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Abstract: Menin inhibitors that disrupt the menin-MLL interaction hold promise for treating specific acute myeloid leukemia (AML) subtypes, including those with KMT2A rearrangements (KMT2A-r), yet resistance remains a challenge. Here, through systematic chromatin-focused CRISPR screens, along with genetic, epigenetic, and pharmacologic studies in a variety of human and mouse KMT2A-r AML models, we uncovered a potential resistance mechanism independent of canonical menin-MLL targets. We show that a group of noncanonical menin targets, which are bivalently cooccupied by active menin and repressive H2AK119ub marks, are typically downregulated after menin inhibition. Loss of polycomb repressive complex 1.1 (PRC1.1) subunits, such as polycomb group ring finger 1 (PCGF1) or BCL6 corepressor (BCOR), leads to menin inhibitor resistance by epigenetic reactivation of these noncanonical targets, including MYC. Genetic and pharmacological inhibition of MYC can resensitize PRC1.1-deficient leukemia cells to menin inhibition. Moreover, we demonstrate that leukemia cells with the loss of PRC1.1 subunits exhibit reduced monocytic gene signatures and are susceptible to BCL2 inhibition, and that combinational treatment with venetoclax overcomes the resistance to menin inhibition in PRC1.1-deficient leukemia cells. These findings highlight the important roles of PRC1.1 and its regulated noncanonical menin targets in modulating the menin inhibitor response and provide potential strategies to treat leukemia with compromised PRC1.1 function., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Abstract: B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, with long-term overall survival rates of ∼85%. However, B-ALL harboring rearrangements of the MLL gene (also known as KMT2A), referred to as MLLr B-ALL, is common in infants and is associated with poor 5-year survival, relapses, and refractoriness to glucocorticoids (GCs). GCs are an essential part of the treatment backbone for B-ALL, and GC resistance is a major clinical predictor of poor outcome. Elucidating the mechanisms of GC resistance in MLLr B-ALL is, therefore, critical to guide therapeutic strategies that deepen the response after induction therapy. Neuron-glial antigen-2 (NG2) expression is a hallmark of MLLr B-ALL and is minimally expressed in healthy hematopoietic cells. We recently reported that NG2 expression is associated with poor prognosis in MLLr B-ALL. Despite its contribution to MLLr B-ALL pathogenesis, the role of NG2 in MLLr-mediated leukemogenesis/chemoresistance remains elusive. Here, we show that NG2 is an epigenetically regulated direct target gene of the leukemic MLL-ALF transcription elongation factor 4 (AF4) fusion protein. NG2 negatively regulates the expression of the GC receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) and confers GC resistance to MLLr B-ALL cells. Mechanistically, NG2 interacts with FLT3 to render ligand-independent activation of FLT3 signaling (a hallmark of MLLr B-ALL) and downregulation of NR3C1 via activating protein-1 (AP-1)-mediated transrepression. Collectively, our study elucidates the role of NG2 in GC resistance in MLLr B-ALL through FLT3/AP-1-mediated downregulation of NR3C1, providing novel therapeutic avenues for MLLr B-ALL., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Nuclear Bcl-xL is found to promote cancer metastasis independently of its mitochondria-based anti-apoptotic activity. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates histone H3 trimethyl Lys4 (H3K4me3) modification have yet to be understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds to Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced invasion and metastasis in mouse models. Furthermore, knockout of CtBP2 not only reduces the nuclear portion of Bcl-xL but also suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of the MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFβ mRNA upregulation, as well as invasion. Moreover, the cleavage under targets and release using nuclease (CUT&RUN) assay coupled with next-generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor regions of genes encoding TGFβ and its signaling pathway members in cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1 and this study offers new therapeutic strategies to treat Bcl-xL-overexpressing cancer., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Treatment of acute leukemia is gradually moving away from a "one-size-fits-all" approach, as scientific and clinical advances expand the arsenal of available targeted therapies. One of the recent additions is the group of menin inhibitors; oral, selective, small molecules that disrupt the interaction between the chromatin adapter menin, and an epigenetic regulator, the lysine methyltransferase 2A (KMT2A) complex. Two susceptible leukemia subtypes have been identified: (i) acute myeloid leukemia with a mutation in nucleophosmin 1 (NPM1), and (ii) any acute leukemia, myeloid or lymphoid, with a translocation resulting in the rearrangement of KMT2A. These leukemias share a distinct genetic expression, maintained by the KMT2A-menin interaction. Together they account for approximately 40% of patients with acute myeloid leukemia and 10% of patients with acute lymphoblastic leukemia. This spotlight review follows the journey of revumenib, as a representative of menin inhibitors, from bench to bedside. It focuses on the pathophysiology of leukemias sensitive to menin inhibition, delineation of how this understanding led to targeted drug development, and data from clinical trials. The important discovery of resistance mechanisms is also explored, as well as future directions in the use of menin inhibitors for treating leukemia.

Chromosomal translocations involving the mixed-lineage leukemia (MLL) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative MLL-rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose MLL oncoproteins promote lineage-switching events through dynamic chromatin binding at lineage-specific target genes, and may support resistance to menin inhibitors through similar changes in chromatin occupancy., (© 2024. The Author(s).)

