In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor-microenvironment interactions.

Functional genomic screens in two-dimensional cell culture models are limited in identifying therapeutic targets that influence the tumor microenvironment. By comparing targeted CRISPR-Cas9 screens in a two-dimensional culture with xenografts derived from the same cell line, we identified MEN1 as the top hit that confers differential dropout effects in vitro and in vivo. MEN1 knockout in multiple solid cancer types does not impact cell proliferation in vitro but significantly promotes or inhibits tumor growth in immunodeficient or immunocompetent mice, respectively. Mechanistically, MEN1 knockout redistributes MLL1 chromatin occupancy, increasing H3K4me3 at repetitive genomic regions, activating double-stranded RNA expression and increasing neutrophil and CD8+ T cell infiltration in immunodeficient and immunocompetent mice, respectively. Pharmacological inhibition of the menin-MLL interaction reduces tumor growth in a CD8+ T cell-dependent manner. These findings reveal tumor microenvironment-dependent oncogenic and tumor-suppressive functions of MEN1 and provide a rationale for targeting MEN1 in solid cancers.

Identification of single-cell blasts in pediatric acute myeloid leukemia using an autoencoder.

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.

KMT2A Rearrangements in Leukemias: Molecular Aspects and Therapeutic Perspectives.

KMT2A (alias: mixed-lineage leukemia [MLL]) gene mapping on chromosome 11q23 encodes the lysine-specific histone N-methyltransferase 2A and promotes transcription by inducing an open chromatin conformation. Numerous genomic breakpoints within the KMT2A gene have been reported in young children and adults with hematologic disorders and are present in up to 10% of acute leukemias. These rearrangements describe distinct features and worse prognosis depending on the fusion partner, characterized by chemotherapy resistance and high rates of relapse, with a progression-free survival of 30-40% and overall survival below 25%. Less intensive regimens are used in pediatric patients, while new combination therapies and targeted immunotherapeutic agents are being explored in adults. Beneficial therapeutic effects, and even cure, can be reached with hematopoietic stem cell transplantation, mainly in young children with dismal molecular lesions; however, delayed related toxicities represent a concern. Herein, we summarize the translocation partner genes and partial tandem duplications of the KMT2A gene, their molecular impact, clinical aspects, and novel targeted therapies.

[Progress of allogeneic hematopoietic stem cell transplantation in KMT2A-rearranged acute leukemia].

KMT2A (lysine methyltransferase 2A) -rearranged acute leukemia is a class of leukemia with unique biological characteristics with moderate or poor prognosis. In recent years, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been increasingly indicated for patients with KMT2A-rearranged acute leukemia. By reviewing the clinical studies of allo-HSCT in KMT2A-rearranged acute leukemia, the efficacy of allo-HSCT in children and adults with KMT2A-rearranged acute myeloid leukemia and acute lymphoblastic leukemia was assessed, the factors affecting the prognosis of allo-HSCT were summarized, and the methods that may improve the outcomes of allo-HSCT were explored.

Rearrangements involving 11q23.3/KMT2A in adult AML: mutational landscape and prognostic implications - a HARMONY study.

Balanced rearrangements involving the KMT2A gene (KMT2Ar) are recurrent genetic abnormalities in acute myeloid leukemia (AML), but there is lack of consensus regarding the prognostic impact of different fusion partners. Moreover, prognostic implications of gene mutations co-occurring with KMT2Ar are not established. From the HARMONY AML database 205 KMT2Ar adult patients were selected, 185 of whom had mutational information by a panel-based next-generation sequencing analysis. Overall survival (OS) was similar across the different translocations, including t(9;11)(p21.3;q23.3)/KMT2A::MLLT3 (p = 0.756). However, independent prognostic factors for OS in intensively treated patients were age >60 years (HR 2.1, p = 0.001), secondary AML (HR 2.2, p = 0.043), DNMT3A-mut (HR 2.1, p = 0.047) and KRAS-mut (HR 2.0, p = 0.005). In the subset of patients with de novo AML < 60 years, KRAS and TP53 were the prognostically most relevant mutated genes, as patients with a mutation of any of those two genes had a lower complete remission rate (50% vs 86%, p < 0.001) and inferior OS (median 7 vs 30 months, p < 0.001). Allogeneic hematopoietic stem cell transplantation in first complete remission was able to improve OS (p = 0.003). Our study highlights the importance of the mutational patterns in adult KMT2Ar AML and provides new insights into more accurate prognostic stratification of these patients.

