Effect of methionine restriction on gene expression of non-KMT2A-rearranged NALM-6 and KMT2A-rearranged SEM BCP-ALL cell lines
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ABSTRACT: Current intensive chemotherapy regimens have improved overall survival in pediatric acute lymphoblastic leukemia but fail to cure some high-risk patient subgroups. While exploring amino acid depletion strategies, we observed that KMT2A-rearranged (KMT2A-r) leukemia, an aggressive subset with a dismal prognosis, is particularly vulnerable to perturbations of the methionine cycle. We demonstrated that this methionine dependency is primarily driven by an increased need for S-adenosylmethionine to maintain the hypermethylated state of KMT2A-r leukemias. Important pro-survival KMT2A-r target genes are repressed under methionine restriction, which, combined with other downstream metabolic changes, results in rapid cell death. FIDAS-5, an orally active methionine adenosyltransferase 2A (MAT2A) inhibitor, successfully impaired leukemia progression in patient-derived xenograft models, and a high-throughput drug screen revealed a strong synergy between MAT2A inhibition and histone deacetylase inhibitors. Our results identified the methionine cycle as a targetable vulnerability in KMT2A-r leukemia, which may increase the efficacy of epigenetic targeting agents.
Project description:Activating mutations in kinase/PI3K/RAS signaling pathways are common in acute leukemia with KMT2A rearrangements (KMT2A-R). These mutations are often subclonal and their biological impact remain unclear. Using a retroviral acute myeloid leukemia model, we demonstrate that NRASG12D, FLT3ITD, and FLT3N676K accelerates KMT2A-MLLT3 leukemia onset. Importantly, also the presence of subclonal FLT3N676K in KMT2A-R leukemic cells shorten disease latency, possibly by providing stimulatory factors such as Mif. Acquired de novo mutations in Braf, Cbl, Kras, and Ptpn11 were identified in KMT2A-MLLT3 driven leukemia and favored clonal expansion. KMT2A-MLLT3 leukemia with an activating mutation enforce Myc- and Myb transcriptional modules, whereas KMT2A-MLLT3 leukemias lacking activating mutations displayed upregulation of signal transduction pathways. Our results provide new insight into the biology of KMT2A-R leukemia and highlights the importance of activated signaling as a contributing driver in this disease.
Project description:MOLM-13 acute myeloid leukemia cells were treated with 3 µM FIDAS-5 methionine S-adenosyltransferase 2A (MAT2A) inhibitor or 0.1% DMSO as control for 48 hours
Project description:We obtained transcriptomic profiles in bovine morulae which had been in vitro treated with a methionine adenosyltransferase 2A (MAT2A) inhibitor from the 8-16 cell stage.
Project description:Bromo- and extra-terminal domain inhibitors (BETi) have exhibited therapeutic activities in many cancers, including KMT2A-rearranged (KMT2A-r) leukemia, in preclinical studies. However, the mechanisms controlling BETi response and resistance are poorly understood. We conducted genome-wide loss-of-function CRISPR screens using BETi-treated KMT2A-r cell lines. SPOP gene deficiency caused significant BETi resistance, which was further validated in cell line and xenograft models. In SPOP-knockout KMT2A-r leukemia cells, TRIM24 was identified as a SPOP substrate that mediates resistance to BETi. Additionally, proteomics analysis and a kinase-vulnerability CRISPR screen indicated that resistant cells are sensitive to GSK3 inhibition. Genetically perturbating TRIM24 or pharmaceutical inhibition of its downstream target GSK3 in SPOP-knockout cells reversed the BETi-resistance phenotype. A combination therapy regimen inhibiting both BET and GSK3 impeded leukemia progression in patient-derived xenografts in vivo. Our results revealed not only novel molecular mechanisms underlying BETi resistance but also a promising strategy for treating KMT2A-r leukemia.
Project description:Activating signaling mutations are common in acute leukemia with KMT2A (previously MLL) rearrangements (KMT2A-R). These mutations are often subclonal and their biological impact remains unclear. Using a retroviral acute myeloid mouse leukemia model, we demonstrate that FLT3ITD, FLT3N676K, and NRAS G12D accelerate KMT2A-MLLT3 leukemia onset. Subclonal FLT3N676K mutations also accelerate disease, possibly by providing stimulatory factors such as Mif. Acquired de novo mutations in Braf, Cbl, Kras, and Ptpn11 were identified in KMT2A-MLLT3 leukemia cells and favored clonal expansion. During clonal evolution, serial genetic changes at the KrasG12D locus was observed, consistent with a strong selective advantage of additional KrasG12D. KMT2A-MLLT3 leukemias with signaling mutations enforced Myc- and Myb transcriptional modules. Our results provide new insight into the biology of KMT2A-R leukemia with subclonal signaling mutations and highlights the importance of activated signaling as a contributing driver in this disease.