(1) Background: The Mixed lineage leukemia 1 (MLL1) gene, located on chromosome 11q23, plays a pivotal role in histone lysine-specific methylation and is consistently associated with various types of leukemia. Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) have been tied to numerous diseases, including cancers, and have become valuable cancer biomarkers. There's a notable gap in studies probing the influence of SNPs on MLL1 protein structure, function, and subsequent modifications., (2) Methods: We utilized an array of bioinformatics tools, including PredictSNP, InterPro, ConSurf, I-Mutant2.0, MUpro, Musitedeep, Project HOPE, RegulomeDB, Mutpred2, and both CScape and CScape Somatic, to meticulously analyze the consequences of nsSNPs in the MLL1 gene., (3) Results: Out of 2,097 nsSNPs analyzed, 62 were determined to be significantly pathogenic by the PredictSNP tool, with ten crucial MLL1 functional domains identified using InterPro. Additionally, 50 of these nsSNPs had high conservation scores, hinting at potential effects on protein structure and function, while 32 were found to undermine MLL1 protein stability. Notably, four nsSNPs were deemed oncogenic, with two identified as cancer drivers. The nsSNP, D2724G, between the MLL1 protein's FY-rich domains, could disrupt proteolytic cleavage, altering gene expression patterns and potentially promoting cancer., (4) Conclusions: Our research provides a comprehensive assessment of nsSNPs' impact in the MLL1 protein structure and function and consequently on leukemia development, suggesting potential avenues for personalized treatment, early detection, improved prognosis, and a deeper understanding of hematological malignancy genesis., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Al-nakhle et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Our current understanding of epigenetic regulation is deeply rooted in the founding contributions of Dr C. David Allis. In 2002, Allis and colleagues first characterized the lysine methyltransferase activity of the mammalian KMT2A (MLL1), a paradigm-shifting discovery that brings epigenetic dysregulation into focus for many human diseases that carry KMT2A mutations. This review will discuss the current understanding of the multifaceted roles of KMT2A in development and disease, which has paved the way for innovative and upcoming approaches to cancer therapy., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Menin is an essential oncogenic cofactor of MLL1 fusion proteins in acute leukemias and inhibitors of the menin-MLL1 interaction are under evaluation in clinical trials. Recent studies found emerging resistance to menin inhibitor treatment in patients with leukemia as a result of somatic mutations in menin. To understand how patient mutations in menin affect the interaction with MLL1, we performed systematic characterization of the binding affinity of these menin mutants (T349M, M327I, G331R and G331D) and the N-terminal fragment of MLL1. We also determined the crystal structures of menin patient mutants and their complexes with MLL1-derived peptides. We found that drug-resistant mutations in menin occur at a site adjacent to the MLL1 binding site, but they do not affect MLL1 binding to menin. On the contrary, our structural analysis shows that all these point mutations in menin generate steric clash with menin inhibitors. We also found that mutation G331D results in a very slow dissociation of MLL1 from menin and this mutant might be particularly difficult to inhibit with small molecule drugs. This work provides structural information to support the development of a new generation of small molecule inhibitors that overcome resistance caused by menin mutations., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Drs. Grembecka and Cierpicki received research support from Kura Oncology, Inc. They have also served as consultants for Kura Oncology, have equity ownership in the company and are co-inventors on patent applications covering menin inhibitors. Drs. Grembecka and Cierpicki receive royalties from the University of Michigan on the patents covering menin inhibitors that were licensed to Kura Oncology., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 (bleximenib) is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) acute myeloid leukemia (AML) cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent antiproliferative activity across several AML and acute lymphoblastic leukemia (ALL) cell lines and patient samples harboring KMT2A or NPM1 alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent antiproliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A cocrystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903)., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The treatment of acute myeloid leukaemia (AML) has changed fundamentally in the last decade with many new targeted therapies entering clinics. Some of the most interesting agents under development are Menin inhibitors which interfere with the interaction of Menin with wild-type (wt) KMT2A or a KMT2A-fusion protein and thereby downregulate the leukaemic gene expression (MEIS1, PBX3, HOX) in NPM1 mutant or KMT2A-rearranged leukaemia. Other HOX and MEIS1 expressing leukaemias may also be sensitive to Menin inhibition. Following the encouraging results as monotherapy in refractory and relapsed AML, the combination of Menin inhibitors with chemotherapeutic agents and other targeted drugs is being investigated clinically., (© 2024 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

The histone acetyltransferase HBO1, also known as KAT7, is a major chromatin modifying enzyme responsible for H3 and H4 acetylation. It is found within two distinct tetrameric complexes, the JADE subunit-containing complex and BRPF subunit-containing complex. The HBO1-JADE complex acetylates lysine 5, 8 and 12 of histone H4, and the HBO1-BRPF complex acetylates lysine 14 of histone H3. HBO1 regulates gene transcription, DNA replication, DNA damage repair, and centromere function. It is involved in diverse signaling pathways and plays crucial roles in development and stem cell biology. Recent work has established a strong relationship of HBO1 with the histone methyltransferase MLL/KMT2A in acute myeloid leukemia. Here, we discuss functional and pathological links of HBO1 to cancer, highlighting the underlying mechanisms that may pave the way to the development of novel anti-cancer therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Immune cell-mediated inflammation is important in normal tissue regeneration but can be pathologic in diabetic wounds. Limited literature exists on the role of CD4+ T cells in normal or diabetic wound repair; however, the imbalance of CD4+ Th17/Tregs has been found to promote inflammation in other diabetic tissues. Here, using human tissue and murine transgenic models, we identified that the histone methyltransferase Mixed-lineage-leukemia-1 (MLL1) directly regulates the Th17 transcription factor RORγ via an H3K4me3 mechanism and increases expression of Notch receptors and downstream Notch signaling. Furthermore, we found that Notch receptor signaling regulates CD4+ Th cell differentiation and is critical for normal wound repair, and loss of upstream Notch pathway mediators or receptors in CD4+ T cells resulted in the loss of CD4+ Th cell differentiation in wounds. In diabetes, MLL1 and Notch-receptor signaling was upregulated in wound CD4+ Th cells, driving CD4+ T cells toward the Th17 cell phenotype. Treatment of diabetic wound CD4+ T cells with a small molecule inhibitor of MLL1 (MI-2) yielded a significant reduction in CD4+ Th17 cells and IL-17A. This is the first study to our knowledge to identify the MLL1-mediated mechanisms responsible for regulating the Th17/Treg balance in normal and diabetic wounds and to define the complex role of Notch signaling in CD4+ T cells in wounds, where increased or decreased Notch signaling both result in pathologic wound repair. Therapeutic targeting of MLL1 in diabetic CD4+ Th cells may decrease pathologic inflammation through regulation of CD4+ T cell differentiation.

Pediatric acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis and high relapse rate. Current challenges in the identification of immunotherapy targets arise from patient-specific blast immunophenotypes and their change during disease progression. To overcome this, we present a new computational research tool to rapidly identify malignant cells. We generated single-cell flow cytometry profiles of 21 pediatric AML patients with matched samples at diagnosis, remission, and relapse. We coupled a classifier to an autoencoder for anomaly detection and classified malignant blasts with 90% accuracy. Moreover, our method assigns a developmental stage to blasts at the single-cell level, improving current classification approaches based on differentiation of the dominant phenotype. We observed major immunophenotype and developmental stage alterations between diagnosis and relapse. Patients with KMT2A rearrangement had more profound changes in their blast immunophenotypes at relapse compared to patients with other molecular features. Our method provides new insights into the immunophenotypic composition of AML blasts in an unbiased fashion and can help to define immunotherapy targets that might improve personalized AML treatment., (© 2024 Driessen et al.)

