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Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase ( IDH1/IDH2 ) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

PARP inhibitors (PARPi) represent a novel class of targeted therapies that have conventionally been used for the treatment of BRCA1/2-mutated solid tumors. PARP1 being an indispensable component of the DNA repair machinery is essential for maintaining genomic integrity. Germline mutations or expression changes in genes compromising homologous recombination (HR)-mediated repair increases dependency on PARP1 and sensitizes these cells to PARP inhibition. Unlike solid tumors, hematologic malignancies do not frequently harbor BRCA1/2 mutations. PARP inhibition as a therapeutic strategy in blood disorders, therefore, did not receive the same importance. However, underlying epigenetic plasticity and leveraging transcriptional dependencies across molecular subtypes of leukemia has invigorated PARP inhibition-guided synthetic lethality in hematologic malignancies. For example, recent studies showing the importance of robust DNA repair machinery in acute myeloid leukemia (AML) increased the evidence of genomic instability associated with leukemia-driven mutations, and compromised repair pathways in certain subgroups of AML has shifted the focus on exploiting PARPi synthetic lethality in leukemia. Single-agent PARPi as well as combination with other targeted therapies has shown promising results in clinical trials involving patients with AML and myelodysplasia. In this study, we evaluated antileukemic potential of PARPi, understood the subtype-dependent differential responses, discussed recent clinical trials, and provided an outlook for future combination therapy strategies. Extensive genetic and epigenetic characterization, utilizing results from completed and ongoing studies will further help to determine specific subset of patients who may respond, and to establish PARPi as a mainstay in leukemia treatment., Competing Interests: Conflict of Interest Disclosure The authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2023 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. Epigenetic deregulation contributes to AML pathogenesis. KDM6 proteins are histone-3-lysine-27-demethylases that play context-dependent roles in AML. We inform that KDM6-demethylase function critically regulates DNA-damage-repair-(DDR) gene expression in AML. Mechanistically, KDM6 expression is regulated by genotoxic stress, with deficiency of KDM6A-(UTX) and KDM6B-(JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations are implicated in chemoresistance, although a significant percentage of relapsed-AML has upregulated KDM6A. Olaparib treatment reduced engraftment of KDM6A-mutant-AML-patient-derived xenografts, highlighting synthetic lethality using Poly-(ADP-ribose)-polymerase-(PARP)-inhibition. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, BCL2A1 associates with venetoclax resistance, and KDM6A loss was accompanied with a downregulated BCL2A1. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support of a novel combination therapy for AML based on subtype-heterogeneity, and establishes KDM6A as a molecular regulator for determining therapeutic efficacy., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Acute myeloid leukemia (AML) is an aggressive, disseminated hematological malignancy associated with clonal selection of aberrant self-renewing hematopoietic stem cells and progenitors and poorly differentiated myeloid blasts. The most prevalent form of leukemia in adults, AML is predominantly an age-related disorder and accounts for more than 10,000 deaths per year in the United States alone. In comparison to solid tumors, AML has an overall low mutational burden, albeit more than 70% of AML patients harbor somatic mutations in genes encoding epigenetic modifiers and chromatin regulators. In the past decade, discoveries highlighting the role of DNA and histone modifications in determining cellular plasticity and lineage commitment have attested to the importance of epigenetic contributions to tumor cell de-differentiation and heterogeneity, tumor initiation, maintenance, and relapse. Orchestration in histone methylation levels regulates pluripotency and multicellular development. The increasing number of reversible methylation regulators being identified, including histone methylation writer, reader, and eraser enzymes, and their implications in AML pathogenesis have widened the scope of epigenetic reprogramming, with multiple drugs currently in various stages of preclinical and clinical trials. AML methylome also determines response to conventional chemotherapy, as well as AML cell interaction within a tumor-immune microenvironment ecosystem. Here we summarize the latest developments focusing on molecular derangements in histone methyltransferases (HMTs) and histone demethylases (HDMs) in AML pathogenesis. AML-associated HMTs and HDMs, through intricate crosstalk mechanisms, maintain an altered histone methylation code conducive to disease progression. We further discuss their importance in governing response to therapy, which can be used as a biomarker for treatment efficacy. Finally we deliberate on the therapeutic potential of targeting aberrant histone methylome in AML, examine available small molecule inhibitors in combination with immunomodulating therapeutic approaches and caveats, and discuss how future studies can enable posited epigenome-based targeted therapy to become a mainstay for AML treatment., Competing Interests: Conflict of interest disclosure The authors declare no conflicts of interest., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Cancer genome sequencing studies have focused on identifying oncogenic mutations. However, mutational profiling alone may not always help dissect underlying epigenetic dependencies in tumorigenesis. Nucleosome remodeling and deacetylase (NuRD) is an ATP-dependent chromatin remodeling complex that regulates transcriptional architecture and is involved in cell fate commitment. We demonstrate that loss of MBD3, an important NuRD scaffold, in human primary acute myeloid leukemia (AML) cells associates with leukemic NuRD. Interestingly, CHD4, an intact ATPase subunit of leukemic NuRD, coimmunoprecipitates and participates with H3K27Me3/2-demethylase KDM6A to induce expression of atypical guanine nucleotide exchange factors, dedicator of cytokinesis (DOCK) 5 and 8 (DOCK5/8), promoting Rac GTPase signaling. Mechanistically, MBD3 deficiency caused loss of histone deacytelase 1 occupancy with a corresponding increase in KDM6A, CBP, and H3K27Ac on DOCK5/8 loci, leading to derepression of gene expression. Importantly, the Cancer Genome Atlas AML cohort reveals that DOCK5/ 8 levels are correlated with MBD3 and KDM6A, and DOCK5/ 8 expression is significantly increased in patients who are MBD3 low and KDM6A high with a poor survival. In addition, pharmacological inhibition of DOCK signaling selectively attenuates AML cell survival. Because MBD3 and KDM6A have been implicated in metastasis, our results may suggest a general phenomenon in tumorigenesis. Collectively, these findings provide evidence for MBD3-deficient NuRD in leukemia pathobiology and inform a novel epistasis between NuRD and KDM6A toward maintenance of oncogenic gene expression in AML.-Biswas, M., Chatterjee, S. S., Boila, L. D., Chakraborty, S., Banerjee, D., Sengupta, A. MBD3/NuRD loss participates with KDM6A program to promote DOCK5/8 expression and Rac GTPase activation in human acute myeloid leukemia.

