H2.0-like Homeobox Regulates Early Hematopoiesis and Promotes Acute Myeloid Leukemia

SummaryHomeobox domain-containing transcription factors are important regulators of hematopoiesis. Here, we report that increased levels of nonclustered H2.0-like homeobox (HLX) lead to loss of functional hematopoietic stem cells and formation of aberrant progenitors with unlimited serial clonogenicity and blocked differentiation. Inhibition of HLX reduces proliferation and clonogenicity of leukemia cells, overcomes the differentiation block, and leads to prolonged survival. HLX regulates a transcriptional program, including PAK1 and BTG1, that controls cellular differentiation and proliferation. HLX is overexpressed in 87% of patients with acute myeloid leukemia (AML) and independently correlates with inferior overall survival (n = 601, p = 2.3 × 10−6). Our study identifies HLX as a key regulator in immature hematopoietic and leukemia cells and as a prognostic marker and therapeutic target in AML

Megakaryopoiesis impairment through acute innate immune signaling activation by azacitidine

Publisher Copyright: © 2022 Okoye-Okafor et al.Thrombocytopenia, prevalent in the majority of patients with myeloid malignancies, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), is an independent adverse prognostic factor. Azacitidine (AZA), a mainstay therapeutic agent for stem cell transplant–ineligible patients with MDS/AML, often transiently induces or further aggravates disease-associated thrombocytopenia by an unknown mechanism. Here, we uncover the critical role of an acute type-I interferon (IFN-I) signaling activation in suppressing megakaryopoiesis in AZA-mediated thrombocytopenia. We demonstrate that megakaryocytic lineage-primed progenitors present IFN-I receptors and, upon AZA exposure, engage STAT1/SOCS1-dependent downstream signaling prematurely attenuating thrombopoietin receptor (TPO-R) signaling and constraining megakaryocytic progenitor cell growth and differentiation following TPO-R stimulation. Our findings directly implicate RNA demethylation and IFN-I signal activation as a root cause for AZA-mediated thrombocytopenia and suggest mitigation of TPO-R inhibitory innate immune signaling as a suitable therapeutic strategy to support platelet production, particularly during the early phases of AZA therapy.Peer reviewe

Enforced Expression of the Transcriptional Coactivator OBF1 Impairs B Cell Differentiation at the Earliest Stage of Development

OBF1, also known as Bob.1 or OCA-B, is a B lymphocyte-specific transcription factor which coactivates Oct1 and Oct2 on B cell specific promoters. So far, the function of OBF1 has been mainly identified in late stage B cell populations. The central defect of OBF1 deficient mice is a severely reduced immune response to T cell-dependent antigens and a lack of germinal center formation in the spleen. Relatively little is known about a potential function of OBF1 in developing B cells. Here we have generated transgenic mice overexpressing OBF1 in B cells under the control of the immunoglobulin heavy chain promoter and enhancer. Surprisingly, these mice have greatly reduced numbers of follicular B cells in the periphery and have a compromised immune response. Furthermore, B cell differentiation is impaired at an early stage in the bone marrow: a first block is observed during B cell commitment and a second differentiation block is seen at the large preB2 cell stage. The cells that succeed to escape the block and to differentiate into mature B cells have post-translationally downregulated the expression of transgene, indicating that expression of OBF1 beyond the normal level early in B cell development is deleterious. Transcriptome analysis identified genes deregulated in these mice and Id2 and Id3, two known negative regulators of B cell differentiation, were found to be upregulated in the EPLM and preB cells of the transgenic mice. Furthermore, the Id2 and Id3 promoters contain octamer-like sites, to which OBF1 can bind. These results provide evidence that tight regulation of OBF1 expression in early B cells is essential to allow efficient B lymphocyte differentiation

H2.0-Like Homeobox (HLX) Induces Unlimited Clonogenicity, Blocks Differentiation, and Cooperates with FLT3-ITD in the Induction of Acute Myeloid Leukemia

