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.

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.

c-Met inhibition in a HOXA9/Meis1 model of CN-AML.

BACKGROUND: Hematopoiesis is a paradigm for developmental processes, hierarchically organized, with stem cells at its origin. Hematopoietic stem cells (HSCs) replenish progenitor and precursor cells of multiple lineages, which normally differentiate into short-lived mature circulating cells. Hematopoiesis has provided insight into the molecular basis of tissue homeostasis and malignancy. Malignant hematopoiesis, in particular acute myeloid leukemia (AML), results from impaired development or differentiation of HSCs and progenitors. Co-overexpression of HOX and TALE genes, particularly the HOXA cluster and MEIS1, is associated with AML. Clinically relevant models of AML are required to advance drug development for an aging patient cohort. RESULTS: Molecular analysis identified altered gene, microRNA, and protein expression in HOXA9/Meis1 leukemic bone marrow compared to normal controls. A candidate drug screen identified the c-Met inhibitor SU11274 for further analysis. Altered cell cycle status, apoptosis, differentiation, and impaired colony formation were shown for SU11274 in AML cell lines and primary leukemic bone marrow. CONCLUSIONS: The clonal HOXA9/Meis1 AML model is amenable to drug screening analysis. The data presented indicate that human AML cells respond in a similar manner to the HOXA9/Meis1 cells, indicating pre-clinical relevance of the mouse model.

Hoxb4 overexpression in CD4 memory phenotype T cells increases the central memory population upon homeostatic proliferation.

Memory T cell populations allow a rapid immune response to pathogens that have been previously encountered and thus form the basis of success in vaccinations. However, the molecular pathways underlying the development and maintenance of these cells are only starting to be unveiled. Memory T cells have the capacity to self renew as do hematopoietic stem cells, and overlapping gene expression profiles suggested that these cells might use the same self-renewal pathways. The transcription factor Hoxb4 has been shown to promote self-renewal divisions of hematopoietic stem cells resulting in an expansion of these cells. In this study we investigated whether overexpression of Hoxb4 could provide an advantage to CD4 memory phenotype T cells in engrafting the niche of T cell deficient mice following adoptive transfer. Competitive transplantation experiments demonstrated that CD4 memory phenotype T cells derived from mice transgenic for Hoxb4 contributed overall less to the repopulation of the lymphoid organs than wild type CD4 memory phenotype T cells after two months. These proportions were relatively maintained following serial transplantation in secondary and tertiary mice. Interestingly, a significantly higher percentage of the Hoxb4 CD4 memory phenotype T cell population expressed the CD62L and Ly6C surface markers, characteristic for central memory T cells, after homeostatic proliferation. Thus Hoxb4 favours the maintenance and increase of the CD4 central memory phenotype T cell population. These cells are more stem cell like and might eventually lead to an advantage of Hoxb4 T cells after subjecting the cells to additional rounds of proliferation.

Entinostat prevents leukemia maintenance in a collaborating oncogene-dependent model of cytogenetically normal acute myeloid leukemia.

The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis, and therapeutic intervention based on improved patient stratification. Relevant preclinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co-overexpressed in cytogenetically normal AML (CN-AML), and a conditional transplantation mouse model was developed that demonstrated oncogene dependency and expression levels comparable to CN-AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN-AML patients using statistically significant connectivity map analysis identified Entinostat as a drug with the potential to alter the leukemic condition toward the normal state. Ex vivo treatment of leukemic cells, but not age-matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation, and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML.

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.

Molecular dissection of Meis1 reveals 2 domains required for leukemia induction and a key role for Hoxa gene activation.

The Hoxa9 and Meis1 genes represent important oncogenic collaborators activated in a significant proportion of human leukemias with genetic alterations in the MLL gene. In this study, we show that the transforming property of Meis1 is modulated by 3 conserved domains, namely the Pbx interaction motif (PIM), the homeodomain, and the C-terminal region recently described to possess transactivating properties. Meis1 and Pbx1 interaction domain-swapping mutants are dysfunctional separately, but restore the full oncogenic activity of Meis1 when cotransduced in primary cells engineered to overexpress Hoxa9, thus implying a modular nature for PIM in Meis1-accelerated transformation. Moreover, we show that the transactivating domain of VP16 can restore, and even enhance, the oncogenic potential of the Meis1 mutant lacking the C-terminal 49 amino acids. In contrast to Meis1, the fusion VP16-Meis1 is spontaneously oncogenic, and all leukemias harbor genetic activation of endogenous Hoxa9 and/or Hoxa7, suggesting that Hoxa gene activation represents a key event required for the oncogenic activity of VP16-Meis1.

Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells.

Overexpression of Hoxb4 in bone marrow cells promotes expansion of hematopoietic stem cell (HSC) populations in vivo and in vitro, indicating that this homeoprotein can activate the genetic program that determines self-renewal. However, this function cannot be solely attributed to Hoxb4 because Hoxb4(-/-) mice are viable and have an apparently normal HSC number. Quantitative polymerase chain reaction analysis showed that Hoxb4(-/-) c-Kit+ fetal liver cells expressed moderately higher levels of several Hoxb cluster genes than control cells, raising the possibility that normal HSC activity in Hoxb4(-/-) mice is due to a compensatory up-regulation of other Hoxb genes. In this study, we investigated the competitive repopulation potential of HSCs lacking Hoxb4 alone, or in conjunction with 8 other Hoxb genes. Our results show that Hoxb4(-/-) and Hoxb1-b9 (-/-) fetal liver cells retain full competitive repopulation potential and the ability to regenerate all myeloid and lymphoid lineages. Quantitative Hox gene expression profiling in purified c-Kit+ Hoxb1-b9(-/-) fetal liver cells revealed an interaction between the Hoxa, b, and c clusters with variation in expression levels of Hoxa4,-a11, and -c4.Together, these studies show a complex network of genetic interactions between several Hox genes in primitive hematopoietic cells and demonstrate that HSCs lacking up to 30% of the active Hox genes remain fully competent.

High incidence of proviral integrations in the Hoxa locus in a new model of E2a-PBX1-induced B-cell leukemia.

Relevant mouse models of E2a-PBX1-induced pre-B cell leukemia are still elusive. We now report the generation of a pre-B leukemia model using E2a-PBX1 transgenic mice, which lack mature and precursor T-cells as a result of engineered loss of CD3epsilon expression (CD3epsilon(-/-)). Using insertional mutagenesis and inverse-PCR, we show that B-cell leukemia development in the E2a-PBX1 x CD3epsilon(-/-) compound transgenic animals is significantly accelerated when compared to control littermates, and document several known and novel integrations in these tumors. Of all common integration sites, a small region of 19 kb in the Hoxa gene locus, mostly between Hoxa6 and Hoxa10, represented 18% of all integrations in the E2a-PBX1 B-cell leukemia and was targeted in 86% of these leukemias compared to 17% in control tumors. Q-PCR assessment of expression levels for most Hoxa cluster genes in these tumors revealed an unprecedented impact of the proviral integrations on Hoxa gene expression, with tumors having one to seven different Hoxa genes overexpressed at levels up to 6600-fold above control values. Together our studies set the stage for modeling E2a-PBX1-induced B-cell leukemia and shed new light on the complexity pertaining to Hox gene regulation. In addition, our results show that the Hoxa gene cluster is preferentially targeted in E2a-PBX1-induced tumors, thus suggesting functional collaboration between these oncogenes in pre-B-cell tumors.

Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion.

Cytogenetic, genetic, and functional studies have demonstrated a direct link between deregulated Hoxa9 expression and acute myeloid leukemia (AML). Hoxa9 overexpression in mouse bone marrow cells invariably leads to AML within 3 to 10 months, suggesting the requirement for additional genetic events prior to AML. To gain further insight into how Hoxa9 affects hematopoietic development at the preleukemic stage, we have engineered its overexpression (1) in hematopoietic stem cells using retrovirus-mediated gene transfer and generated bone marrow transplantation chimeras and (2) in lymphoid cells using transgenic mice. Compared with controls, recipients of Hoxa9-transduced cells had an about 15-fold increase in transplantable lymphomyeloid long-term repopulating cells, indicating the capacity for this oncogene to confer a growth advantage to hematopoietic stem cells. In addition, overexpression of Hoxa9 in more mature cells enhanced granulopoiesis and partially blocked B lymphopoiesis at the pre-B-cell stage but had no detectable effect on T lymphoid development. Interestingly, despite specifically directing high expression of Hoxa9 in T and B lymphoid lineages, none of the Hoxa9 transgenic mice developed lymphoid malignancies for the observation period of more than 18 months.