HOXA9 Overexpression Research Articles

Upregulation ofHOXA10Protein Expression Predicts Poor Prognosis for Colorectal Cancer

The homeobox (HOX) genes function as transcriptional factors that can promote tumorigenesis. However, the expression profile of HOXA10 and the role this protein plays in solid tumors are unclear. Here we examined HOXA10 protein expression in samples from colorectal cancer (CRC) patients to address the clinical significance of this protein. Seven independent investigations from the Oncomine database were retrieved. A total of 85 patients who underwent radical excision followed by 5-fluorouracil (5-FU)-based adjuvant chemotherapy were enrolled. Immunohistochemistry was performed on pairs of cancerous and normal tissues to detect the expression of both HOXA10, and the phosphatase and tensin homolog deleted on chromosome ten (PTEN). Lentivirus-mediated RNA interference was used to knock down HOXA10 expression in LoVo and HT-29 cell lines, then cells' proliferation, apoptosis, and tumor growth in vivo were detected. Oncomine data showed that HOXA10 expression was significantly upregulated in CRC tissues compared with relevant normal controls. In our study, 58 cases (68.2%) showed positive HOXA10 protein expression in tumor tissue and negative expression in normal tissues. HOXA10 protein upregulation was consistent with PTEN downregulation. Although not related to clinicopathological parameters, a significant correlation was found between HOXA10 upregulation and a decreased 5-year disease-free survival (DFS). A Cox proportional hazards model further suggested that HOXA10 overexpression was an independent factor to predict DFS of CRC patients. Furthermore, HOXA10 knockdown significantly increased sensitivity to 5-FU chemotherapy in vitro and in vivo. Significant HOXA10 overexpression in CRC may be a potential biomarker indicating poor prognosis and 5-FU resistance.

HOXA9 Cooperates with Activated JAK/STAT Signaling to Drive Leukemia Development.

Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells.Significance: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. Cancer Discov; 8(5); 616-31. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Methylation of the HOXA10 Promoter Directs miR-196b-5p-Dependent Cell Proliferation and Invasion of Gastric Cancer Cells.

The cross-talk between epigenetics and miRNA expression plays an important role in human tumorigenesis. Herein, the regulation and role of miR-196b-5p in gastric cancer was investigated. qRT-PCR demonstrated that miR-196b-5p is significantly overexpressed in human gastric cancer tissues (P < 0.01). In addition, it was determined that HOXA10, a homeobox family member and host gene for miR-196b-5p, is overexpressed and positively correlated with miR-196b-5p expression levels (P < 0.001). Quantitative pyrosequencing methylation analysis demonstrated significantly lower levels of DNA methylation at the HOXA10 promoter in gastric cancer, as compared with nonneoplastic gastric mucosa specimens. 5-Aza-2'-deoxycytidine treatment confirmed that demethylation of HOXA10 promoter induces the expression of HOXA10 and miR-196b-5p in gastric cancer cell model systems. Using the Tff1 knockout mouse model of gastric neoplasia, hypomethylation and overexpression of HOXA10 and miR-196b-5p in gastric tumors was observed, as compared with normal gastric mucosa from Tff1 wild-type mice. Mechanistically, reconstitution of TFF1 in human gastric cancer cells led to an increased HOXA10 promoter methylation with reduced expression of HOXA10 and miR-196b-5p. Functionally, miR-196b-5p reconstitution promoted human gastric cancer cell proliferation and invasion in vitro In summary, the current data demonstrate overexpression of miR-196b-5p in gastric cancer and suggest that TFF1 plays an important role in suppressing the expression of miR-196b-5p by mediating DNA methylation of the HOXA10 promoter. Loss of TFF1 expression may promote proliferation and invasion of gastric cancer cells through induction of promoter hypomethylation and expression of the HOXA10/miR-196b-5p axis.Implications: This study indicates that loss of TFF1 promotes the aberrant overexpression of HOXA10 and miR-196b-5p by demethylation of the HOXA10 promoter, which provides a new perspective of TFF1/HOXA10/miR-196b-5p functions in human gastric cancer. Mol Cancer Res; 16(4); 696-706. ©2018 AACR.

Hoxa9 and Hoxa10 induce CML myeloid blast crisis development through activation of Myb expression.