The Core Complex of Yeast COMPASS and Human Mixed-Lineage Leukemia (MLL), Structure, Function, and Recognition of the Nucleosome.

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.

Histone H3K18 & H3K23 acetylation directs establishment of MLL-mediated H3K4 methylation.

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.

MLL-AF9 regulates transcriptional initiation in mixed lineage leukemic cells.

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.

HBO1, a MYSTerious KAT and its links to cancer.

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.

Synergistic effects of the KDM4C inhibitor SD70 and the menin inhibitor MI-503 against MLL::AF9-driven acute myeloid leukaemia.

MLL-rearranged (MLL-r) leukaemia is observed in approximately 10% of acute myeloid leukaemia (AML) and is associated with a relatively poor prognosis, highlighting the need for new treatment regimens. MLL fusion proteins produced by MLL rearrangements recruit KDM4C to mediate epigenetic reprogramming, which is required for the maintenance of MLL-r leukaemia. In this study, we used a combinatorial drug screen to selectively identify synergistic treatment partners for the KDM4C inhibitor SD70. The results showed that the drug combination of SD70 and MI-503, a potent menin-MLL inhibitor, induced synergistically enhanced apoptosis in MLL::AF9 leukaemia cells without affecting normal CD34+ cells. In vivo treatment with SD70 and MI-503 significantly prolonged survival in AML xenograft models. Differential gene expression analysis by RNA-seq following combined pharmacological inhibition of SD70 and MI-503 revealed changes in numerous genes, with MYC target genes being the most significantly downregulated. Taken together, these data provide preclinical evidence that the combination of SD70 and MI-503 is a potential dual-targeted therapy for MLL::AF9 AML.

KMT2A and chronic inflammation as potential drivers of sporadic parathyroid adenoma.

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.

Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A-Rearranged Acute Lymphoblastic and Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) exhibit favorable survival rates. However, for AML and ALL patients carrying KMT2A gene translocations clinical outcome remains unsatisfactory. Key players in KMT2A-fusion-driven leukemogenesis include menin and DOT1L. Recently, menin inhibitors like revumenib have garnered attention for their potential therapeutic efficacy in treating KMT2A-rearranged acute leukemias. However, resistance to menin inhibition poses challenges, and identifying which patients would benefit from revumenib treatment is crucial. Here, we investigated the in vitro response to revumenib in KMT2A-rearranged ALL and AML. While ALL samples show rapid, dose-dependent induction of leukemic cell death, AML responses are much slower and promote myeloid differentiation. Furthermore, we reveal that acquired resistance to revumenib in KMT2A-rearranged ALL cells can occur either through the acquisition of MEN1 mutations or independently of mutations in MEN1. Finally, we demonstrate significant synergy between revumenib and the DOT1L inhibitor pinometostat in KMT2A-rearranged ALL, suggesting that such drug combinations represent a potent therapeutic strategy for these patients. Collectively, our findings underscore the complexity of resistance mechanisms and advocate for precise patient stratification to optimize the use of menin inhibitors in KMT2A-rearranged acute leukemia.

Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia.

BACKGROUND: Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence. RESULTS: Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers. CONCLUSIONS: Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.

Myeloid Targeted Human MLL-ENL and MLL-AF9 Induces cdk9 and bcl2 Expression in Zebrafish Embryos.

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.

Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes.

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.

DDB2 promotes melanoma cell growth by transcriptionally regulating the expression of KMT2A and predicts a poor prognosis.

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.