Abstract Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF‐E2‐related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT‐199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine‐increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase‐1 and NADP‐quinone oxidoreductase‐1. Venetoclax treatment also leads to dissociation of B‐cell lymphoma 2 from the Nrf2/Keap‐1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.
<p>Fig S1. SF3B1 mutant CLL samples had pervasive changes in 3’ splice site. Fig S2. Omics analyses identify widespread post-transcriptional upregulation of splicing factors in CLL. Fig S3. Abundance of spliceosome complexes and RNA-binding proteins are associated with clinical outcomes in CLL. Fig S4. METTL3 is consistently upregulated along with differential m6A modification on transcripts of RNA splicing process in CLL. Fig S5. KO or pharmacological inhibition of METTL3 impacts cell growth. Fig S6. KO or pharmacological inhibition of METTL3 impacts apoptosis, cell cycle, and splicing factor abundance. Fig S7. Splicing factors are either direct or indirect targets of METTL3. Fig S8. Validation of association between m6A and splicing factor abundance using dCasRx-METTL3.</p>
5'-Deoxy-5'-methylthioadenosine and 5'-deoxy-5'-methylthioinosine, which are metabolized to the methionine precursor, 5-methylthioribose-1-phosphate, by 5'-deoxy-5'-methylthioadenosine phosphorylase and purine nucleoside phosphorylase, respectively, can serve as sources of methionine for cultured HL-60 promyelocytic leukemia cells. CCRF-CEM T-cell leukemia cells, which lack 5'-deoxy-5'-methylthioadenosine phosphorylase, convert 5'-deoxy-5'-methylthioinosine (but not 5'-deoxy-5'-methylthioadenosine) to methionine; this conversion is blocked by purine nucleoside phosphorylase inhibitors. Therefore, the pathway for the conversion of 5-methylthioribose-1-phosphate to methionine is present in both human leukemic lines.
Abstract RNA splicing dysregulation is a hallmark of cancers and underlies the onset and progression of diseases. Chronic lymphocytic leukemia (CLL) is one of the most common adult leukemia in western countries. Spliceosome mutations occur in ~20% of patients. However, the mechanism for splicing defects in spliceosome unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis of primary CLL samples, we discovered proteins involved in RNA splicing are post-transcriptionally upregulated. Coupled with clinical annotation, we found spliceosome protein abundance is an independent risk factor and associated with poor prognosis. Splice variants found in CLL are highly overlapped with those driven by high spliceosome abundance but not splicing factor mutations, indicating high spliceosome abundance contributes to genetic lesion-independent splicing defects. To identify potential regulators for spliceosome, we proteome-widely analyzed the proteins that highly correlated with splicing factors expression. Analysis of 113 CLL samples has consistently identified METTL3 upregulation with positive correlation with 77.6% of detected splicing factors. METTL3 is an RNA methyltransferase that modifies N6-methyladenosine (m6A) on mRNA and regulates the translation of m6A-installed transcripts. m6A level on mRNA is increased in CLL cells with differential m6A highly enriched on splicing related transcripts. Moreover, high METTL3 expression in CLL is also associated with poor clinical outcomes. These results suggested that METTL3 translationally controls splicing factors through m6A and plays a role in CLL progression. Toward this end, we demonstrated that METTL3 is essential for CLL growth in both in vitro and in vivo studies. Knock out (KO) and pharmaceutical inhibition of METTL3 decreased cell growth and splicing factor expression. Overexpression of wildtype but not catalytic mutant METTL3 restored both defects in METTL3 KO cells, indicating that the regulation of splicing factor is m6A-dependent. To dissect the underlying mechanism, we performed an integrated Ribo-seq, RNA-seq, and MeRIP-seq on CLL cells with or without METTL3. KO of METTL3 decreased overall translation efficiency (TE) with RNA splicing as the most significantly affected pathway. Splicing factors with reduced TE displayed either hypo-m6A at stop codon region or hyper-m6A at CDS regions upon METTL3 KO, as direct or indirect targets of METTL3. Moreover, we found that m6A at stop codon and CDS regions regulates splicing factor translation via ribosome recycling and ribosome pausing, respectively. Taken together, our results uncovered a novel regulatory axis for METTL3 that controls splicing factor translation and contributes to CLL progression. Our study highlights a post-transcriptional layer of m6A modification as a major contributor to genetic lesion-independent splicing defects in CLL. Citation Format: Yiming Wu, Meiling Jin, Mike Fernandez, Kevyn Hart, Aijun Liao, Stacey M. Fernandes, Tinisha McDonald, Zhenhua Chen, Daniel Röth, Lucy Ghoda, Guido Marcucci, Markus Kalkum, Raju K. Pillai, Alexey V. Danilov, Jianjun Chen, Jennifer R. Brown, Steven T. Rosen, Tanya Siddiqi, Lili Wang. METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3482.
<p>Table S1 shows CLL sample information; Table S2 shows protein expression detected in CLL and normal B cells using TMT proteomics; Table S3 shows top 150 transcripts with highest m6A density in B cells; Table S4 shows sequences of shRNAs, sgRNAs, and primers used in our study; Table S5 shows cellular pathways enriched in our study.</p>
<p>Fig S1. SF3B1 mutant CLL samples had pervasive changes in 3’ splice site. Fig S2. Omics analyses identify widespread post-transcriptional upregulation of splicing factors in CLL. Fig S3. Abundance of spliceosome complexes and RNA-binding proteins are associated with clinical outcomes in CLL. Fig S4. METTL3 is consistently upregulated along with differential m6A modification on transcripts of RNA splicing process in CLL. Fig S5. KO or pharmacological inhibition of METTL3 impacts cell growth. Fig S6. KO or pharmacological inhibition of METTL3 impacts apoptosis, cell cycle, and splicing factor abundance. Fig S7. Splicing factors are either direct or indirect targets of METTL3. Fig S8. Validation of association between m6A and splicing factor abundance using dCasRx-METTL3.</p>
<p>Table S1 shows CLL sample information; Table S2 shows protein expression detected in CLL and normal B cells using TMT proteomics; Table S3 shows top 150 transcripts with highest m6A density in B cells; Table S4 shows sequences of shRNAs, sgRNAs, and primers used in our study; