Meis1 Expression Research Articles

Meis1 Targets Protein Tyrosine Phosphatase Receptor J in Fibroblast to Retard Chronic Kidney Disease Progression.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) with the proliferation and activation of myofibroblasts being definite effectors and drivers. Here, increased expression of Meis1 (myeloid ecotropic viral integration site 1) is observed, predominantly in the nucleus of the kidney of CKD patients and mice, and negatively correlates with serum creatinine. Fibroblast-specific knock-in of Meis1 inhibits myofibroblast activation and attenuates renal fibrosis and kidney dysfunction in CKD models. Overexpression of Meis1 in NRK-49F cells suppresses the pro-fibrotic response induced by transforming growth factor-β1 but accelerates by its knockdown. Mechanistically, Meis1 targets protein tyrosine phosphatase receptor J (Ptprj) to block renal fibrosis by inhibiting the proliferation and activation of fibroblasts. Finally, a new activator of Ptprj is identified through computer-aided virtual screening, which has the effect of alleviating renal fibrosis. Collectively, these results illustrate that the Meis1/Ptprj axis has therapeutic potential for clinically treating CKD.

Disparity of gene expression in coronary artery disease: insights from MEIS1, HIRA, and Myocardin.

Coronary artery disease (CAD) in young adults can have devastating consequences. The cardiac developmental gene MEIS1 plays important roles in vascular networks and heart development. This gene effects on the regeneration capacity of the heart. Considering role of MEIS1 in cardiac tissue development and the progression of myocardial infarction this study investigated the expression levels of the MEIS1, HIRA, and Myocardin genes in premature CAD patients compared to healthy subjects and evaluated the relationships between these genes and possible inflammatory factors. The study conducted a case-control design involving 35 CAD patients and 35 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were collected, and gene expression analysis was performed using real-time PCR. Compared with control group, the number of PBMCs in the CAD group exhibited greater MEIS1 and HIRA gene expression, with fold changes of 2.45 and 3.6. The expression of MEIS1 exhibited a negative correlation with IL-10 (r= -0.312) expression and positive correlation with Interleukin (IL)-6 (r = 0.415) and tumor necrosis factor (TNF)-α (r = 0.534) gene expression. Moreover, there was an inverse correlation between the gene expression of HIRA and that of IL-10 (r= -0.326), and a positive correlation was revealed between the expression of this gene and that of the IL-6 (r = 0.453) and TNF-α (r = 0.572) genes. This research demonstrated a disparity in expression levels of MEIS1, HIRA, and Myocardin, between CAD and healthy subjects. The results showed that, MEIS1 and HIRA play significant roles in regulating the synthesis of proinflammatory cytokines, namely, TNF-α and IL-6.

MEIS2 suppresses breast cancer development by downregulating IL10.

Breast cancer (BC) is the most commonly diagnosed female cancer. Homeobox protein MEIS2, a key transcription factor, is involved in the regulation of many developmental and cellular processes. However, the role of MEIS2 in the development of breast cancer is still unclear. We aimed to examine the role of myeloid ecotropic insertion site (MEIS2) in breast cancer and the association of MEIS2 with breast cancer clinical stages and pathological grades. We revealed the underlying mechanism by which MEIS2 affected breast cancer cell growth and tumor development. Using human BC cell lines, clinical samples and animal xenograft model, we reveal that MEIS2 functions as a tumor suppressor in breast cancer. The expression of MEIS2 is inversely correlated with BC clinical stages and pathological grades. MEIS2 knockdown (MEIS2-KD) promotes while MEIS2 overexpression suppresses breast cancer cell proliferation and tumor development in vitro and in animal xenograft models, respectively. To determine the biological function of MEIS2, we screen the expression of a group of MEIS2 potential targeting genes in stable-established cell lines. Results show that the knockdown of MEIS2 in breast cancer cells up-regulates the IL10 expression, but MEIS2 overexpression opposed the effect on IL10 expression. Furthermore, the suppressive role of MEIS2 in breast cancer cell proliferation is associated with the IL10 expression and myeloid cells infiltration. Our study demonstrates that the tumor suppressor of MEIS2 in breast cancer progression is partially via down regulating the expression of IL10 and promoting myeloid cells infiltration. Targeting MEIS2 would be a potentially therapeutic avenue for BC.

A Comprehensive Analysis of HOXB13 Expression in Hepatocellular Carcinoma.

Background and objectives: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and is caused by multiple factors. To explore novel targets for HCC treatment, we comprehensively analyzed the expression of HomeoboxB13 (HOXB13) and its role in HCC. Materials and Methods: The clinical significance of HCC was investigated using open gene expression databases, such as TIMER, UALCAN, KM, OSlihc, and LinkedOmics, and immunohistochemistry analysis. We also analyzed cell invasion and migration in HCC cell lines transfected with HOXB13-siRNA and their association with MMP9, E2F1, and MEIS1. Results: HOXB13 expression was higher in fibrolamellar carcinoma than in other histological subtypes. Its expression was associated with lymph node metastasis, histological stage, and tumor grade. It was positively correlated with immune cell infiltration of B cells (R = 0.246), macrophages (R = 0.182), myeloid dendritic cells (R = 0.247), neutrophils (R = 0.117), and CD4+ T cells (R = 0.258) and negatively correlated with immune cell infiltration of CD8+ T cells (R = -0.107). A positive correlation was observed between HOXB13, MMP9 (R = 0.176), E2F1 (R = 0.241), and MEIS1 (R = 0.189) expression (p < 0.001). The expression level of HOXB13 was significantly downregulated in both HepG2 and PLC/PFR/5 cell lines transfected with HOXB13-siRNA compared to that in cells transfected with NC siRNA (p < 0.05). Additionally, HOXB13 significantly affected cell viability and wound healing. Conclusions: HOXB13 overexpression may lead to poor prognosis in patients with HCC. Additional in vivo studies are required to improve our understanding of the biological role and the exact mechanism of action of HOXB13 in HCC.

Abstract 1227: Novel combination therapies to overcome non-genetic/adaptive menin inhibitor resistance in AML with MLL1r or mtNPM1

Abstract In AML with MLL1 rearrangement or MLL1 fusion proteins (FP), the N-terminus of MLL1 becomes fused to over 80 partner proteins resulting in an aberrant leukemic transcriptional program including dysregulated expression of HOXA9, MEIS1, PBX3, FLT3, MEF2C and CDK6. On the other hand, in mutant NPM1 AML cells, wild type MLL1 is the main regulator of HOXA9, MEIS1 and FLT3, promoting self-renewal of myeloid progenitor cells. Menin inhibitors have been developed that disrupt the binding of Menin to MLL1 leading to reduced activity of HOXA9 and MEIS1 and repression of MLL1 or MLL1-FP target genes. Clinical trials have demonstrated that Menin inhibitors (MI) are well-tolerated and clinical remissions have been observed in patients with relapsed/refractory AML harboring MLLr. Unfortunately, most patients either fail to respond or eventually relapse. MI-resistance in AML with MLL1r or mtNPM1 is either due to recently described mutations in the MI-binding domain of Menin or is non-genetic/adaptive, due to dysregulated epigenome/transcriptome/proteome. In the present studies, through repeated shocks with LD90 dosing of MI, followed by drug washout and recovery, we have generated MLL1r (MV4-11-MITR) and mtNPM1 (OCI-AML3-MITR) AML cell lines that are tolerant/resistant (TR) to MI with LD50 values greater than 1 μM. Whole exome sequencing confirmed that these cells were lacking in new driver mutations. We determined that resistance in the MITR cells was not due to presence of hotspot mutations in Menin. ChIP-Seq analysis of H3K27Ac showed that the activities of super enhancers and core transcriptional regulatory circuitry was altered in both MITR cell types. Non-genetic/adaptive resistance to MI also concordantly reduced genome wide ATAC-Seq and RNA-Seq peaks in the OCI-AML3-MITR cells with significant depletion of MEIS1, IGF2BP2, BCL2, and PBX3, with concomitant induction of leukemia stem cell associated CLEC12A and CD244 expressions. A domain-specific CRISPR screen in MV4-11-MITR cells identified several druggable co-dependencies (e.g. EP300 and MOZ) and co-enrichments (SMARCA4, CREBBP and BRD4) with MI, suggesting them as potential mechanisms of non-genetic/adaptive MI resistance. Consistent with these findings, co-treatment with SMARCA2/SMARCA4 (chromatin remodeler ATPases) inhibitor FHD-286 and the BETi OTX015 or the MI SNDX-50469 exerted synergistic lethality in MV4-11-MITR and OCI-AML3-MITR cells as well as in PD MITR AML cells with MLL1r or mtNPM1. Co-treatment with CBP/p300 inhibitor GNE049 or GNE781 was also synergistic with MI in these cells. In vivo treatment with FHD-286 and OTX015 or SNDX-5613 significantly reduced the AML burden in mice bearing OCI-AML3-MITR xenografts. These findings underscore preclinical activity of epigenetically-targeted agent-based combinations and highlight their promise in overcoming MI resistance in AML with MLL1r or mtNPM1. Citation Format: Warren C. Fiskus, Christopher P. Mill, Jessica Piel, Murphy Hentemann, Christine E. Birdwell, Kaberi Das, John A. Davis, Hanxi Hou, Tapan M. Kadia, Naval Daver, Sanam Loghavi, Branko Cuglievan, Courtney D. DiNArdo, Kapil N. Bhalla. Novel combination therapies to overcome non-genetic/adaptive menin inhibitor resistance in AML with MLL1r or mtNPM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1227.

ERN1 knockdown modifies the hypoxic regulation of homeobox gene expression in U87MG glioblastoma cells.

