Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia

Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the KMT2A gene, encoding the KMT2A lysine methyltransferase (also known as mixed-lineage leukemia-1), and produce in-frame fusions of KMT2A to other chromatin-regulatory proteins. Here we map fusion-specific targets across the genome for diverse KMT2A oncofusion proteins in cell lines and patient samples. By modifying CUT&Tag chromatin profiling for full automation, we identify common and tumor-subtype-specific sites of aberrant chromatin regulation induced by KMT2A oncofusion proteins. A subset of KMT2A oncofusion-binding sites are marked by bivalent (H3K4me3 and H3K27me3) chromatin signatures, and single-cell CUT&Tag profiling reveals that these sites display cell-to-cell heterogeneity suggestive of lineage plasticity. In addition, we find that aberrant enrichment of H3K4me3 in gene bodies is sensitive to Menin inhibitors, demonstrating the utility of automated chromatin profiling for identifying therapeutic vulnerabilities. Thus, integration of automated and single-cell CUT&Tag can uncover epigenomic heterogeneity within patient samples and predict sensitivity to therapeutic agents.

Adult Acute Myeloid Leukemia with the KMT2A-Mixed Lineage Leukemia T10 Fusion: An Analysis of 10 Cases Showed Common Features and Frequent Mutations in the RAS Signaling Pathway

Mixed lineage leukemia (<i>MLL) T10</i> is a relatively rare partner for the <i>KMT2A</i> lysine (K)-specific methyltransferase 2A gene. The common features and coexisting mutations of acute myeloid leukemia (AML) patients with <i>KMT2A-MLLT10</i> remain unknown. In this study, 10 adult AML patients with <i>KMT2A-MLLT10</i> fusions were picked up from 496 AML patients by using RT-polymerase chain reaction (PCR) and/or fluorescence in situ hybridization, and then screened for mutations in the 49 genes panel with next-generation sequencing and PCR, followed by direct Sanger sequencing. Of the 10 unique individuals identified, 6 were male and 4 were female (M:F ratio, 1.5:1) with ages ranging from 19 to 52 years (median 39.5 years). Most (90%, 9/10) patients with <i>KMT2A-MLLT10</i> were accompanied by additional mutations. Twelve mutated genes were detected, averaging 2.1 mutations per patient (range, 0–4). The most frequently mutated gene was <i>NRAS</i> (<i>n</i> = 5). Clinical and laboratory data pointed to common features: French American British-M5 subtype (<i>n</i> = 7), a high rate of relapse, and biomarkers CD33 (<i>n</i> = 10), CD117 (<i>n</i> = 9), CD13 (<i>n</i> = 8), and CD64 (<i>n</i> = 8). Overall, most patients harbored at least one mutation. A high incidence of mutations affecting the RAS signaling pathway or RAS regulating components was found in 50% (5/10) patients. The overall survival is about 12.0 months. Allogeneic-hematopoietic stem cell transplantation trends to improve survival in selected patients.

Synthesis and Biological Activity of a Cytostatic Inhibitor of MLLr Leukemia Targeting the DOT1L Protein

Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.

Multi-omics of MLL-rearranged B-cell precursor acute lymphoblastic leukemia reveals broad glycoproteome remodelling

B-cell precursor acute lymphoblastic leukemia (pre-B ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are needed. To identify changes that could distinguish leukemic from normal precursor B-cells, we performed integrated multi-omics on primary patient samples. MLL-r cells feature an extensive remodelling of their glycoprotein glycocalyx, with increased Core 2 type O-glycans and complex N-glycans as well as significant changes in sialylation and fucosylation levels. Notably, a glycosaminoglycan remodelling from chondroitin sulfate to heparan sulfate was also observed. A survival screen to determine if glycan remodelling enzymes are redundant identified MGAT1 and NGLY1, components of the N-glycosylation/degradation pathway, as essential. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation, were also indispensable. Transcriptomics and proteomics further identified Fes and GALNT7-mediated glycosylation as possible therapeutic targets. Finally, next to well-known MLL-r pre-B ALL glycoprotein markers, our integrated multi-omics workflow identified previously unidentified diagnostic/therapeutic protein candidates.

