa gene expression to proliferation of adult HSPCs.

HOXA4 provides stronger engraftment potential to short-term repopulating cells than HOXB4.

Genes of the HOX4 paralog group have been shown to expand hematopoietic stem cells (HSCs). Endogenous expression of HOXA4 is 10-fold higher than HOXB4 in embryonic primitive hematopoietic cells undergoing self-renewal suggesting a more potent capacity of HOXA4 to expand HSC. In this study, we provide evidence by direct competitive bone marrow cultures that HOXA4 and HOXB4 induce self-renewal of primitive hematopoietic cells with identical kinetics. Transplantation assays show that short-term repopulation by HOXA4-overexpressing multilineage progenitors was significantly greater than HOXB4-overexpressing progenitors in vivo, indicating differences in the sensitivity of the cells to external signals. Small array gene expression analysis showed an increase in multiple Notch and Wnt signaling -associated genes, including receptors and ligands, as well as pluripotency genes, for both HOXA4- and HOXB4-overexpressing cells, which was more pronounced for HOXA4, suggesting that both HOX proteins may assert their affects through intrinsic and extrinsic pathways to induce self-renewal of primitive hematopoietic cells. Thus, HOXA4 increases short-term repopulation to higher levels than HOXB4, which may involve Notch signaling.

Hoxa9 collaborates with E2A-PBX1 in mouse B cell leukemia in association with Flt3 activation and decrease of B cell gene expression.

BACKGROUND: The fusion protein E2A-PBX1 induces pediatric B cell leukemia in human. Previously, we reported oncogenic interactions between homeobox (Hox) genes and E2A-PBX1 in murine T cell leukemia. A proviral insertional mutagenesis screen with our E2A-PBX1 B cell leukemia mouse model identified Hoxa genes as potential collaborators to E2A-PBX1. Here we studied whether Hoxa9 could enhance E2A-PBX1 leukemogenesis. RESULTS: We show that Hoxa9 confers a proliferative advantage to E2A-PBX1 B cells. Transplantation experiments with E2A-PBX1 transgenic B cells overexpressing Hoxa9 isolated from bone marrow chimeras showed that Hoxa9 accelerates the generation of E2A-PBX1 B cell leukemia, but Hoxa9 is unable to transform B cells alone. Quantitative-reverse transcriptase polymerase chain reaction analysis demonstrated a strong repression of B cell specific genes in these E2A-PBX1/Hoxa9 leukemias in addition to Flt3 activation, indicating inhibition of B cell differentiation in combination with enhanced proliferation. Overexpression of Hoxa9 in established E2A-PBX1 mouse leukemic B cells resulted in a growth advantage in vitro, which was also characterized by an enhanced expression of Flt3. CONCLUSIONS: we show for the first time that Hoxa9 collaborates with E2A-PBX1 in the oncogenic transformation of B cells in a mouse model that involves Flt3 signaling, which is potentially relevant to human disease.

HOXA4 induces expansion of hematopoietic stem cells in vitro and confers enhancement of pro-B-cells in vivo.

Members of the homeobox (Hox) gene family are known to mediate expansion of hematopoietic stem cells (HSCs) and progenitors. The absence of oncogenic properties promoted HOXB4 as prime candidate in the quest to expand HSCs for clinical purposes. Despite its potential to expand HSCs, studies with mutant mice showed that Hoxb4 is not essential for HSC generation and function under physiological conditions. Expression studies and the existence of functional redundancy in particular between paralog Hox genes suggest that HOXA4 might have potent properties to expand HSCs. Here we measured the ability of HOXA4 to promote ex vivo expansion of HSCs and progenitors using retrovirus-mediated overexpression. Our results provide evidence that HOXA4-transduced HSCs and primitive progenitors expand in culture conditions and demonstrate that the potential of expanded HOXA4 HSCs to give rise to mature myeloid and lymphoid progeny in normal proportions remained intact. Interestingly, constitutive overexpression of HOXA4 resulted in an unbalanced expansion of lymphoid/myeloid progenitors in bone marrow chimeras favorable to B-cell progenitors responsive to interleukin-7. This expansion was specific for these progenitors and not for the more primitive Whitlock-Witte-initiating cells. These data indicate that early stages of B-cell development associated with proliferation are in particular sensitive to HOXA4. Thus, this study supports the potential use of HOXA4 to expand both HSCs and B-cell progenitor populations for therapeutic strategies.

HoxA cluster is haploinsufficient for activity of hematopoietic stem and progenitor cells.

OBJECTIVE: Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knockouts. Because HoxB genes are dispensable for hematopoiesis, and most HoxA genes are expressed an order of magnitude higher than other cluster genes in hematopoietic stem cell (HSC)-enriched populations, we hypothesize that maintenance of HoxA cluster expression is important for adult hematopoiesis and that global decrease of HoxA gene expression levels affects steady-state hematopoiesis. MATERIALS AND METHODS: Expression levels of HoxA cluster genes have been determined in primitive hematopoietic populations derived from adult mice using quantitative reverse transcriptase polymerase chain reaction. Furthermore, the functional effect of single allelic deletion of the entire HoxA cluster on hematopoietic cells was analyzed by competitive repopulation assays using HoxA(+/-) mice. RESULTS: We show that the HoxA cluster is predominantly expressed in long-term HSCs and that expression declines with progression to short-term HSCs and early progenitors in a quantifiable manner. Monoallelic deletion of the HoxA cluster caused a general increase in primitive hematopoietic cell populations, but a decrease in side populations. In addition exhaustion of B-cell progenitors with age was observed, resulting in less mature B cells. Moreover, bone marrow of HoxA(+/-) mice had a significant larger population of Mac1/Gr1 neutrophils, which might be caused by accelerated maturation of myeloid progenitors. Transplantation assays demonstrated that HoxA(+/-) HSCs were less competitive in long-term repopulation of myeloablated recipients, which appeared intrinsic to HSCs. CONCLUSION: These results show for the first time that maintenance of adult HSCs and progenitors is particularly sensitive to HoxA gene levels, suggesting a specific role for the HoxA cluster in primary regulation of definitive hematopoiesis.