Homeobox genes play an important role in health and disease including oncogenesis. The present investigation aimed to study ERN1-dependent hypoxic regulation of the expression of genes encoding homeobox proteins MEIS (zinc finger E-box binding homeobox 2) and LIM homeobox 1 family, SPAG4 (sperm associated antigen 4) and NKX3-1 (NK3 homeobox 1) in U87MG glioblastoma cells in response to inhibition of ERN1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioblastoma growth. The expression level of homeobox genes was studied in control (transfected by vector) and ERN1 knockdown U87MG glioblastoma cells under hypoxia induced by dimethyloxalylglycine (0.5 mM for 4 h) by quantitative polymerase chain reaction and normalized to ACTB. It was found that hypoxia down-regulated the expression level of LHX2, LHX6, MEIS2, and NKX3-1 genes but up-regulated the expression level of MEIS1, LHX1, MEIS3, and SPAG4 genes in control glioblastoma cells. At the same time, ERN1 knockdown of glioblastoma cells significantly modified the sensitivity of all studied genes to a hypoxic condition. Thus, ERN1 knockdown of glioblastoma cells removed the effect of hypoxia on the expression of MEIS1 and LHX1 genes, but increased the sensitivity of MEIS2, LHX2, and LHX6 genes to hypoxia. However, the expression of MEIS3, NKX3-1, and SPAG4 genes had decreased sensitivity to hypoxia in ERN1 knockdown glioblastoma cells. Moreover, more pronounced changes under the conditions of ERN1 inhibition were detected for the pro-oncogenic gene SPAG4. The results of the present study demonstrate that hypoxia affected the expression of homeobox genes MEIS1, MEIS2, MEIS3, LHX1, LHX2, LHX6, SPAG4, and NKX3-1 in U87MG glioblastoma cells in gene-specific manner and that the sensitivity of all studied genes to hypoxia condition is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling, and possibly contributed to the control of glioblastoma growth. A fundamentally new results of this work is the establishment of the fact regarding the dependence of hypoxic regulation of SPAG4 gene expression on ER stress, in particular ERN1, which is associated with suppression of cell proliferation and tumor growth.

Myeloid Ecotropic Viral Integration Site (MEIS) Proteins: Illuminating Cancer Diagnosis and Biomarker Significance

Context: The MEIS proteins, originally described as the "helper groups" of HOX proteins, are now becoming recognized as significant and incredibly diverse controllers of cellular behavior. The MEIS agents can incorporate a variety of epigenetic modulators into chromatin to aid in embryonic growth or tissue homeostasis by adult stem cells. The MEIS gene's expression, binding, stability, post-translational protein modification, and controlled intracellular availability are all fully regulated, and they all react to external stimuli. Objectives: We explored the diverse roles of MEIS proteins in cellular behavior and epigenetic regulation and investigated the potential of MEIS proteins as diagnostic and therapeutic biomarkers in cancers. Results: The MEIS proteins can be diagnostic and therapeutic biomarkers for cancer. The MEIS-PBX heterotrimeric is involved in neurogenesis processes, and the formation of the MEIS2 heterodimer with PAX6 and DLX2 can proliferate periplomerular dopaminergic neurons in the olfactory bulb. The estrogen receptor in breast cancer upregulates MEIS1 and FOXP3. The estrogen receptor pathway's cancer-related genes can express themselves more strongly when MEIS1-FOXP3 interacts with a positive feedback mechanism. Because MEIS1 expression is decreased in clear renal cell carcinoma (ccRCC) cell lines, MEIS1 may function as a tumor suppressor in the development of ccRCC. Conclusions: MEIS1 is recognized as one of the main contributors to the development of leukemias. The MEIS proteins influence the c-MYC signaling pathway, cell proliferation, and invasion of PC by contributing to its development.

Targeting Epigenetic Resistance Mechanisms to PI3 Kinase Inhibition in Leukemic Stem Cells