Project description:The KMT2A rearranged B lineage infant ALL cell line SEM was treated with the proteasome inhibitor bortezomib followed by a multiplexed mass spectrometry-based proteomic analysis at multiple time points (0, 6, 12, 16, and 20 hours) over 20 hours to further understand the cellular response of these cells to proteasome inhibition.
Project description:Acute myeloid leukemias (AML) patients bearing chromosomal rearrangements of KMT2A or MLL gene (KMT2A -r) have poor overall survival. Recently, compounds targeting the KMT2A fusion protein complex, such as DOT1L and Menin inhibitors, have shown promising pre-clinical efficacy. Yet, molecular regulators of the anti-tumor activities of these agents remain poorly studied. UTX is a histone H3K27 demethylase with recurrent loss-of-function mutations in human cancers including leukemia. UTX-null leukemia shows greater resistance to chemotherapy agents. However, the impact of UTX on drug resistance of KMT2A -r AML has not been explored. Through a epigenetic compound screen, we identified a unique role of UTX in the regulation of DOT1L and Menin therapies in KMT2A-r AML. Loss of UTX confers resistance to DOT1L and Menin inhibition in an MLL target gene-independent manner. Mechanistically, We show that UTX is required for activation of myeloid differentiation programs induced by DOT1L inhibition. We also revealed BCL2A1 as a target of UTX. Depletion of UTX increased vulnerability to BCL2 inhibitor venetoclax in vitro and in vivo, and combinational treatment of venetoclax could overcome the therapeutic resistance to DOT1L inhibition caused by UTX loss. Our study provides new insights into the role of UTX in therapeutic responses in KMT2A-r AML.
Project description:Acute myeloid leukemia (AML) is a disease with poor outcome but patients harbouring certain chromosomal rearrangements or complex karyotypes have particularly adverse prognosis. For these patients, targeted therapies have not yet made a significant clinical impact. To understand the molecular landscape of poor risk AML we profiled 55 poor risk AML patients using a multiomics approach that included transcriptomics (n=39), proteomics (n=55), phosphoproteomics (n=55) and an ex vivo drug sensitivity screening (482 compounds tested in at least 30 patients). We identified a phosphoproteomics signature that define two biologically distinct groups of KMT2A rearranged leukaemia, which we term MLLGA and MLLGB. MLLGA presented increased DOT1L phosphorylation, HOXA gene expression, CDK1 activity and phosphorylation of proteins involved in RNA metabolism, replication and DNA damage when compared to MLLGB and no KMT2A rearranged samples. MLLGA was particularly sensitive to 15 compounds including genotoxic drugs and inhibitors of mitotic kinases and IMPDH relative to other cases. The expression of IMPDH2 and multiple nucleolar proteins was higher in MLLGA and correlated with the response to IMPDH inhibition in KMT2A rearranged leukaemia, suggesting a role of the nucleolar activity in sensitivity to IMPDH inhibition. In summary, our multilayer molecular profiling of poor risk AML matched to the response to hundreds of compounds identified a phosphoproteomics signature that define two biologically and phenotypically distinct groups of KMT2A rearranged leukaemia. These data provide a rationale for the development of specific therapies for KMT2A subgroups characterised by the MLLGA phosphoproteomics signature identified in this study.
Project description:Background: Targeting the metabolic dependencies of acute myeloid leukemia (AML) cells is a promising therapeutical strategy. In particular, the cysteine and methionine metabolism pathway (C/M) is significantly altered in AML cells compared to healthy blood cells. Moreover, methionine has been identified as one of the dominant amino acid dependencies of AML cells. Methods: Through RNA-seq, we found that the two nucleoside analogs 8-chloro-adenosine (8CA) and 8-amino-adenosine (8AA) significantly suppress the C/M pathway in AML cells, and methionine-adenosyltransferase-2A (MAT2A) is one of most significantly downregulated genes. Additionally, mass spectrometry analysis revealed that Venetoclax (VEN), a BCL-2 inhibitor recently approved by the FDA for AML treatment, significantly decreases the intracellular level of methionine in AML cells. Based on these findings, we hypothesized that combining 8CA or 8AA with VEN can efficiently target the Methionine-MAT2A-S-adenosyl-methionine (SAM) axis in AML. Results: Our results demonstrate that VEN and 8CA/8AA synergistically decrease the SAM biosynthesis and effectively target AML cells both in vivo and in vitro. Conclusions: These findings suggest the promising potential of combining 8CA/8AA and VEN for AML treatment by inhibiting Methionine-MAT2A-SAM axis and provide a strong rationale for our recently activated clinical trial.