Abstract: In acute myeloid leukemia (AML), leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) interact with various cell types in the bone marrow (BM) microenvironment, regulating their expansion and differentiation. To study the interaction of CD4+ and CD8+ T cells in the BM with LSCs and LPCs, we analyzed their transcriptome and predicted cell-cell interactions by unbiased high-throughput correlation network analysis. We found that CD4+ T cells in the BM of patients with AML were activated and skewed toward T-helper (Th)1 polarization, whereas interleukin-9 (IL-9)-producing (Th9) CD4+ T cells were absent. In contrast to normal hematopoietic stem cells, LSCs produced IL-9, and the correlation modeling predicted IL9 in LSCs as a main hub gene that activates CD4+ T cells in AML. Functional validation revealed that IL-9 receptor signaling in CD4+ T cells leads to activation of the JAK-STAT pathway that induces the upregulation of KMT2A and KMT2C genes, resulting in methylation on histone H3 at lysine 4 to promote genome accessibility and transcriptional activation. This induced Th1-skewing, proliferation, and effector cytokine secretion, including interferon gamma (IFN-γ) and tumor necrosis factor α (TNF-α). IFN-γ and, to a lesser extent, TNF-α produced by activated CD4+ T cells induced the expansion of LSCs. In accordance with our findings, high IL9 expression in LSCs and high IL9R, TNF, and IFNG expression in BM-infiltrating CD4+ T cells correlated with worse overall survival in AML. Thus, IL-9 secreted by AML LSCs shapes a Th1-skewed immune environment that promotes their expansion by secreting IFN-γ and TNF-α., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin "switch" governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression., Competing Interests: Conflict of interest EpiCypher is a commercial developer and supplier of fully defined semi-synthetic nucleosomes as used in this study. N. N. R., B. G., and B. D. S. own shares in EpiCypher with BDS also a board member of the same. All other authors declare that they have no other conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mixed lineage leukemia-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce MLL through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here, we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II in HEL, a human erythroleukemia cell line without MLL1 rearrangement. MLL1 and AF9 only coregulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and MLL-AF9 occupy promoters and distal intergenic regions, exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely believed elongation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers of Myb form a hub of interactions with the Myb promoter. We identified a long non-coding RNA (Myrlin) originating from the -81-kb murine Myb enhancer. Myrlin and Myb are coordinately regulated during erythroid differentiation. Myrlin TSS deletion using CRISPR-Cas9 reduced Myrlin and Myb expression and LDB1 complex occupancy at the Myb enhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing of Myrlin left LDB1 and the Myb enhancer hub unperturbed, although Myrlin and Myb expressions were downregulated, decoupling transcription and chromatin looping. Myrlin interacts with the KMT2A/MLL1 complex. Myrlin CRISPRi compromised KMT2A occupancy in the Myb locus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in the Myb first exon/intron. Thus, Myrlin directly participates in activating Myb transcription by recruiting KMT2A., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear protein that attaches negatively charged poly (ADP-ribose) (PAR) to itself and other target proteins. While its function in DNA damage repair is well established, its role in target chromatin recognition and regulation of gene expression remains to be better understood. This study showed that PARP1 interacts with SET1/MLL complexes by binding directly to WDR5. Notably, although PARP1 does not modulate WDR5 PARylation or the global level of H3K4 methylation, it exerts locus-specific effects on WDR5 binding and H3K4 methylation. Interestingly, PARP1 and WDR5 show extensive co-localization on chromatin, with WDR5 facilitating the recognition and expression of target genes regulated by PARP1. Furthermore, we demonstrated that inhibition of the WDR5 Win site impedes the interaction between PARP1 and WDR5, thereby inhibiting PARP1 from binding to target genes. Finally, the combined inhibition of the WDR5 Win site and PARP shows a profound inhibitory effect on the proliferation of cancer cells. These findings illuminate intricate mechanisms underlying chromatin recognition, gene transcription, and tumorigenesis, shedding light on previously unrecognized roles of PARP1 and WDR5 in these processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Identification of potential key targets of melanoma, a fatal skin malignancy, is critical to the development of new cancer therapies. Lysine methyltransferase 2A (KMT2A) promotes melanoma growth by activating the human telomerase reverse transcriptase (hTERT) signaling pathway; however, the exact mechanism remains elusive. This study aimed to reveal new molecular targets that regulate KMT2A expression and melanoma growth. Using biotin-streptavidin-agarose pull-down and proteomics, we identified Damage-specific DNA-binding protein 2 (DDB2) as a KMT2A promoter-binding protein in melanoma cells and validated its role as a regulator of KMT2A/hTERT signaling. DDB2 knockdown inhibited the expression of KMT2A and hTERT and inhibited the growth of melanoma cells in vitro. Conversely, overexpression of DDB2 activated the expression of KMT2A and promoted the growth of melanoma cells. Additionally, we demonstrated that DDB2 expression was higher in tumor tissues of patients with melanoma than in corresponding normal tissues and was positively correlated with KMT2A expression. Kaplan-Meier analysis showed a poor prognosis in patients with high levels of DDB2 and KMT2A. Overall, our data suggest that DDB2 promotes melanoma cell growth through the transcriptional regulation of KMT2A expression and predicts poor prognosis. Therefore, targeting DDB2 may regulate the effects of KMT2A on melanoma growth and progression, providing a new potential therapeutic strategy for melanoma., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches., Competing Interests: Competing interests statement:The authors declare no competing interest.