Acute myeloid leukemia (AML) is a heterogeneous disease associated with poor survival and frequent relapse rates due to the inability to effectively target leukemic stem cells (LSCs) and acquired resistance to existing treatments. Development of treatments targeting LSCs and overcoming acquired resistance is crucial to improve the survival of AML patients. The PI3 kinase (PI3K)/AKT signaling pathway is activated in 80% of AML patients, across various AML subtypes, making PI3K an attractive therapeutic target for this disease. Several PI3K inhibitors, such as copanlisib, are clinically approved for other indications, with a reasonable toxicity profile. The effects of PI3K inhibitors on LSCs however, remains unknown. Several catalytic isoforms of PI3K are expressed in hematopoietic cells. To determine whether these PI3K isoforms play a role in LSC self-renewal, we performed colony assays on KMT2A-AF9 retrovirally transduced hematopoietic stem and progenitor cells (HSPCs) with either genetic deletion or pharmacologic inhibition of PI3K isoforms. We found that disruption of PI3K signaling reduces the self-renewal of LSCs and promotes myeloid differentiation in both mouse LSCs and human AML cell lines. KMT2A-AF9 induces leukemic transformation via aberrant upregulation of key self-renewal genes in hematopoietic progenitors, such as HoxA9 and Meis1 (PMID: 16862118). We found that pharmacologic inhibition of PI3K isoforms leads to a reduction in HoxA9 and Meis1 expression in LSC colonies. In the KMT2A-AF9 retroviral bone marrow transplantation model of AML, genetic deletion of the PI3K isoform Pik3ca leads to a reduction in LSC numbers in the bone marrow and prolonged survival (Fig 1A), although it is not curative. In addition, we observed significant prolongation of survival upon secondary transplantation of Pik3ca leukemic cells, suggesting a reduction in functional LSCs. Transcriptome analysis on LSCs isolated from pre-clinical bone marrow aspirates of KMT2A-AF9 primary transplant mice revealed downregulation of the target genes of HoxA9, an important driver of LSC self-renewal. We also observed enrichment of the targets of EZH2, a histone methyltransferase that catalyzes the H3K27 trimethylation repressive mark. AKT is known to phosphorylate EZH2 at Ser21, leading to inhibition of EZH2 function (PMID: 16224021). Therefore, we hypothesized that Pik3ca deletion can promote LSC differentiation by de-repressing EZH2 activation via AKT inactivation. To determine whether PI3K inactivation differentiates KMT2A-AF9 leukemic cells via dephosphorylation of EZH2, we performed Western blot analysis on bone marrow cells from Pik3ca KO and WT leukemic mice. As expected, Pik3ca deletion significantly reduced AKT phosphorylation. Unexpectedly, a reduction of total EZH2 protein levels was also observed in Pik3ca KO leukemic cells (Fig 1B), suggesting that EZH2 downregulation may be a non-genetic acquired resistance mechanism to PI3K inactivation. Histone proteomics analysis at two different timepoints after treatment with the PI3K inhibitor copanlisib revealed rapid and dynamic changes in histone modifications, including H3K27me3 and H3K4me3, consistent with epigenetic mechanisms of resistance to PI3K inhibition. EZH1, a homolog of EZH2, has been reported to compensate for EZH2 function (PMID: 26219303, PMID: 31189531). Thus, we hypothesize that PI3K inhibition may sensitize leukemic cells to EZH1/2 dual inhibition by reducing EZH2 levels. We found that dual inhibition with copanlisib and the EZH1/2 inhibitor valemetostat leads to a combinatorial effect to strongly decrease the proliferation of AML cell lines and AML patient samples. Additionally, we found that treatment of KMT2A-AF9 AML mice with these compounds induces myeloid differentiation and leads to a reduction in leukemia initiating cell capacity, as demonstrated by prolonged survival upon secondary transplantation. Overall, our data suggests that PI3K plays an important role in leukemic stem cells, and that EZH2 downregulation is a non-genetic mechanism of resistance to PI3K inhibition. Additionally, our findings indicate that combining PI3K inhibitors with EZH1/2 dual inhibitors could be a promising therapeutic approach to reduce LSCs, prevent resistance, and prolong survival in AML patients.

Targeting Oncogenic Enhancers in ASXL1-Mutant Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR&amp;lt;2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

Investigating the Role of the SETDB1 Target TANGO6 (TMCO7) in Leukemia

Despite advancements in AML therapies over the last 2 decades, 1 out of 3 patients diagnosed with AML succumb to their disease. The mainstay of treatment remains systemic chemotherapy, however better understanding of the genetic drivers in AML has generated new therapeutic opportunities. Targeted therapies have emerged to more precisely tailor leukemic treatment based on genetic and epigenetic drivers. We previously demonstrated that SETDB1, a H3K9 methyltransferase involved in heterochromatin regulation, negatively regulates the expression of proleukemic genes Hoxa9 and Meis1. Further, overexpression of SETDB1 correlates with improved overall AML patient survival and extends leukemic disease latency in mouse models (Ropa et al. Haematologica 2020). Here we describe a novel SETDB1 target, TANGO6, and aim to explore its role in leukemogenesis. We mined our genome wide expression and epigenetic datasets generated upon overexpression of SETDB1 to identify targets that may be important for leukemic progression (Ropa et al. Haematologica 2020). TANGO6 was found to be transcriptionally and epigenetically regulated by SETDB1, with little known about its role in leukemia. We performed a targeted CRISPR-Cas9 mediated knock out of TANGO6 to investigate its importance in several human leukemic cell lines: MV4;11, K562 and KOPN8. We established the above cell lines with Cas9 constitutively expressed and introduced sgRNAs targeting TANGO6 by lentivirus. We then performed a competitive growth assay using flow cytometry to assess cell fitness on days 3, 14, 10, 21. We observed that loss of TANGO6 resulted in decreased fitness across all three leukemic cell lines compared to the negative control. This phenotype was more striking in the K562 and KOPN8 cell lines. In lieu of having a reliable antibody to visualize changes in protein expression, we assessed mutational changes in the DNA sequence to evaluate efficiency of our CRISPR/Cas9 system. Genomic DNA was prepared from sg TANGO6 transduced K562 cells on days 5 and 10. The targeted region of TANGO6 was PCR amplified and TOPO cloned for direct Sanger sequencing. 92% (12 out of 13 samples) showed a mutation involving the target sequence. Of these mutations, ~67% resulted in frameshift mutations leading to early truncation of the protein. The remaining mutations involved deletions ranging from 1 to 28 amino acids, with one sample resulting in two single point mutations conferring a change of leucine to serine and lysine to arginine. These mutations may or may not affect the folding of the protein. This data demonstrates we are successfully targeting TANGO6, which produces the biological phenotype in our competitive growth assay. To further understand the potential importance of TANGO6 in human leukemia, we performed qPCR on 5 leukemic cell lines (2 MLL-rearranged AML cell lines, 2 B-lymphoblastic cell lines, and 1 CML cell line). Our data showed that TANGO6 was expressed at higher levels across all leukemic cell lines compared to normal CD34+ hematopoietic stem/progenitor cells, consistent with a role in leukemogenesis. Our preliminary data suggests that TANGO6 plays an important role in leukemogenesis. Ongoing work focuses on validating this hypothesis using in vivo leukemic mouse models and protein localization studies. Given the lack of available information on human TANGO6, characterizing its location and mechanism is essential to understanding its role in disease. Our work has established the need for further exploration of TANGO6 as a potential therapeutic target for AML.