Mechanisms underlying the progression of Chronic Myeloid Leukemia (CML) from chronic phase to myeloid blast crisis are poorly understood. Our previous studies have suggested that overexpression of SETBP1 can drive this progression by conferring unlimited self-renewal capability to granulocyte macrophage progenitors (GMPs). Here we show that overexpression of Hoxa9 or Hoxa10, both transcriptional targets of Setbp1, is also sufficient to induce self-renewal of primary myeloid progenitors, causing their immortalization in culture. More importantly, both are able to cooperate with BCR/ABL to consistently induce transformation of mouse GMPs and development of aggressive leukemias resembling CML myeloid blast crisis, suggesting that either gene can drive CML progression by promoting the self-renewal of GMPs. We further identify Myb as a common critical target for Hoxa9 and Hoxa10 in inducing self-renewal of myeloid progenitors as Myb knockdown significantly reduced colony-forming potential of myeloid progenitors immortalized by the expression of either gene. Interestingly, Myb is also capable of immortalizing primary myeloid progenitors in culture and cooperating with BCR/ABL to induce leukemic transformation of mouse GMPs. Significantly increased levels of MYB transcript also were detected in all human CML blast crisis samples examined over chronic phase samples, further suggesting the possibility that MYB overexpression may play a prevalent role in driving human CML myeloid blast crisis development. In summary, our results identify overexpression of HOXA9, HOXA10, and MYB as critical drivers of CML progression, and suggest MYB as a key therapeutic target for inhibiting the self-renewal of leukemia-initiating cells in CML myeloid blast crisis patients.

RETRACTED: Physcion blocks cell cycle and induces apoptosis in human B cell precursor acute lymphoblastic leukemia cells by downregulating HOXA5

Acute lymphoblastic leukemia (ALL) presents the most common type of malignancy in children and ranks the third most common cancer in adults. This study is aimed to investigate the anti-leukemia activity of physcion in ALL. Our results have showed that physcion could significantly suppress cell growth, induce apoptosis and blocked cell cycle progression in vitro. Mechanistically, we found that physcion downregulated the expression of HOXA5, which is responsible for the anti-leukemia activity of physcion. To verify this finding, siRNA targeting HOXA5 and overexpressing plasmid were used to repress HOXA5 expression and introduce ectopic overexpression of HOXA5 in ALL cell lines, respectively. Our results showed that overexpression of HOXA5 significantly abrogated the inducing effect of physcion on apoptosis and cell cycle blockasde. In contrast, knockdown of HOXA5 by siRNA enhanced the anti-tumor effect of physcion on ALL cell lines. Our results provided experimental base for the use of physcion in the treatment of ALL.

Overexpression of HOXA4 and HOXA9 genes promotes self-renewal and contributes to colon cancer stem cell overpopulation.

Because HOX genes encode master regulatory transcription factors that regulate stem cells (SCs) during development and aberrant expression of HOX genes occurs in various cancers, our goal was to determine if dysregulation of HOX genes is involved in the SC origin of colorectal cancer (CRC). We previously reported that HOXA4 and HOXD10 are expressed in the colonic SC niche and are overexpressed in CRC. HOX gene expression was studied in SCs from human colon tissue and CRC cells (CSCs) using qPCR and immunostaining. siRNA-mediated knockdown of HOX expression was used to evaluate the role of HOX genes in modulating cancer SC (CSC) phenotype at the level of proliferation, SC marker expression, and sphere formation. All-trans-retinoic-acid (ATRA), a differentiation-inducing agent was evaluated for its effects on HOX expression and CSC growth. We found that HOXA4 and HOXA9 are up-regulated in CRC SCs. siRNA knockdown of HOXA4 and HOXA9 reduced: (i) proliferation and sphere-formation and (ii) gene expression of known SC markers (ALDH1, CD166, LGR5). These results indicate that proliferation and self-renewal ability of CRC SCs are reduced in HOXA4 and HOXA9 knockdown cells. ATRA decreased HOXA4, HOXA9, and HOXD10 expression in parallel with reduction in ALDH1 expression, self-renewal, and proliferation. Overall, our findings indicate that overexpression of HOXA4 and HOXA9 contributes to self-renewal and overpopulation of SCs in CRC. Strategies designed to modulate HOX expression may provide ways to target malignant SCs and to develop more effective therapies for CRC.