Human mixed lineage leukemia 4 (MLL4), also known as KMT2D, regulates cell type specific transcriptional programs through enhancer activation. Along with the catalytic methyltransferase domain, MLL4 contains seven less characterized plant homeodomain (PHD) fingers. Here, we report that the sixth PHD finger of MLL4 (MLL4 PHD6 ) binds to the hydrophobic motif of ten-eleven translocation 3 (TET3), a dioxygenase that converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4 PHD6 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site of MLL4 PHD6 , and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3 PHD7 ). Analysis of genomic localization of endogenous MLL4 and ectopically expressed TET3 in mouse embryonic stem cells reveals a high degree overlap on active enhancers and suggests a potential functional relationship of MLL4 and TET3., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Acute myeloid leukemia (AML) accounts for greater than twenty thousand new cases of leukemia annually in the United States. The average five-year survival rate is approximately 30%, pointing to the need for developing novel model systems for drug discovery. In particular, patients with chromosomal rearrangements in the mixed lineage leukemia (MLL) gene have higher relapse rates with poor outcomes. In this study we investigated the expression of human MLL-ENL and MLL-AF9 in the myeloid lineage of zebrafish embryos. We observed an expansion of MLL positive cells and determined these cells colocalized with the myeloid markers spi1b, mpx, and mpeg. In addition, expression of MLL-ENL and MLL-AF9 induced the expression of endogenous bcl2 and cdk9, genes that are often dysregulated in MLL-r-AML. Co-treatment of lyz: MLL-ENL or lyz:MLL-AF9 expressing embryos with the BCL2 inhibitor, Venetoclax, and the CDK9 inhibitor, Flavopiridol, significantly reduced the number of MLL positive cells compared to embryos treated with vehicle or either drug alone. In addition, cotreatment with Venetoclax and Flavopiridol significantly reduced the expression of endogenous mcl1a compared to vehicle, consistent with AML. This new model of MLL-r-AML provides a novel tool to understand the molecular mechanisms underlying disease progression and a platform for drug discovery., Competing Interests: The authors have declared that no competing interests., (Copyright: © 2024 Belt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Sporadic parathyroid adenoma (PA) is the most common cause of hyperparathyroidism, yet the mechanisms involved in its pathogenesis remain incompletely understood., Methods: Surgically removed PA samples, along with normal parathyroid gland (PG) tissues that were incidentally dissected during total thyroidectomy, were analysed using single-cell RNA-sequencing with the 10× Genomics Chromium Droplet platform and Cell Ranger software. Gene set variation analysis was conducted to characterise hallmark pathway gene signatures, and single-cell regulatory network inference and clustering were utilised to analyse transcription factor regulons. Immunohistochemistry and immunofluorescence were performed to validate cellular components of PA tissues. siRNA knockdown and gene overexpression, alongside quantitative polymerase chain reaction, Western blotting and cell proliferation assays, were conducted for functional investigations., Results: There was a pervasive increase in gene transcription in PA cells (PACs) compared with PG cells. This is associated with high expression of histone-lysine N-methyltransferase 2A (KMT2A). High KMT2A levels potentially contribute to promoting PAC proliferation through upregulation of the proto-oncogene CCND2, which is mediated by the transcription factors signal transducer and activator of transcription 3 (STAT3) and GATA binding protein 3 (GATA3). PA tissues are heavily infiltrated with myeloid cells, while fibroblasts, endothelial cells and macrophages in PA tissues are commonly enriched with proinflammatory gene signatures relative to their counterparts in PG tissues., Conclusions: We revealed the previously underappreciated involvement of the KMT2A‒STAT3/GATA3‒CCND2 axis and chronic inflammation in the pathogenesis of PA. These findings underscore the therapeutic promise of KMT2A inhibition and anti-inflammatory strategies, highlighting the need for future investigations to translate these molecular insights into practical applications., Highlights: Single-cell RNA-sequencing reveals a transcriptome catalogue comparing sporadic parathyroid adenomas (PAs) with normal parathyroid glands. PA cells show a pervasive increase in gene expression linked to KMT2A upregulation. KMT2A-mediated STAT3 and GATA3 upregulation is key to promoting PA cell proliferation via cyclin D2. PAs exhibit a proinflammatory microenvironment, suggesting a potential role of chronic inflammation in PA pathogenesis., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the β-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The long-term maintenance of leukaemia stem cells (LSCs) is responsible for the high degree of malignancy in MLL (mixed-lineage leukaemia) rearranged acute myeloid leukaemia (AML). The DNA damage response (DDR) and DOT1L/H3K79me pathways are required to maintain LSCs in MLLr-AML, but little is known about their interplay. This study revealed that the DDR enzyme ATM regulates the maintenance of LSCs in MLLr-AML with a sequential protein-posttranslational-modification manner via CBP-DOT1L. We identified the phosphorylation of CBP by ATM, which confers the stability of CBP by preventing its proteasomal degradation, and characterised the acetylation of DOT1L by CBP, which mediates the high level of H3K79me2 for the expression of leukaemia genes in MLLr-AML. In addition, we revealed that the regulation of CBP-DOT1L axis in MLLr-AML by ATM was independent of DNA damage activation. Our findings provide insight into the signalling pathways involoved in MLLr-AML and broaden the understanding of the role of DDR enzymes beyond processing DNA damage, as well as identigying them as potent cancer targets., (© 2024. The Author(s).)

The activation and specialization of regulatory T cells (Tregs) are crucial for maintaining immune self-tolerance; however, the regulation of these processes by histone modifications is not fully understood. Here, we show that T cell-specific deletion of the lysine methyltransferase MLL1 results in a spontaneous lymphocyte proliferation phenotype in aged mice without disturbing the development of conventional T cells and Tregs. Treg-specific MLL1 ablation leads to a systemic autoimmune disease associated with Treg dysfunction. Moreover, RNA sequencing demonstrates that the induction of multiple genes involved in Treg activation, functional specialization, and tissue immigration is defective in MLL1-deficient Tregs. This dysregulation is associated with defects in H3K4 trimethylation at these genes' transcription start sites. Finally, using a T-bet fate-mapping mouse system, we determine that MLL1 is required to establish stable Th1-type Tregs. Thus, MLL1 is essential in optimal Treg function by providing a coordinated chromatin context for activation and specialization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: In leukemogenesis, genotoxic stress in hematopoietic stem and progenitor cells (HSPCs) drives individual context-dependent programs of malignant transformation. In light of the various differentiation stages of HSPCs based on a recently revised definition using CD150/CD48, our analyses showed that a subpopulation of long-term repopulating HSCs was most susceptible to MLL-ENL-mediated transformation. An analysis of the molecular mechanism identified Bromo-adjacent homology domain and coiled-coil containing 1 (Bahcc1), which encodes a reader molecule of trimethylated histone H3 lysine 27 (H3K27me3), as a candidate gene involved in distinct susceptibility to leukemic transformation. Interestingly, Bahcc1 was previously reported to be highly expressed in acute myeloid leukemia (AML) with an unfavorable prognosis, including some cases of MLL-rearranged AML. We found that MLL-ENL upregulated Bahcc1 through binding to its promoter, and that Bahcc1 was involved in MLL-ENL-mediated immortalization at least partly through repression of H3K27me3-marked Cdkn1c. Analyses using bone marrow transplantation in mice showed that depletion of Bahcc1 suppressed the leukemogenic activity of MLL-ENL. In a public database, high BAHCC1 expression was found to be associated with a poor prognosis in pediatric AML, in which BAHCC1 expression was significantly lower in MLL-AF9-AML than in other MLL-fusion-AML. These findings shed light on the distinct immortalization potential of HSPCs and suggest a novel MLL-fusion-Bahcc1 axis, which may lead to development of molecular targeted therapy against MLL-fusion-mediated leukemia., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C-C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA., (© 2024. The Author(s).)