Discovery, Structure-Activity Relationship and In Vitro Anticancer Activity of Small-Molecule Inhibitors of the Protein-Protein Interactions between AF9/ENL and AF4 or DOT1L.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause 5-10% acute leukemias with poor clinical outcomes. Protein-protein interactions (PPI) between the most frequent MLL fusion partner proteins AF9/ENL and AF4 or histone methyltransferase DOT1L are drug targets for MLL-rearranged (MLL-r) leukemia. Several benzothiophene-carboxamide compounds were identified as novel inhibitors of these PPIs with IC50 values as low as 1.6 μM. Structure-activity relationship studies of 77 benzothiophene and related indole and benzofuran compounds show that a 4-piperidin-1-ylphenyl or 4-pyrrolidin-1-ylphenyl substituent is essential for the activity. The inhibitors suppressed expression of MLL target genes HoxA9, Meis1 and Myc, and selectively inhibited proliferation of MLL-r and other acute myeloid leukemia cells with EC50 values as low as 4.7 μM. These inhibitors are useful chemical probes for biological studies of AF9/ENL, as well as pharmacological leads for further drug development against MLL-r and other leukemias.

MEIS inhibitors reduce the viability of primary leukemia cells and Stem cells by inducing apoptosis

Leukemia stem cells (LSCs) exhibit self-renewal, resistance to standard treatments, and involvement in leukemia relapse. Higher Myeloid Ecotropic Integration Site-1 (MEIS1) expression in leukemic blast samples has been linked to resistance to conventional treatment. We studied the MEIS1 and associated factors in relapsed LSCs and assessed the effect of recently developed MEIS inhibitors (MEISi). Meis1 gene expression was found to be higher in patients with leukemia and relapsed samples. The majority of CD123+ and CD34+ LSCs demonstrated higher MEIS1/2/3 content. Depending on the patient chemotherapy regimen, Meis1 expression increased in relapsed samples. Although there are increased Meis2, Meis3, Hoxa9, Pbx1, or CD34 expressions in the relapsed patients, they are not correlated with Meis1 content in every patient or regimen. MEISi has reduced MEIS1 transcriptional activity and LSC cell survival by apoptosis. Pharmacological targeting with MEISi in LSCs could have a potential effect in limiting leukemia relapse and chemotherapeutic resistance.

Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

P-376 Restoration of mRNA expression level of endometrial MEIS1, as a co-factor of HOXA10, after laparoscopic salpingectomy of women with hydrosalpinx

Abstract Study question Do salpingectomy could restore HOXA10 co-factor MEIS1 expression in endometrial tissues of hydrosalpinx patients? Summary answer Laparoscopic salpingectomy could restore endometrial HOXA10-cofactor- MEIS1 expression level, in addition to HOXA10, in hydrosalpinx women. What is known already Hydrosalpinx is defined as a distally blocked and dilated fallopian tube which has been filled with serous fluid. Evidence suggests that hydrosalpinx is associated with adverse effect on endometrial receptivity by abnormal expression of key molecules such as HOXA10 in the pre-implantation endometrium. Previous data showed salpingectomy results in statistically significant increase in endometrial HOXA10 expression. MEIS1 is a three-amino-acid loop extension (TALE) family homeobox gene which has been proven to be a novel coactivator of HOXA10 during human and mouse endometrial decidualization. MEIS1-HOXA10 complex binds to the promoters of the target genes which are beneficial for human stromal cell decidualization. Study design, size, duration This prospective study was conducted from January 2021 to January 2022 to determine whether salpingectomy would reverse HOXA10-cofactor- MEIS1 expression levels after salpingectomy. All women signed the informed consent and did not receive any hormonal medication during the last three months. In this study 10 infertile women with moderate to severe hydrosalpinx proven by hysterosalpingography or laparoscopy undergoing salpingectomy were recruited. Ten healthy fertile age-matched women considered as control group. Participants/materials, setting, methods Ten infertile women, aged 20-40, with sonovisible hydrosalpinx (diameter &amp;gt;10mm), AMH&amp;gt;1.2 ng/dL, BMI (18–28 kg/m2) and regular menstrual cycle indicated by mid-luteal progesterone levels of &amp;gt; 10 ng/mL were included. Ten healthy fertile age-matched women with a history of successful pregnancy considered as control group. Mid–luteal-phase endometrial sampling (by pipelle) was performed at the time of surgery and second sampling was obtained forth-post treatment cycle. RNA extraction and cDNA synthesis were done. Main results and the role of chance Quantitative mRNA expression of MEIS1 was determined by real-time PCR technique. Gene expression data were analyzed based on 2 –ΔΔCT to estimate the relative fold change value. The non-parametric Wilcoxon signed rank test was used for data analysis. P value less than 0.05 was considered statistically significant.There was a significant reduction in the expression levels of MEIS1 gene in the endometrial samples of hydrosalpinx group before surgical removal. The obtained data showed that salpingectomy resulted in a statistically significant increase (about 6-fold) in endometrial MEIS1 expression (P = 0.02). Moreover, there was not significant difference in MEIS1 mRNA expression of hydrosalpinx group after salpingectomy and the control group (P &amp;gt; 0.05). Limitations, reasons for caution Larger sample size for the confirmation of these data is needed. Epigenetic evaluation of hydrosalpinx endometrial tissues especially in HOXA10 promoter is recommended. Wider implications of the findings This study suggests that the restoration of MEIS1 gene expression observed in hydrosalpinx women after salpingectomy can be considered as a molecular mechanism by which salpingectomy results in improvement of pregnancy rate in IVF cycle. Trial registration number not applicable