Abstract 1373: The role of the ASH1L SET domain in leukemogenesis

Abstract ASH1L (absent, small, or homeotic-like 1) is a histone lysine methyltransferase (KMTase) specific for histone 3, lysine 36 that has an emerging role in multiple types of cancer. During development, ASH1L activates HOX genes and regulates self-renewal potential of long-term hematopoietic stem cells. Interestingly, HOX genes are oncogenic drivers in a variety of cancer types. For example, overexpression of HOXA9 is highly associated with a poor prognosis in acute myeloid leukemia (AML). In addition, ASH1L is overexpressed in a variety of cancers, including breast and thyroid cancer, but its role in cancer remains poorly characterized. Recent data suggest that ASH1L promotes leukemogenesis in mixed-lineage leukemia by recruiting the MLL epigenetic regulator to target genes. However, ASH1L is a large protein with multiple chromatin-interacting domains, and which regions of the ASH1L protein are important in leukemia has not been reported. Specifically, the role of ASH1L’s KMTase activity in leukemogenesis remains unknown. To further investigate the function of ASH1L in leukemia, we knocked down expression of ASH1L in leukemia cell lines. We found that ASH1L knockdown slowed cell growth and caused a decrease in expression of HOXA genes and their collaborator MEIS1. To specifically investigate the role of ASH1L KMTase activity in leukemia, we used a mouse model with a targeted in-frame deletion of the ASH1L SET domain (ΔSET). ΔSET cells express a shorter version of ASH1L that does not contain the SET domain. We transduced bone marrow cells (BMCs) isolated from ΔSET and WT mice with several oncogenes relevant to AML to determine the role of the ASH1L SET domain in leukemic transformation. We found that ΔSET BMCs were significantly impaired in leukemic transformation compared to WT BMCs, as manifested by decreased colony-forming ability and reduced expression of HOXA genes. These findings suggest that the ASH1L SET domain plays an important role in leukemogenesis mediated by multiple different oncogenes. Future studies will further characterize ASH1L’s role in leukemia by identifying the complete set of genetic loci that it methylates and regulates, and will determine whether the ASH1L SET domain represents a viable therapeutic target. Citation Format: David Rogawski, Hongzhi Miao, Katarzyna Kempinska, Lili Chen, Jen Chase, Ann Friedman, Xiaomei Yan, Kenichi Nishioka, Gang Huang, Patricia Ernst, Andy Muntean, Ivan Maillard, Tomasz Cierpicki, Jolanta Grembecka. The role of the ASH1L SET domain in leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1373. doi:10.1158/1538-7445.AM2017-1373

Functional screen of MSI2 interactors identifies an essential role for SYNCRIP in myeloid leukemia stem cells.

The identity of the RNA binding proteins (RBPs) that govern cancer stem cell remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomics analysis of the MSI2 interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia and SYNCRIP was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell gene associated program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. We validated SYNCRIP as a novel RBP that controls the myeloid leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in leukemia.

Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

SummaryThe transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia.

Mesenchymal Stem Cells Promoted Lung Wound Repair through Hox A9 during Endotoxemia-Induced Acute Lung Injury

Objectives. Acute lung injury (ALI) is a common clinical critical disease. Stem cells transplantation is recognized as an effective way to repair injured lung tissues. The present study was designed to evaluate the effects of mesenchymal stem cells (MSCs) on repair of lung and its mechanism. Methods. MSCs carrying GFP were administrated via trachea into wild-type SD rats 4 hours later after LPS administration. The lung histological pathology and the distribution of MSCs were determined by HE staining and fluorescence microscopy, respectively. Next, differentially expressed HOX genes were screened by using real-time PCR array and abnormal expression and function of Hox A9 were analyzed in the lung and the cells. Results. MSCs promoted survival rate of ALI animals. The expression levels of multiple HOX genes had obvious changes after MSCs administration and HOX A9 gene increased by 5.94-fold after MSCs administration into ALI animals. HOX A9 was distributed in endothelial cells and epithelial cells in animal models and overexpression of Hox A9 can promote proliferation and inhibit inflammatory adhesion of MSCs. Conclusion. HoxA9 overexpression induced by MSCs may be closely linked with lung repair after endotoxin shock.