DNA, as a target for therapeutic intervention, remains largely unexplored. DLX-4, a homeodomain containing transcription factor, and its spliced isoforms play crucial roles in many aspects of cellular biochemistry and important roles in many diseases. A smaller peptide mimicking the homeodomain of the transcription factor DLX-4 was designed and synthesized by suitable conjoining of its modified DNA-binding elements. The peptide binds to DLX-4 target sites on the regulatory region of the globin gene cluster with native-like affinity and specificity in vitro. When conjugated to cell penetrating and nuclear localization sequences, it upregulated some of the genes repressed by DLX-4 or its isoforms, such as β- and γ-globin genes in erythropoietin-induced differentiating CD34 + human hematopoietic stem/progenitor cells with high specificity by competing with the respective binding sites. Engineered peptides mimicking DNA-binding domains of transcription factors offer the potential for creating synthetic molecules for directly targeting DNA sites with high specificity.

SWI/SNF is an evolutionarily conserved multi-subunit chromatin remodeling complex that regulates epigenetic architecture and cellular identity. Although SWI/SNF genes are altered in approximately 25% of human malignancies, evidences showing their involvement in tumor cell-autonomous chromatin regulation and transcriptional plasticity are limiting. This study demonstrates that human primary acute myeloid leukemia (AML) cells exhibit near complete loss of SMARCB1 (BAF47 or SNF5/INI1) and SMARCD2 (BAF60B) associated with nucleation of SWI/SNF Δ SMARCC1 (BAF155), an intact core component of SWI/SNF Δ , colocalized with H3K27Ac to target oncogenic loci in primary AML cells. Interestingly, gene ontology (GO) term and pathway analysis suggested that SMARCC1 occupancy was enriched on genes regulating Rac GTPase activation, cell trafficking, and AML-associated transcriptional dysregulation. Transcriptome profiling revealed that expression of these genes is upregulated in primary AML blasts, and loss-of-function studies confirmed transcriptional regulation of Rac GTPase guanine nucleotide exchange factors ( GEF ) by SMARCB1. Mechanistically, loss of SMARCB1 increased recruitment of SWI/SNF Δ and associated histone acetyltransferases (HAT) to target loci, thereby promoting H3K27Ac and gene expression. Together, SMARCB1 deficiency induced GEFs for Rac GTPase activation and augmented AML cell migration and survival. Collectively, these findings highlight tumor suppressor role of SMARCB1 and illustrate SWI/SNF Δ function in maintaining an oncogenic gene expression program in AML. Implications: Loss of SMARCB1 in AML associates with SWI/SNF Δ nucleation, which in turn promotes Rac GTPase GEF expression, Rac activation, migration, and survival of AML cells, highlighting SWI/SNF Δ downstream signaling as important molecular regulator in AML. Mol Cancer Res; 16(5); 791-804. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Acute myeloid leukemia (AML) remains an aggressive hematopoietic malignancy that is caused by proliferation of immature myeloid cells and is frequently characterized by perturbations in chromatin-modifying enzymes. Emerging evidence indicates that histone demethylases play a role in tumorigenesis. However, due to the complexity of this enormous family of histone-modifying enzymes, substrate redundancy, and context-specific roles, the contribution of each member remains ambiguous and targeting them remains challenging. Here, we analyzed expression of histone-3-lysine (H3K) demethylases and their cognate substrates in a cohort of de novo AML patients, which demonstrated that the expression of H3K27Me3/2-demethylases and selected members of H3K9Me3/2/1-demethylases are significantly increased in AML. KDM6 upregulation is associated with a global decrease in H3K27Me3 level. Importantly, our data show that pharmacological inhibition of H3K27Me3/2-demethylases or H3K9Me3/2-demethylases, either alone or in combination, could be considered an interesting molecular therapeutic modality in human AML independent of its subtype., (Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)