Abstract A45: UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus

Abstract Pediatric acute myeloid leukemia (AML) is often refractory to chemotherapy and remains a devastating disease with poor outcomes. Consequently, there is an urgent need to characterize molecular drivers of pediatric AML with the goal of developing more effective molecular therapies. Our group recently identified somatic tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) as a subtype-defining genomic alteration in pediatric AML that is associated with poor outcomes, high relapse rate, and measurable residual disease (MRD) positivity. However, how these alterations drive leukemogenesis is yet to be investigated. Wild-type UBTF regulates rRNA transcription, ribosome biogenesis, and nucleolar formation. Our recent work suggests that UBTF-TD retains these functions. However, we found that expression of UBTF-TD in cord blood CD34+ cells, but not wild-type UBTF, leads to their increased proliferation, self-renewal, and CD117/CD33 surface marker expression, suggesting a potential gain of function as well. Furthermore, we also observed increased expression of HOXB locus genes, PRDM16, MEIS1, and NKX2-3 in the CD34+ UBTF-TD overexpression model relative to our WT control. These expression patterns are similar to what we see in patients, collectively implicating UBTF-TD driving leukemic phenotypes. Based on our localization studies of UBTF-TD to DNA, we hypothesized that UBTF-TD protein may gain the ability to interact with genomic loci that characterize UBTF-TD AML, like the HOXB locus. We therefore wanted to test if UBTF-TD localized to the HOXB cluster and if this lead direct transcriptional upregulation. To test this, we expressed HA-tagged UBTF-TD in cord-blood CD34+ cells and mapped UBTF-TD genomic occupancy using CUT&amp;RUN. We found that UBTF-TD localized to the promoters and gene bodies of key genes, including PRDM16, MEIS1, NKX2-3, and the HOXB cluster. These sites were accompanied by H3K4me3 and H3K27ac active marks without the H3K27me3 repressive mark. To validate these as bone-fide UBTF-TD-occupied regions, we utilized a expressed FKBP12F36V-HA-UBTF-TD fusion in CD34+ cells. This degradation tag (dTAG) system allows for rapid degradation of UBTF-TD protein upon dTAG-13 treatment. We treated our FKBP12F36V-HA-UBTF-TD expressing cord blood CD34+ cells with 1uM of dTAG-13 or DMSO. We found that a 3-day treatment with dTAG-13 led to downregulation of HOXB gene locus expression, but loss of CD117 expression or cell growth required prolonged treatment. Furthermore, our CUT&amp;RUN data showed that dTAG-13 treatment resulted in loss of UBTF-TD protein at the HOXB gene cluster. Collectively, these data show that UBTF-TD interacts with genomic loci at the HOXB gene cluster to directly upregulate their expression. This loss of HOX gene expression leads then leads to loss of a leukemic phenotype. Our data highlights a potential mechanism for UBTF-TD leukemogenesis and sets a foundation where inhibiting the UBTF-TD molecular signature may be a viable therapeutic option. Citation Format: Juan M Barajas, Masayuki Umeda, Ryan Hiltenbrand, Reethu Krishnan, Tamara Westover, Michael P Walsh, Jing Ma, Sherif Abdelhamed, Jeffery M Klco. UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A45.