Histone H3 lysine 4 mono-methylation (H3K4me1) marks poised or active enhancers. KMT2C (MLL3) and KMT2D (MLL4) catalyze H3K4me1, but their histone methyltransferase activities are largely dispensable for transcription during early embryogenesis in mammals. To better understand the role of H3K4me1 in enhancer function, we analyze dynamic enhancer-promoter (E-P) interactions and gene expression during neural differentiation of the mouse embryonic stem cells. We found that KMT2C/D catalytic activities were only required for H3K4me1 and E-P contacts at a subset of candidate enhancers, induced upon neural differentiation. By contrast, a majority of enhancers retained H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, H3K4me1 signals at these KMT2C/D-independent sites were reduced after acute depletion of KMT2B, resulting in aggravated transcriptional defects. Our observations therefore implicate KMT2B in the catalysis of H3K4me1 at enhancers and provide additional support for an active role of H3K4me1 in enhancer-promoter interactions and transcription in mammalian cells., Competing Interests: Declaration of interests B.R. is a co-founder of Arima Genomics, Inc. and Epigenome Technologies, Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Infant and adult MLL1/KMT2A-rearranged (MLLr) leukemia represents a disease with a dismal prognosis. Here, we present a functional and proteomic characterization of in utero-initiated and adult-onset MLLr leukemia. We reveal that fetal MLL::ENL-expressing lymphomyeloid multipotent progenitors (LMPPs) are intrinsically programmed towards a lymphoid fate but give rise to myeloid leukemia in vivo, highlighting a complex interplay of intra- and extracellular factors in determining disease subtype. We characterize early proteomic events of MLL::ENL-mediated transformation in fetal and adult blood progenitors and reveal that whereas adult pre-leukemic cells are mainly characterized by retained myeloid features and downregulation of ribosomal and metabolic proteins, expression of MLL::ENL in fetal LMPPs leads to enrichment of translation-associated and histone deacetylases signaling proteins, and decreased expression of inflammation and myeloid differentiation proteins. Integrating the proteome of pre-leukemic cells with their secretome and the proteomic composition of the extracellular environment of normal progenitors highlights differential regulation of Igf2 bioavailability, as well as of VLA-4 dimer and its ligandome, upon initiation of fetal- and adult-origin leukemia, with implications for human MLLr leukemia cells' ability to communicate with their environment through granule proteins. Our study has uncovered opportunities for targeting ontogeny-specific proteomic vulnerabilities in in utero-initiated and adult-onset MLLr leukemia., (© 2024. The Author(s).)

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies., Competing Interests: GH, JW, KR, CJ, PP, TT, AF, LV, SL, BG, BS, MS, ER, SG, FM, QL No competing interests declared, MS Receives research funding from ALX Oncology, Astex, Incyte, Takeda and TG Therapeutics; has stock in Karyopharm and Ryvu; serves on advisory boards or consults for BMS, CTI, Forma, Geron, GSK, Karyopharm, Rigel, Ryvu, Taiho and Treadwell, TL, SF Patents: Lee T, Alvarado J, Tian J, Meyers KM, Han C, Mills JJ, Teuscher KB, Stauffer SR, Fesik SW. WDR5 inhibitors and modulators. WO 2020086857. 30 April 2020; Lee T, Han C, Mills JJ, Teuscher KB, Tian J, Meyers KM, Chowdhury S, Fesik SW. WDR5 inhibitors and modulators. WO 2020247679. 10 December 2020; Lee T, Teuscher KB, Tian J, Meyers KM, Chowdhury S, Fesik SW. WDR5 Inhibitors and modulators. WO 2021092525. 14 May 2021; Lee T, Teuscher KB, Chowdhury S, Tian J, Meyers KM, Fesik SW. WDR5 Inhibitors and modulators. WO2022236101. 10 November 2022, WT Patents: Fesik SW, Stauffer SR, Salovich JM, Tansey WP, Wang F, Phan J, Olejniczak ET, inventors. WDR5 inhibitors and modulators. United States Patent US 10,501,466. 10 December 2019; Fesik SW, Stauffer SR, Tansey WP, Olejniczak ET, Phan J, Wang F, Jeon K, Gogliotti RD, inventors. WDR5 inhibitors and modulators. United States Patent US 10,160,763. 25 December 2018, (© 2023, Howard et al.)

The escalating prevalence of diabetes mellitus underscores the need for a comprehensive understanding of pancreatic beta cell function. Interest in glucose effectiveness has prompted the exploration of novel regulatory factors. The myeloid/lymphoid or mixed-lineage leukaemia gene ( MLL ) is widely recognised for its role in leukemogenesis and nuclear regulatory mechanisms through its histone methyltransferase activity in active chromatin. However, its function within pancreatic endocrine tissues remains elusive. Herein, we unveil a novel role of MLL in glucose metabolism and insulin secretion. MLL knockdown in βHC-9 pancreatic beta cells diminished insulin secretion in response to glucose loading, paralleled by the downregulation of the glucose-sensitive genes SLC2a1 and SLC2a2 . Similar observations were made in MLL heterozygous knockout mice ( MLL +/-), which exhibited impaired glucose tolerance and reduced insulin secretion without morphological anomalies in pancreatic endocrine cells. The reduction in insulin secretion was independent of changes in beta cell mass or insulin granule morphology, suggesting the regulatory role of MLL in glucose-sensitive gene expression. The current results suggest that MLL interacts with circadian-related complexes to modulate the expression of glucose transporter genes, thereby regulating glucose sensing and insulin secretion. Our findings shed light on insulin secretion control, providing potential avenues for therapeutics against diabetes.