MIR142 Loss-of-Function Mutations Promote Leukemogenesis through Derepression of ASH1L Resulting in Increased HOX Gene Expression

Mutations of MIR142 have been identified in approximately 2% of de novo AML and in 20% of diffuse large B cell lymphoma (DLBCL). In AML, all of the mutations in MIR142 localize to the seed sequence of miRNA-142-3p, which is crucial in determining its target specificity. Previously, we showed that these mutations disrupt both miRNA-142-3p and miRNA-142-5p function, suggesting that loss of MIR142 plays a role in leukemic transformation (Yao et al, ASH abstract 1254, 2015). To test this hypothesis, we first characterized hematopoiesis in Mir142-/-mice. We previously reported that loss of Mir142 results in an expansion of myeloid progenitors with impaired erythropoiesis and lymphopoiesis (Yao, ASH abstract 1254, 2015). We now extend these analyses to investigate how loss of Mir142 promotes leukemic transformation.In the TCGA AML cohort, all 4 cases with somatic mutations in MIR142 also harbored mutations in either IDH1 or IDH2. To assess the functional importance of this association, we transduced wild-type or Mir142-/-hematopoietic stem/progenitor cells (HSPCs) with a retrovirus expressing the canonical IDH2 mutation, R172K. These cells were transplanted into lethally irradiated recipients and a tumor watch established. Loss of Mir142 alone was associated with mild splenomegaly, anemia, and leukopenia, but it was not sufficient to induce AML. Consistent with a prior report (Sasaki et al, Nature 2012), expression of IDH2 R172K alone induced a myeloproliferative disorder (MPD) characterized by increased myeloid cells, anemia, and splenomegaly. Concomitant loss of Mir142 did not affect the latency or penetrance of this MPD. However, the MPD in the double mutant mice was characterized by an increased percentage of CD34+ Gr1+ myeloblasts in the bone marrow and spleen plus a more severe anemia. To assess leukemia-initiating activity, we transplanted one million splenic cells into secondary recipients. Whereas IDH2 R172K alone cells rarely engrafted, Mir142-/- x IDH2 R172K cells efficiently engrafted and produced an MPD-like phenotype. These data suggest that loss of function mutations in MIR142 cooperate with IDH1/2 mutations to induce AML, possibly by increasing leukemic cell self-renewal.We examined several putative miR-142 target genes, eventually focusing on ASH1L. ASH1L is a member of the trithorax family of histone methyltransferases that has been recently implicated in MLL-associated leukemogenesis. The 3' UTR of ASH1L contains 4 putative binding sites for miRNA-142-3p, suggesting that this miRNA is critical in its post-transcriptional regulation. Indeed, in a luciferase assay with the ASH1L 3' UTR, MIR142 overexpression decreased translation by 80 percent. Consequently, Ash1l protein levels were 3 fold higher in Mir142-/- mice bone marrow compared to control mice. Since ASH1L is a key regulator of HOX gene expression, we examined HoxA9 and HoxA10 expression in Mir142-/- hematopoietic progenitor subsets. While HoxA9 and HoxA10 expression were not different in hematopoietic stem cells, they were markedly upregulated in myeloid progenitors. For example, in granulocyte-macrophage progenitors (GMPs), HoxA9 and HoxA10 expression were increased 2.86-fold and 34.4-fold, respectively in Mir142-/- versus control cells. Likewise, in megakaryocyte-erythroid progenitors (MEPs), HoxA9 and HoxA10 expression were increased 5.3-fold and 21.4-fold. Dysregulated HoxA9 and HoxA10 expression have been implicated in enhanced self-renewal capacity, and HoxA9 overexpression has been shown to cooperate with mutant IDH1 to induce AML in mice (Chaturvedi et al, Blood 2013). Collectively, these data suggest a model in which MIR142 mutations contribute to leukemogenesis by derepressing ASH1L expression, which, in turn, increases expression of HoxA9/10 and enhances self-renewal. Inhibitors targeting ASH1L may have therapeutic benefit in AML characterized by increased HOX gene expression. DisclosuresNo relevant conflicts of interest to declare.