Abstract A19: Targeting epigenetic regulators to override cellular identity programs and induce therapeutic differentiation in MLL-rearranged acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a poor-prognosis disease that is universally characterized by a prominent differentiation block. Chemotherapeutic regimens and disease outcomes have not changed in decades, and there is great need for novel therapeutic treatments and approaches. Differentiation therapy is a fundamentally different therapy that aims to reactivate latent maturation programs to induce proliferation arrest and apoptosis. This approach is curative in the promyelocytic AML subtype but not others. It is therefore critical to identify the enforcers of differentiation arrest in non-APL AML. Here, we performed a cell-fate selection CRISPR screen for chromatin factors whose inhibition promotes AML cell differentiation. We found that both genetic and chemical inhibition of the histone acetyltransferase KAT6A induces myeloid maturation, most prominently in MLL-rearranged AML. KAT6A loss markedly reduces self-renewal and extinguishes the proliferative capacity of AML cells. Through ChIP-seq, RNA-seq, and clinical dataset analyses, we found that KAT6A is the likely initiator of a newly-described "writer-reader" module that drives oncogene expression in AML. KAT6A catalyzes promoter H3K9 acetylation, which is bound by the H3K9ac reader ENL, leading to Super-elongation complex recruitment and release of paused RNA PolII at oncogenic loci such as MYC. KAT6A is elevated in human AMLs compared to matched normal tissue, and its downregulation correlates with monocytic differentiation in clinical AML datasets. These findings suggest the potential of targeting KAT6A and/or ENL to disrupt this writer-reader module and ablate downstream MYC transcriptional programs in MLL-rearranged AML. This strategy may be viable in the near future, as a recently developed KAT6A/B inhibitor has already entered clinical trials for breast cancer. In follow-up work, we performed differentiation-specific "synergy screens," in which we delivered sgRNA libraries to U937 cells and ER-Hoxb8 cells treated with or without an LSD1 inhibitor (LSD1i). We identified multiple genes whose knockout synergized with LSD1i treatment to induce complete, terminal differentiation with low toxicity. Genetic results were confirmed with small molecule inhibitors of selected hits, and the most synergistic drug combination was pursued further. We found that AML cells dependent on hyperactivity of both Hoxa9 and Meis1 are most sensitive to this drug combination. Preliminary data suggest that LSD1i treatment alone fails to downregulate Meis1 expression, and that the ability of the second drug to fully ablate Meis1 is the likely mechanism underlying the observed synergy. Importantly, the second drug is also in clinical trials and will be discussed in the abstract presentation. Altogether, this work advances our understanding of the AML differentiation block and nominates a specific drug combination for AML differentiation therapy. Citation Format: Andres Blanco. Targeting epigenetic regulators to override cellular identity programs and induce therapeutic differentiation in MLL-rearranged acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A19.

CircDHRS3 inhibits prostate cancer cell proliferation and metastasis through the circDHRS3/miR-421/MEIS2 axis

ABSTRACT Prostate cancer is the most prevalent type of cancer among men worldwide. The importance of circular RNA (circRNA) in prostate cancer and its connection to malignancy has been steadily recognized. circRNA expression was obtained by circRNA sequencing of prostate cancer. circRNA and its function were further analysed. The results were verified by qRT-PCR, RIP assay, FISH, RNA pulldown, WB, CCK-8, colony formation assay and wound-healing assay. BALB/c Nude mice were used for xenograft hosts. Low expression of circDHRS3 was assessed in prostate cancer. Overexpression of circDHRS3 inhibited prostate cancer growth and migration in vitro. Additionally, miR-421 was shown to be the downstream target of circDHRS3, as shown by fluorescence in situ hybridization and dual-luciferase experiments. The rescue assay results for the PC3 and Du145 cell lines demonstrated that circDHRS3 inhibits prostate cancer cell lines’ ability to proliferate and metastasize by modulating MEIS2 expression through the circDHRS3/miR-421/MEIS2 axis. In vivo investigations confirmed that the overexpression of circDHRS3 could inhibit both the lung and bone metastasis of prostate cancer cells. circDHRS3 has the potential to become a biomarker and a targeted therapeutic site for prostate cancer, particularly in the malignant stage. Our study indicates that circDHRS3 inhibits prostate cancer cell proliferation and metastasis through the circDHRS3/miR-421/MEIS2 axis.

MEIS1 Is a Common Transcription Repressor of the miR-23a and NORHA Axis in Granulosa Cells.

MicroRNA-23a (miR-23a) is an endogenous small activating RNA (saRNA) involved in ovarian granulosa cell (GC) apoptosis and sow fertility by activating lncRNA NORHA transcription. Here, we reported that both miR-23a and NORHA were repressed by a common transcription factor MEIS1, which forms a small network regulating sow GC apoptosis. We characterized the pig miR-23a core promoter, and the putative binding sites of 26 common transcription factors were detected in the core promoters of both miR-23a and NORHA. Of them, transcription factor MEIS1 expression was the highest in the ovary, and widely distributed in various ovarian cells, including GCs. Functionally, MEIS1 is involved in follicular atresia by inhibiting GC apoptosis. Luciferase reporter and ChIP assays showed that transcription factor MEIS1 represses the transcription activity of miR-23a and NORHA through direct binding to their core promoters. Furthermore, MEIS1 represses miR-23a and NORHA expression in GCs. Additionally, MEIS1 inhibits the expression of FoxO1, a downstream of the miR-23a/NORHA axis, and GC apoptosis by repressing the miR-23a/NORHA axis. Overall, our findings point to MEIS1 as a common transcription repressor of miR-23a and NORHA, and develop the miR-23a/NORHA axis into a small regulatory network regulating GC apoptosis and female fertility.