Abstract: Small molecules that target the menin-KMT2A protein-protein interaction (menin inhibitors) have recently entered clinical trials in lysine methyltransferase 2A (KMT2A or MLL1)-rearranged (KMT2A-r) and nucleophosmin-mutant (NPM1c) acute myeloid leukemia (AML) and are demonstrating encouraging results. However, rationally chosen combination therapy is needed to improve responses and prevent resistance. We have previously identified IKZF1/IKAROS as a target in KMT2A-r AML and shown in preclinical models that IKAROS protein degradation with lenalidomide or iberdomide has modest single-agent activity yet can synergize with menin inhibitors. Recently, the novel IKAROS degrader mezigdomide was developed with greatly enhanced IKAROS protein degradation. In this study, we show that mezigdomide has increased preclinical activity in vitro as a single-agent in KMT2A-r and NPM1c AML cell lines, including sensitivity in cell lines resistant to lenalidomide and iberdomide. Further, we demonstrate that mezigdomide has the greatest capacity to synergize with and induce apoptosis in combination with menin inhibitors, including in MEN1 mutant models. We show that the superior activity of mezigdomide compared with lenalidomide or iberdomide is due to its increased depth, rate, and duration of IKAROS protein degradation. Single-agent mezigdomide was efficacious in 5 patient-derived xenograft models of KMT2A-r and 1 NPM1c AML. The combination of mezigdomide with the menin inhibitor VTP-50469 increased survival and prevented and overcame MEN1 mutations that mediate resistance in patients receiving menin inhibitor monotherapy. These results support prioritization of mezigdomide for early phase clinical trials in KMT2A-r and NPM1c AML, either as a single agent or in combination with menin inhibitors., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

To interfere the Menin-MLL interaction using small molecular inhibitors has been shown as new treatment of several special hematological malignancies. Herein, a series of Menin-MLL interaction inhibitors with pyrrolo[2,3-d]pyrimidine scaffold were designed, synthesized and evaluated. Among them, compound A6 exhibited potent binding affinity with an IC 50 value of 0.38 μM, and strong anti-proliferative activity against MV4-11 cells with an IC 50 value of 1.07 μM. Further study showed A6 reduced the transcriptional levels of HOXA9 and MEIS1 genes. Moreover, A6 induced cellular apoptosis, arrested the cell cycle in G0/G1 phase, and reversed the differentiation arrest in a concentration-dependent manner. This study suggested compound A6 was as a novel potent Menin-MLL interaction inhibitor, and it proved that introduction of 4-amino pyrrolo[2,3-d]pyrimidine to occupy the P10 hydrophobic pocket was new idea for design of novel Menin-MLL interaction inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Cell migration is a critical contributor to metastasis. Cytokine production and its role in cancer cell migration have been traditionally associated with immune cells. We find that the histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) controls 3D cell migration via cytokines, IL-6, IL-8, and TGF-β1, secreted by the cancer cells themselves. MLL1, with its scaffold protein Menin, controls actin filament assembly via the IL-6/8/pSTAT3/Arp3 axis and myosin contractility via the TGF-β1/Gli2/ROCK1/2/pMLC2 axis, which together regulate dynamic protrusion generation and 3D cell migration. MLL1 also regulates cell proliferation via mitosis-based and cell cycle-related pathways. Mice bearing orthotopic MLL1-depleted tumors exhibit decreased lung metastatic burden and longer survival. MLL1 depletion leads to lower metastatic burden even when controlling for the difference in primary tumor growth rates. Combining MLL1-Menin inhibitor with paclitaxel abrogates tumor growth and metastasis, including preexistent metastasis. These results establish MLL1 as a potent regulator of cell migration and highlight the potential of targeting MLL1 in patients with metastatic disease.