Abstract Abstract 651 Acute myeloid leukemia (AML) is an aggressive disease which is associated with poor clinical outcome. Less than one third of patients achieve durable remission with current treatment regimens, and prognostication and risk stratification are challenging. Identification and functional studies of genes and pathways which regulate leukemic transformation and maintenance is instrumental to understanding the pathogenesis of AML and for development of novel therapeutic strategies. Several members of the Hox (class I homeobox) family of transcription factors have been implicated in the regulation of normal hematopoiesis and leukemogenesis. Less is known about the role of non-clustered (class II) homeobox genes. We found that a new non-clustered homeobox gene, H2.0-like homeobox (HLX), regulates early hematopoiesis and promotes AML in mice and humans. HLX is 2 to 16 fold overexpressed in more than 80% of patients with AML, across all major disease subtypes. Higher levels of HLX are associated with poor overall survival in 3 different, large cohorts of AML patients (N=601, p=2.3×10−6), and HLX holds up as an independent prognostic factor in a multivariate analysis. ShRNA-mediated inhibition of HLX in both murine and human AML cells significantly inhibits leukemic growth and clonogenic capacity, and overcomes the differentiation block of AML cells. When we analyzed pre-leukemic hematopoietic stem and progenitor cells (HSPC) in a PU.1 URED/D AML mouse model, we found a 4-fold elevation of Hlx, suggesting that Hlx is involved in malignant transformation. Overexpression of HLX in wildtype HSPC in a competitive, congenic transplantation model led to near complete depletion of long-term HSC and 16-fold enrichment of myeloid progenitors with a surface phenotype slightly past the GMP stage (CD45+Kit−CD34−CD44highCD49bhighCD11bmid). Overexpression of HLX in HSPC in vitro led to a myeloid differentiation block and to formation of aberrant, CD34−Kit− progenitors with unlimited serial clonogenicity. The mechanism of action of Hlx is so far unknown. The presence of a C-terminal homeobox domain suggests Hlx may directly interact with DNA, however, no studies have shown DNA binding by Hlx or identified direct Hlx target genes. We find that mutation of only two residues of the Hlx homeodomain is sufficient to completely abrogate the differentiation block induced by HLX overexpression in HSPC, indicating Hlx is acting through the DNA-binding ability of its homeodomain. Furthermore, we have now identified direct HLX target genes in both HSPC and AML cells using a combination of expression microarrays and chromatin-immunoprecipitation (chIP). We find that HLX regulates a set of genes which mediate its leukemia-promoting functions, such as BTG1, and we have used chIP to identify a subset of these genes, including PAK1, that are direct targets of HLX. Internal tandem duplications of FLT3 (FLT3-ITD) are seen in approximately 25% of all AML patients, and confer a poor prognosis. Correlative analyses showed that AML patients with mutant FLT3 and low HLX have overall survival similar to WT FLT3 patients, and survive significantly longer than patients with mutant FLT3 and high HLX (p=0.005), demonstrating that FLT3 mutations confer poor prognosis only if HLX is highly expressed, and suggesting that HLX and mutant FLT3 functionally cooperate. We find that co-expression of HLX and FLT3-ITD leads to dramatically enhanced cytokine independent growth and clonogenicity of 32D cells as well as primary murine HSPC in vitro. When we retrovirally co-expressed HLX and FLT3-ITD, or FLT3-ITD alone (plus an empty control), in primary Lin−Kit+ cells and transplanted them into congenic recipient animals, we found that four weeks after transplantation donor chimerism was 4-fold increased on average in the peripheral blood (PB) and bone marrow (BM), and by 12 weeks post-transplantation mice expressing FLT3-ITD and HLX developed AML with large numbers of leukemic blasts in the PB and BM. We have generated knock-in mice conditionally overexpressing Hlx from the Rosa26 locus and ongoing studies include crossing these mice into FLT3-ITD knock-in animals. In summary, our studies have identified HLX as a novel key transcription factor involved in the regulation of early hematopoiesis and AML pathogenesis, and suggest HLX and downstream pathways as promising new therapeutic targets in AML. Disclosures: No relevant conflicts of interest to declare