Modeling Clonal Hematopoietic Disorders in Zebrafish Using Color Barcoding

Myeloid neoplasms, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), are clonal hematopoietic disorders that arise from abnormal hematopoietic stem and progenitor cells (HSPCs)with acquired somatic mutations. To follow the hematopoietic output of HSPC clones endogenously, we have developed a novel marking strategy in which each HSPC clone is labeled in a unique fluorescent hue. The transgenic zebrafish line Zebrabow uses a Cre/lox system where stochastic recombination of multiple transgenic insertions harboring three fluorescent proteins (dTomato, YFP and CFP) provide stable and heritable color barcodes. Tissue-specificity is ensured by tamoxifen-induced Cre activation under the draculin promoter which is highly expressed in zebrafish HSPCs throughout development. The result of Zebrabow labeling is a collection of colored blood cells, which are grouped by their hue, and largely cluster in various groups, each representing a clonal population of cells that arise from a specific HSPC. To induce clonal hematopoietic states in zebrafish assessed by color dominance in hematopoietic cells, we introduced combinations of known genetic mutations founds in MDS and AML into Zebrabow embryos. Embryos at the 1-cell stage were injected with combinations of mutations and HSPC color labeling was performed at 36 to 48 hours post fertilization, during peak emergence of definitive blood stem cells from the dorsal aorta. The injected mutations were grouped by known co-occurence in patients with MDS and AML. The transgenic overexpression of Jak2V617F, FLT3ITD, IDH2R140Q, HOXA9 or HOXB4 under the zebrafish blood-specific promoter draculin, was combined with somatic mutagenesis of endogenous tet2, asxl1, dnmt3a, ezh2, ptena/b and tp53 via the CRISPR/Cas9 system. Mosaic combinations of these mutations resulted in the expansion of single color clones, contributing to ≥30% of granulocytes, with concomittant myeloid expansion in a third of these zebrafish. The majority of zebrafish exhibiting clonal expansion harbored frame-shift mutations in asxl1 with >25% variant allelic frequency identified by next-generation sequencing. Similarly, combinations including Jak2V617F, tet2, or asxl1 mutations lead to single color expansion in kidney marrow cells in 30% of injected zebrafish, with the occasional presence of a blast-like population of cells in the marrow and spleen. We have successfully modeled early stages of clonal hematopoietic expansion with myeloproliferation in the presence of Jak2V617F and asxl1 deletion using the unique color barcoding system in zebrafish. DisclosuresZon:Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

LSD1 Inhibition Induces Differentiation and Reduces the Frequency of Leukemia-Initiating Cells in Hoxa9/Meis1-Induced Acute Myeloid Leukemia

The histone demethylating enzyme LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). Inhibition of LSD1 has been shown to induce differentiation and facilitate the responsiveness of AML cells to all-trans retinoic acid (ATRA). As it remains unclear how LSD1 inhibition induces differentiation, we have now used a murine leukemia model based on retroviral overexpression of Hoxa9 and Meis1 to study the effect of of both genetic inactivation as well as pharmacological inhibition of LSD1.We transformed bone marrow cells from conditional LSD1 knock-out (KO) mice to confirm the responsiveness of Hoxa9/Meis1-induced leukemias to an inhibition of LSD1. Marked granulocytic differentiation was observed in these cells upon induction of the LSD1 KO. The cells also showed a decrease in expression of c-Kit by 53 %. Furthermore the cells acquired the mature granulocytic marker Ly6G. We also found an increased sensitivity to a treatment with ATRA upon induction of the LSD1 KO. We then transplanted Hoxa9/Meis1-transformed cells from conditional LSD1 KO mice into lethally irradiated mice and induced the LSD1 KO 20 days after transplantation by treatment with tamoxifen. This extended the survival of mice from a median of 48 days for control-treated mice (N=5) to a median of 78 days for tamoxifen-treated mice (N=5) (p=0.0021).We then treated Hoxa9/Meis1-transformed myeloid progenitors with three irreversible (tranylcypromine, trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (ORY86, AW69), (1S,2R)-2-phenyl-N-(piperidin-4-ylmethyl)cyclopropanamine (AW84)) and three reversible LSD1 inhibitors (MS120 (=Namoline), MS142, SP2509) alone or in combination with ATRA. We observed signs of granulocytic differentiation with the irreversible inhibitors and a reduction in c-Kit expression from 92% to 25% after treatment with AW69. Colony formation of Hoxa9/Meis1-transformed myeloid progenitors was reduced by 70% compared to control cells after treatment with AW69 for 96 hrs. Treatment with AW69 and AW84 also enhanced the response of leukemia cells to ATRA by enhancing the anti-proliferative effect and by reducing colony formation by 99% in Hoxa9/Meis1 cells in the combination. The reversible LSD1 inhibitors exhibited only minor effects on differentiation even when used at effective concentrations.We also analyzed the effect of LSD1 inhibition on leukemia-initiating cell frequency. We therefore treated Hoxa9/Meis1-transformed myeloid progenitors with AW69 in vitro for a total of 96 hrs and then assessed the LIC frequency in a limiting-dilution assay. In this assay, AW69 induced a striking reduction in LIC frequency from 1/40 (95 % CI 1/10.7 to 1/175) to 1/3200 (95 % CI 1/835.5 to 1/12101) (p < 0.001).As the differentiating effect of LSD1 inhibition in our model was limited to the irreversible inhibitors we then wanted to investigate if the effect was due to interference with the enzymatic activity of LSD1. We therefore used the combination of Hoxa9 and Meis1 to transform cells from mice carrying a conditional knock-in (KI) of an enzymatically inactive form of LSD1 (LSD1mut) and assessed its consequence both in vitro and in vivo. Similar to the effect seen with the conditional KO we observed granulocytic differentiation upon induction of the conditional LSD1mut KI as well as a downregulation of c-Kit expression. However, we did not find a survival benefit in vivo upon induction of the conditional LSD1mut KI, but observed a more differentiated immunophenotype in the developing leukemias.In summary, LSD1 depletion or inhibition using tranylcypromine or its derivatives induces granulocytic differentiation, reduces LIC frequency and induces a survival benefit in Hoxa9/Meis1-driven AML. Our data further suggest that these effects of LSD1 inhibition are mediated by interfering with both enzymatic and scaffolding functions of LSD1. DisclosuresLübbert:Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding. Berg:Astellas: Other: Travel Funding; Alexion: Other: Travel Funding; Celgene: Other: Travel Funding.