Ectopic activation of rearranged during transfection (RET) has been reported to facilitate lineage differentiation and cell proliferation in different cytogenetic subtypes of acute myeloid leukemia (AML). Herein, we demonstrate that RET is significantly (p < 0.01) upregulated in AML subtypes containing rearrangements of the lysine methyltransferase 2A gene (KMT2A), commonly referred to as KMT2A-rearranged (KMT2A-r) AML. Integrating multi-epigenomics data, we show that the KMT2A-MLLT3 fusion induces the development of CCCTC-binding (CTCF)-guided de novo extrusion enhancer loop to upregulate RET expression in KMT2A-r AML. Based on the finding that RET expression is tightly correlated with the selective chromatin remodeler and mediator (MED) proteins, we used a small-molecule inhibitor having dual inhibition against RET and MED12-associated cyclin-dependent kinase 8 (CDK8) in KMT2A-r AML cells. Dual inhibition of RET and CDK8 restricted cell proliferation by producing multimodal oxidative stress responses in treated cells. Our data suggest that epigenetically enhanced RET protects KMT2A-r AML cells from oxidative stresses, which could be exploited as a potential therapeutic strategy., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Purpose of Review: We provide an update on the successes and ongoing challenges of Menin inhibition as a novel approach for the treatment of patients with acute leukemias that express HOXA cluster genes including leukemias with KMT2A -rearrangements, NPM1 mutations or NUP98 -rearrangements. Initial clinical trials show promising response rates in heavily pretreated patients suggesting these inhibitors may have a significant impact on patient outcome. Furthermore, the development of resistance mutations that decrease drug binding affinity, validates Menin as a therapeutic target in human cancers. Therapeutic strategies aiming at overcoming and preventing resistance, are of high clinical relevance., Recent Findings: Several Menin inhibitor chemotypes have entered clinical trials. Acquired point mutations have recently been described as a mechanism of resistance towards Menin inhibitors. However, resistance can develop in absence of these mutations. Combination therapies are currently being investigated in preclinical models and in early phase clinical trials., Summary: Given the remarkable overall response rates, shedding light on treatment options for patients whose leukemias develop resistance to Menin inhibitors is an imminent clinical need. Studying the underlying mechanisms to inform clinical decision making, and to potentially prevent the development of resistance is of outmost importance., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Systemic iron overload is a common clinical challenge leading to significantly serious complications in patients with acute myeloid leukemia (AML), which affects both the quality of life and the overall survival of patients. Symptoms can be relieved after iron chelation therapy in clinical practice. However, the roles and mechanisms of iron overload on the initiation and progression of leukemia remain elusive. Here we studied the correlation between iron overload and AML clinical outcome, and further explored the role and pathophysiologic mechanism of iron overload in AML by using two mouse models: an iron overload MLL-AF9-induced AML mouse model and a nude xenograft mouse model. Patients with AML had an increased ferritin level, particularly in the myelomonocytic (M4) or monocytic (M5) subtypes. High level of iron expression correlated with a worsened prognosis in AML patients and a shortened survival time in AML mice. Furthermore, iron overload increased the tumor load in the bone marrow (BM) and extramedullary tissues by promoting the proliferation of leukemia cells through the upregulation of FOS. Collectively, our findings provide new insights into the roles of iron overload in AML. Additionally, this study may provide a potential therapeutic target to improve the outcome of AML patients and a rationale for the prospective evaluation of iron chelation therapy in AML., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Abnormal placental development is a significant factor contributing to perinatal morbidity and mortality, affecting approximately 5-7% of pregnant women. Trophoblast syncytialization plays a pivotal role in the establishment and maturation of the placenta, and its dysregulation is closely associated with several pregnancy-related disorders, including preeclampsia and intrauterine growth restriction. However, the underlying mechanisms and genetic determinants of syncytialization are largely unknown., Methods: We conducted a systematic drug screen using an epigenetic compound library to systematically investigate the epigenetic mechanism essential for syncytialization, and identified mixed lineage leukemia 1 (MLL1), a histone 3 lysine 4 methyltransferase, as a crucial regulator of trophoblast syncytialization. BeWo cells were utilized to investigate the role of MLL1 during trophoblast syncytialization. RNA sequencing and CUT&Tag were further performed to search for potential target genes and the molecular pathways involved. Human placenta tissue was used to investigate the role of MLL1 in TEA domain transcription factor 4 (TEAD4) expression and the upstream signaling during syncytialization. A mouse model was used to examine whether inhibition of MLL1-mediated H3K4me3 regulated placental TEAD4 expression and fetoplacental growth., Results: Genetic knockdown of MLL1 or pharmacological inhibition of the MLL1 methyltransferase complex (by MI-3454) markedly enhanced syncytialization, while overexpression of MLL1 inhibited forskolin (FSK)-induced syncytiotrophoblast formation. In human placental villous tissue, MLL1 was predominantly localized in the nuclei of cytotrophoblasts. Moreover, a notable upregulation in MLL1 expression was observed in the villus tissue of patients with preeclampsia compared with that in the control group. Based on RNA sequencing and CUT&Tag analyses, depletion of MLL1 inhibited the Hippo signaling pathway by suppressing TEAD4 expression by modulating H3K4me3 levels on the TEAD4 promoter region. TEAD4 overexpression significantly reversed the FSK-induced or MLL1 silencing-mediated trophoblast syncytialization. Additionally, decreased hypoxia-inducible factor 1A (HIF1A) enrichment at the MLL1 promoter was observed during syncytialization. Under hypoxic conditions, HIF1A could bind to and upregulate MLL1, leading to the activation of the MLL1/TEAD4 axis. In vivo studies demonstrated that the administration of MI-3454 significantly enhanced fetal vessel development and increased the thickness of the syncytial layer, thereby supporting fetoplacental growth., Conclusions: These results revealed a novel epigenetic mechanism underlying the progression of syncytialization with MLL1, and suggest potential avenues for identifying new therapeutic targets for pregnancy-related disorders., (© 2024. The Author(s).)

Abstract: RNA-binding proteins (RBPs) are emerging as a novel class of therapeutic targets in cancer, including in leukemia, given their important role in posttranscriptional gene regulation, and have the unexplored potential to be combined with existing therapies. The RBP insulin-like growth factor 2 messenger RNA-binding protein 3 (IGF2BP3) has been found to be a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Here, we study the combined effects of targeting IGF2BP3 and menin-MLL interaction in MLL-AF4-driven leukemia in vitro and in vivo, using genetic inhibition with CRISPR-Cas9-mediated deletion of Igf2bp3 and pharmacologic inhibition of the menin-MLL interaction with multiple commercially available inhibitors. Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the effects of menin-MLL inhibition on cell growth and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and menin-MLL inhibition led to increased differentiation in vitro and in vivo, seen in functional readouts and by gene expression analyses. IGF2BP3 knockdown had a greater effect on increasing survival and attenuating disease than pharmacologic menin-MLL inhibition with small molecule MI-503 alone and showed enhanced antileukemic effects in combination. Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4-mediated leukemogenesis and a potent therapeutic target, providing a paradigm for targeting leukemia at both the transcriptional and posttranscriptional level., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Lymphoid-primed multipotent progenitor (LMPP)-like and granulocyte-monocyte progenitor (GMP)-like leukemia stem cells (LSCs) co-exist in the blood of most patients with acute myeloid leukemia (AML). Complete elimination of both types of LSCs is required to cure AML. Using an MLL-AF9-induced murine AML model, we studied the role of hematopoietic cytokines in the survival of LMPP- and GMP-like LSCs. We found that SCF or FLT3L promotes the survival of LMPP-like LSCs by stimulating Stat5-mediated Mcl1 expression, whereas interleukin-3 (IL-3) or IL-6 induces the survival of GMP-like LSCs by stimulating Stat3/nuclear factor κB (NF-κB)-mediated Bcl2 expression. Functional study demonstrated that, compared to AML cells cultured in IL-3 and IL-6 medium, AML cells in SCF- or Flt3L-only culture are highly clonogenic in in vitro culture and are highly leukemogenic in vivo. Our study suggests that co-inhibition of both STAT5-MCL1 and STAT3/NF-κB-BCL2 signaling might represent an improved treatment strategy against AML, specifically AML cases with a monocytic phenotype and/or FLT3 mutations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Yeast COMPASS (complex of proteins associated with Set1) and human MLL (mixed-lineage leukemia) complexes are histone H3 lysine 4 methyltransferases with critical roles in gene regulation and embryonic development. Both complexes share a conserved C-terminal SET domain, responsible for catalyzing histone H3 K4 methylation on nucleosomes. Notably, their catalytic activity toward nucleosomes is enhanced and optimized with assembly of auxiliary subunits. In this review, we aim to illustrate the recent X-ray and cryo-EM structures of yeast COMPASS and human MLL1 core complexes bound to either unmodified nucleosome core particle (NCP) or H2B mono-ubiquitinated NCP (H2Bub.NCP). We further delineate how each auxiliary component of the complex contributes to the NCP and ubiquitin recognition to maximize the methyltransferase activity., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