The characterization of MCEF : an AFF transcription factor associated with acute lymphoglastic leukemia and HIV-1

Acute Lymphoblastic Leukemia (ALL) results from environmentally-triggered in utero translocations between the Mixed Lineage Leukemia (MLL) gene and partner genes. In the most frequent cases of ALL, this partner gene is one of the AF4 family (AFF) of transcription factors. The newest AFF member to be discovered and cloned is AFF-4/AF5q31/MCEF. MCEF interacts with a transcription factor necessary for transcription of HIV-1. In addition, evidence suggests that male knockout mice are azoospermic. Therefore, the characterization of MCEF is clinically and theoretically important. The purpose of my research was to further characterize MCEF. In this paper, I first review the AFF members, focusing on MCEF. I then show a series of experimental results addressing MCEF isoforms and HIV-1 repression domains, as well as the generation of anti-MCEF antisera. Finally, I highlight intriguing results with live virus replication assays that suggest how MCEF could be exploited as a therapeutic target for AIDS.

The characterization of MCEF : an AFF transcription factor associated with acute lymphoglastic leukemia and HIV-1

Acute Lymphoblastic Leukemia (ALL) results from environmentally-triggered in utero translocations between the Mixed Lineage Leukemia (MLL) gene and partner genes. In the most frequent cases of ALL, this partner gene is one of the AF4 family (AFF) of transcription factors. The newest AFF member to be discovered and cloned is AFF-4/AF5q31/MCEF. MCEF interacts with a transcription factor necessary for transcription of HIV-1. In addition, evidence suggests that male knockout mice are azoospermic. Therefore, the characterization of MCEF is clinically and theoretically important. The purpose of my research was to further characterize MCEF. In this paper, I first review the AFF members, focusing on MCEF. I then show a series of experimental results addressing MCEF isoforms and HIV-1 repression domains, as well as the generation of anti-MCEF antisera. Finally, I highlight intriguing results with live virus replication assays that suggest how MCEF could be exploited as a therapeutic target for AIDS.

Structure, function and inhibition of critical protein–protein interactions involving mixed lineage leukemia 1 and its fusion oncoproteins

Mixed lineage leukemia 1 (MLL1, also known as MLL or KMT2A) is an important transcription factor and histone-H3 lysine-4 (H3K4) methyltransferase. It is a master regulator for transcription of important genes (e.g., Hox genes) for embryonic development and hematopoiesis. However, it is largely dispensable in matured cells. Dysregulation of MLL1 leads to overexpression of certain Hox genes and eventually leukemia initiation. Chromosome translocations involving MLL1 cause ~ 75% of acute leukemia in infants and 5–10% in children and adults with a poor prognosis. Targeted therapeutics against oncogenic fusion MLL1 (onco-MLL1) are therefore needed. Onco-MLL1 consists of the N-terminal DNA-interacting domains of MLL1 fused with one of > 70 fusion partners, among which transcription cofactors AF4, AF9 and its paralog ENL, and ELL are the most frequent. Wild-type (WT)- and onco-MLL1 involve numerous protein–protein interactions (PPI), which play critical roles in regulating gene expression in normal physiology and leukemia. Moreover, WT-MLL1 has been found to be essential for MLL1-rearranged (MLL1-r) leukemia. Rigorous studies of such PPIs have been performed and much progress has been achieved in understanding their structures, structure–function relationships and the mechanisms for activating gene transcription as well as leukemic transformation. Inhibition of several critical PPIs by peptides, peptidomimetic or small-molecule compounds has been explored as a therapeutic approach for MLL1-r leukemia. This review summarizes the biological functions, biochemistry, structure and inhibition of the critical PPIs involving MLL1 and its fusion partner proteins. In addition, challenges and perspectives of drug discovery targeting these PPIs for the treatment of MLL1-r leukemia are discussed.