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals.

SUMMARYThe functionality of stem cells declines during aging thereby contributing to aging-associated impairments in tissue regeneration and function1. Alterations in developmental pathways have been associated with declines in stem cell function during aging2–6 but the nature of this process remains poorly defined. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in aging7,8. This study identifies an altered epigenetic stress response in muscle stem cells (also known as satellite cells = SCs) of aged compared to young mice. This includes aberrant global and site-specific induction of active chromatin marks in activated SCs from aged mice resulting in the specific induction of Hoxa9 among all Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor separating gene expression of SCs from aged compared to young mice. This includes most of the currently known inhibitors of SC function in aging muscle such as Wnt-, TGFß-, JAK/STAT- and senescence signaling2–4,6. Inhibition of aberrant chromatin activation or deletion of Hoxa9 suffices to improve SC function and muscle regeneration in aged mice, while overexpression of Hoxa9 mimics aging-associated defects in SCs from young mice, which can be rescued by inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated SCs from aged mice, which limits SC function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

Upregulation of Flt3 is a passive event in Hoxa9/Meis1-induced acute myeloid leukemia in mice.

HOXA9, MEIS1 and FLT3 are genes frequently upregulated in human acute myeloid leukemia. Hoxa9 and Meis1 also cooperate to induce aggressive AML with high Flt3 expression in mice, suggesting an important role for Flt3 in Hoxa9/Meis1-induced leukemogenesis. To define the role of Flt3 in AML with high Hoxa9/Meis1, we treated mice with Hoxa9/Meis1-induced AML with the Flt3 inhibitor AC220, used an Flt3-ligand (FL-/-) knockout model, and investigated whether overexpression of Flt3 could induce leukemia together with overexpression of Hoxa9. Flt3 inhibition by AC220 did not delay AML development in mice transplanted with bone marrow cells overexpressing Hoxa9 and Meis1. In addition, Hoxa9/Meis1 cells induced AML in FL-/- mice as rapid as in wild-type mice. However, FL-/- mice had reduced organ infiltration compared with wild-type mice, suggesting some Flt3-dependent effect on leukemic invasiveness. Interestingly, leukemic Hoxa9/Meis1 cells from sick mice expressed high levels of Flt3 regardless of presence of its ligand, showing that Flt3 is a passive marker on these cells. In line with this, combined engineered overexpression of Flt3 and Hoxa9 did not accelerate the progression to AML. We conclude that the Hoxa9- and Meis1-associated upregulation of Flt3 is not a requirement for leukemic progression induced by Hoxa9 and Meis1.

Distal regulation of c-myb expression during IL-6-induced differentiation in murine myeloid progenitor M1 cells.