In the ever evolving field of functional genomics, CRISPR-based screening technologies have become pivotal tools for elucidating gene function across various cell types. A recent study by Gilan and colleagues advances this technological frontier by introducing CRISPR-ChIP, a platform designed to investigate the complex dynamics of epigenetic regulation of chromatin. In proof-of-concept experiments, the authors demonstrate the potential of this tool to identify key molecular regulators of two major histone modifications associated with active transcription, H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 79 dimethylation (H3K79me2). They further unveiled a previously unknown functional partitioning of the H3K79-specific methyltransferase DOT1L into an oncogenic complex with MLL-AF9 and a native complex with MLLT10, which cooperatively regulate mixed lineage leukemia fusion protein (MLL-FP) target gene expression. This novel epigenomic approach integrates high-throughput CRISPR screening with chromatin immunoprecipitation-based direct readout of chromatin modifications in situ, offering a powerful tool to investigate the epigenetic regulatory layers across a diverse spectrum of biological processes and disease states., (©2023 American Association for Cancer Research.)

Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML., (© 2023. The Author(s).)

KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3β, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia., (© 2023. The Author(s).)

Background: Enhancer dysregulation is one of the important features for cancer cells. Enhancers enriched with H3K4me3 have been implicated to play important roles in cancer. However, their detailed features and regulatory mechanisms have not been well characterized., Results: Here, we profile the landscape of H3K4me3-enriched enhancers (m3Es) in 43 pairs of colorectal cancer (CRC) samples. M3Es are widely distributed in CRC and averagely possess around 10% of total active enhancers. We identify 1322 gain variant m3Es and 367 lost variant m3Es in CRC. The target genes of the gain m3Es are enriched in immune response pathways. We experimentally prove that repression of CBX8 and RPS6KA5 m3Es inhibits target gene expression in CRC. Furthermore, we find histone methyltransferase MLL1 is responsible for depositing H3K4me3 on the identified Vm3Es. We demonstrate that the transcription factor AP1/JUN interacts with MLL1 and regulates m3E activity. Application of a small chemical inhibitor for MLL1 activity, OICR-9429, represses target gene expression of the identified Vm3Es, enhances anti-tumor immunity and inhibits CRC growth in an animal model., Conclusions: Taken together, our study illustrates the genome-wide landscape and the regulatory mechanisms of m3Es in CRC, and reveals potential novel strategies for cancer treatment., (© 2023. The Author(s).)

Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation (H3K4me3) and partially exerts its untoward functional effects by interacting with multiple subunits including menin and WD repeat-containing protein 5 (WDR5). In this study, we investigated the role and mechanisms of MLL1 in murine models of acute kidney injury induced by folic acid (FA) and ischemia-reperfusion. Injury to the kidney elevated the expression of MLL1, menin, WDR5, and H3K4Me3, which was accompanied by increased serum creatinine and blood urea nitrogen, renal tubular injury, and apoptosis. Pharmacological inhibition of MLL1 activity with MI503 to disrupt the interaction between MLL1 with menin further increased serum creatinine and blood urea nitrogen levels, enhanced expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and induced more apoptosis in the kidney following FA and ischemia-reperfusion injury. In contrast, MI503 treatment decreased the expression of vimentin and proliferating cell nuclear antigens. Similarly, treatment with MM102 to disrupt the interaction between MLL1 and WDR5 also worsened renal dysfunction, aggravated tubular cell injury, increased apoptosis, and inhibited cellular dedifferentiation and proliferation in mice following FA injection. Moreover, MI503 inhibited FA-induced phosphorylation of epidermal growth factor receptor, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase-1/2 in injured kidneys. Collectively, these data suggest that MLL1 contributes to renal protection and functional recovery and promotes renal regeneration through a mechanism associated with activation of the epidermal growth factor receptor signaling pathway. NEW & NOTEWORTHY Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation and exerts its functional roles by interacting with multiple subunits. In this study, we demonstrated that inhibition of MLL1 activity by MI503 or MM102 aggravated renal injury and apoptosis and suppressed renal tubular cell dedifferentiation and proliferation, suggesting that MLL1 activation during acute kidney injury acts as an intrinsic protective mechanism to mediate renal tubular cell survival and regeneration.

Enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation are crucial for proper cellular differentiation and are frequently mutated in cancer. The Mixed lineage leukemia (MLL) family of enzymes deposit H3K4 mono-, di-, or trimethylation at distinct genomic locations, requiring precise spatial and temporal control. Despite evidence that the degree of H3K4 methylation is controlled in part by a hierarchical assembly pathway with key subcomplex components, we previously found that the assembled state of the MLL1 core complex is not favored at physiological temperature. To better understand this paradox, we tested the hypothesis that increasing the concentration of subunits in a biomolecular condensate overcomes this thermodynamic barrier via mass action. Here, we demonstrate that MLL1 core complex phase separation stimulates enzymatic activity up to 60-fold but not primarily by concentrating subunits into droplets. Instead, we found that stimulated activity is largely due to the formation of an altered oligomeric scaffold that greatly reduces substrate K m . We posit that phase separation-induced scaffolding of the MLL1 core complex is a potential "switch-like" mechanism for spatiotemporal control of H3K4 methylation through the rapid formation or dissolution of biomolecular condensates within RNA Pol II transcription factories., Competing Interests: Conflicts of interest M. S. C. owns stock and serves on the Consultant Advisory Board for Kathera Bioscience Inc, the makers of antifungal technologies. M. S. C. also hold US patents (8133690), (8715678), and (10392423) for compounds and methods for inhibiting SET1/MLL family complexes. The other authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)