Abstract PR004: High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia

Abstract Several leukemia-associated oncoproteins activate transcriptional circuits resembling a stem-like state in acute myeloid leukemia (AML). This activation of “stemness” genes is achieved by enlisting the activity of specialized components of the epigenetic machinery. We sought to comprehensively map epigenetic regulators critical for perpetuating these stemness networks in AML, as they may represent new therapeutic targets. To this end, we used a GFP-reporter knocked into the endogenous locus of the key leukemia oncogene and self-renewal-associated gene MEIS1 and conducted a pooled domain-focused CRISPR screen targeting &amp;gt;600 epigenetic modifiers. Through this screen, we identified and validated multiple members of eight distinct chromatin-modifying complexes that were required for sustaining MEIS1 expression in diverse AML subtypes. These included several novel MEIS1 regulators such as TAF6, LDB1, KAT2A, AFF2, JADE3, casein kinase 2 (CK2), ENY2 and SGF29, in addition to previously characterized regulators such as DOT1L, AF10, ENL and HBO1. A secondary pooled CRISPR screen, coupled with a single-cell transcriptome readout (CROP-seq) revealed that the deletion of several of these MEIS1 activators not only reversed MEIS1 activation but also reduced expression of stem-cell associated genes, including genes of the HOXA cluster, BMI1, SATB1 and Musashi 2, and concomitantly activated expression of differentiation-associated genes. Of particular interest to us was the TUDOR domain chromatin reader SGF29, a key component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. CRISPR-ko of SGF29 significantly reduced the proliferation of cells with distinct HOX-activating mutations including KMT2A rearranged and AF10-rearranged AML cells. ChIP-seq studies showed that SGF29 occupied the promoters and enhancers of key leukemia-oncogenes in a TUDOR-domain-dependent manner and SGF29 deletion selectively attenuated their transcription. Importantly, chromatin proteomics showed that SGF29 deletion led to the eviction of the SAGA complex subunit KAT2A from chromatin and into the cytoplasm. Further, SGF29 knockout impaired blast colony formation and induced differentiation in the KMT2A-MLLLT3, KMT2A-AF10, and CALM-AF10 mouse AML models but did not affect normal hematopoietic colony formation. Lastly, SGF29 deletion delayed disease latency in two distinct human AML cell line models as well as a patient-derived xenograft model in vivo and resulted in striking antiproliferative effects. Our study revealed several new attractive nodes for therapeutic targeting of leukemia stem cells in AML, including the chromatin reader SGF29, and provides a framework to identify vulnerabilities against recalcitrant oncogenic networks. Citation Format: Karina O. Barbosa Guerra, Anagha Deshpande, Ping Xiang, Anna Minkina, Fiorella Schischlik, Adam Brown, Neil A. Robertson, John Doench, Peter D. Adams, Keith Humphries, Eytan Ruppin, Jay Shendure, Prashant Mali, Aniruddha Deshpande. High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR004.

Combination of Menin and IDH Mutant Inhibition in Acute Myeloid Leukemia

The interaction between menin and mixed lineage leukemia 1 (MLL1) protein contributes to abnormal proliferation in the MLL1-(KMT2A) rearranged and nucleophosmin 1 (NPM1)-mutated acute myeloid leukemia (AML). Indeed, small molecule inhibition of the menin/MLL1 interaction was shown to inhibit leukemia in both AML subtypes. Approximately 25% of NPM1 mutated AML also carry an IDH 1 or 2 mutation. Carrying both NPM1- and IDH mutations is associated with worse overall survival and relapse free survival, compared to having only the NPM1 mutation. MI-3454, a small molecule inhibitor of MLL1/menin interaction, has been shown to be effective in AML with KMT2A rearrangements and NPM1 mutations. A close analogue of this compound, KO-539 (Ziftomenib) is now being tested in clinical trials in refractory and relapsed AML patients (NCT 04067336). The aim of this study is to determine the efficacy of the combination of MI-3454 and IDH mutant inhibitors in AML carrying concurrent NPM1 and IDH1/2 mutations. Blood or bone marrow aspirate samples from patients with AML harboring both NPM1 and IDH1 and/or IDH2 mutations were obtained and used to assess the effect of combinatorial treatment of menin and IDH mutant inhibitors. Colony formation assays with these AML patient samples were performed in methylcellulose media with cells treated with a concentration range of MI-3454, ivosidenib (IDH1 inhibitor) or enasidenib (IDH2 inhibitor) as single agents and in combinations. At the end point of the experiments, colonies were counted. Cells were collected for 1) cytohistology to assess cell morphology 2) flow cytometry to evaluate markers of differentiation and apoptosis and 3) gene expression studies to assess the effect of the combinations on the expression level of genes implicated in leukemogenesis and differentiation. In the samples with concurrent NPM1 and IDH1 mutations, the combination of MI-3454 and ivosidenib caused synergistic reduction in colony number compared to the single agents, with combination indices (CI) ranging from 0.44 to 0.94. Furthermore, cells treated with this combination demonstrated more differentiating phenotypes than single agent treated cells. In the samples with concurrent NPM1 and IDH2 mutations, the combination of MI-3454 and enasidenib caused synergistic reduction in colony formation versus either single agent MI-3454 or enasidenib, with CI values 0.12-0.64. The combination of these agents also yielded stronger differentiation than single agents alone based on both morphologic assessment and enhanced expression of CD11B differentiation marker. Mechanistically, the combination of MI-3454 and IDH1/2 mutant inhibitors also more significantly decreased the expression of HOXA9, MEIS1, and HOXB2, which are oncogenes implicated in leukemogenesis, over single agents. The expression of MNDA was also significantly enhanced by these combinations, supporting differentiation. Overall, our study demonstrates that combination of MI-3454 and IDH mutant inhibitors outperforms the effect of single agents with respect to their anti-leukemic effects in clinically relevant pre-clinical models. These results support that combination of menin and IDH mutant inhibitors could be utilized in clinical trials to improve the ability of patients with AML to achieve remission.