The c-Myb transcription factor is a major regulator that controls differentiation and proliferation of hematopoietic progenitor cells, which is frequently deregulated in hematological diseases, such as lymphoma and leukemia. Understanding of the mechanisms regulating the transcription of c-myb gene is challenging as it lacks a typical promoter and multiple factors are involved. Our previous studies identified some distal regulatory elements in the upstream regions of c-myb gene in murine myeloid progenitor M1 cells, but the detailed mechanisms still remain unclear. In the present study, we found that a cell differentiation-related DNase1 hypersensitive site is located at a −28k region upstream of c-myb gene and that transcription factors Hoxa9, Meis1 and PU.1 bind to the −28k region. Circular chromosome conformation capture (4C) assay confirmed the interaction between the −28k region and the c-myb promoter, which is supported by the enrichment of CTCF and Cohesin. Our analysis also points to a critical role for Hoxa9 and PU.1 in distal regulation of c-myb expression in murine myeloid cells and cell differentiation. Overexpression of Hoxa9 disrupted the IL-6-induced differentiation of M1 cells and upregulated c-myb expression through binding of the −28k region. Taken together, our results provide an evidence for critical role of the −28k region in distal regulatory mechanism for c-myb gene expression during differentiation of myeloid progenitor M1 cells.

Hoxa-10 and Cyclin D3 Overexpression in the Decidual Reaction in a Superovulation Protocol in Young Adult C57BL/6J Mice.

Following the performance of a superovulation protocol, multiple nodules were observed bilaterally in the uterine horns of 31 of 276 (11.2%) C57BL/6 J female mice aged 8.5 ± 0.6 (mean and standard error of mean) weeks. These lesions prevented embryo collection, and the uterine decidual reaction was suspected. Samples of pathological uteri (n = 20) and the normal genital tracts of donors treated with a similar superovulation protocol (control group, n = 10) were collected. Immunohistochemistry was performed to evaluate pancytokeratin, desmin, vimentin, progesterone receptor (PR), estrogen receptor α (ERα), Ki-67, cyclin D3 and c-Myc expression, as well as quantitative polymerase chain reaction to assess cyclin D3, Hoxa-10 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA expression. The uterine decidual reaction presented a high degree of structural organization and specifically affected the antimesometrial region of the endometrium. The abnormal decidual cells were large polygonal cells that were frequently polyploid or binucleated and strongly positive for desmin. Immunohistochemistry showed higher Ki-67 proliferation index and higher expression of PR and cyclin D3 in decidual cells in the antimesometrial aspect of the endometrium, compared to nondecidualized endometrial stromal cells in the mesometrial aspect of affected uteri, and compared to endometrial stromal cells in healthy uteri. High expression of cyclin D3 and Hoxa-10 mRNA was also observed in uteri affected by the decidual reaction. These results suggest that PR overexpression in endometrial stromal cells, likely due to high progesterone levels, triggers cyclin D3 and Hoxa-10 overexpression, which may be involved in the pathological mechanisms of the mouse uterine decidual reaction.

Abstract 883: NUP98-fusion proteins interact with the NSL/MLL1 complexes to drive leukemogenesis

Abstract The Nucleoporin 98 gene (NUP98) is fused to a variety of partner genes in an array of hematopoietic malignancies via chromosomal translocations involving 11p15. NUP98-rearranged leukemias show elevated HOXA and HOXB cluster genes and mouse model systems have recapitulated this high-level expression independent of whether the leukemias are derived from mouse or human bone marrow. However, the molecular mechanisms of NUP98-fusion mediated leukemogenesis and elevated HOX gene expression in this leukemia are unclear. Recent studies in Drosophila show that nucleoplasmic Nup98 functions as a potential transcriptional activator, and physically interacts with the non-specific lethal (NSL) and trithorax (Trx)/mixed lineage leukemia (MLL) complex (Kalverda et al., 2010; Pascual-Garcia et al., 2014). To test whether the intranuclear localized NUP98-fusion proteins interact with NSL/MLL1 complexes, coimmunoprecipitation (co-IP) experiments using 293T cells with overexpression of NUP98 fusions and wild type (WT) NUP98 were performed. NUP98-fusion proteins NUP98-HOXA9, NUP98-HOXD13, NUP98-NSD1 and NUP98-PHF23, but not the full-length WT NUP98, show physical interaction with MLL1 and the NSL histone acetyltransferase (HAT) complexes. Genome-wide chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) illustrated that NUP98-HOXA9 and MLL1 co-localize at Hoxa and Hoxb gene promoters, which correlates with the presence of activating chromatin modifications such as H4K16ac and H3K4me3. In vitro and in vivo functional assays further showed that Mll1 is crucial for the growth of NUP98-HOXA9-transformed cells, and for the initiation and maintenance of NUP98-HOXA9 driven AML. These findings were further supported by transcriptome analyses performed in mouse NUP98-HOXA9 transformed cells lacking Mll1. We found a significant enrichment of co-bound targets of MLL1 and NUP98-HOXA9 and genes downregulated in the absence of Mll1. Gene set enrichment analysis (GSEA) demonstrated strong similarity between Mll1-dependent gene expression signature in our murine NUP98-HOXA9 AML model and the expression profile of human NUP98-fusion AML, indicating that our findings in murine AML models can be extended to human AML. Finally, the overexpression of Hoxa9 and Meis1, direct binding targets of NUP98-HOXA9 and MLL1 that are downregulated upon Mll1 loss, rescues the in vitro transformation defects in NUP98-HOXA9 Mll1-/- cells. In conclusion, our findings support a model where NUP98-fusion proteins recruit the NSL/MLL1 complex as an important step during leukemogenesis. The deregulated HOX gene expression in NUP98 fusion transformed cells remains dependent on MLL1. The discovery of this common leukemogenic pathway for NUP98-fusion proteins opens up new avenues for potential therapeutic opportunities to treat patients with leukemia that harbor various NUP98 rearrangements. Citation Format: Haiming Xu, Daria G. Valerio, Meghan E. Eisold, Amit Sinha, Richard. P. Koche, Wenhuo Hu, Chun-Wei Chen, Scott H. Chu, Gerard L. Brien, Scott A. Armstrong. NUP98-fusion proteins interact with the NSL/MLL1 complexes to drive leukemogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 883.

Deregulation of the HOXA9/MEIS1 axis in acute leukemia.

HOXA9 is a homeodomain transcription factor that plays an essential role in normal hematopoiesis and acute leukemia, in which its overexpression is strongly correlated with poor prognosis. The present review highlights recent advances in the understanding of genetic alterations leading to deregulation of HOXA9 and the downstream mechanisms of HOXA9-mediated transformation. A variety of genetic alterations including MLL translocations, NUP98-fusions, NPM1 mutations, CDX deregulation, and MOZ-fusions lead to high-level HOXA9 expression in acute leukemias. The mechanisms resulting in HOXA9 overexpression are beginning to be defined and represent attractive therapeutic targets. Small molecules targeting MLL-fusion protein complex members, such as DOT1L and menin, have shown promising results in animal models, and a DOT1L inhibitor is currently being tested in clinical trials. Essential HOXA9 cofactors and collaborators are also being identified, including transcription factors PU.1 and C/EBPα, which are required for HOXA9-driven leukemia. HOXA9 targets including IGF1, CDX4, INK4A/INK4B/ARF, mir-21, and mir-196b and many others provide another avenue for potential drug development. HOXA9 deregulation underlies a large subset of aggressive acute leukemias. Understanding the mechanisms regulating the expression and activity of HOXA9, along with its critical downstream targets, shows promise for the development of more selective and effective leukemia therapies.

Hoxa10 null animals exhibit reduced platelet biogenesis.

The transcription factor HOXA10 is an important regulator of myelopoiesis. Engineered over-expression of Hoxa10 in mice results in a myeloproliferative disorder that progresses to acute myeloid leukaemia (AML) over time, and in humans over-expression is associated with poor outcomes in AML. Here, we report that loss of Hoxa10 expression in mice results in reduced platelet count and platelet production, but does not affect clotting efficiency. About 40% fewer platelets were found in Hoxa10 null animals in comparison to wild type littermates. We found a nearly 50% reduction in the percentage of reticulated platelets in Hoxa10 null mice, suggesting deficient platelet production. Furthermore, Hoxa10 null animals recovered less efficiently from induced thrombocytopenia, supporting our hypothesis of defective platelet production. This also correlated with reduced colony formation potential of stem and progenitor cells seeded in megakaryocyte-enhancing conditions invitro. Together, our results indicate that HOXA10 is important for megakaryopoiesis and platelet biogenesis.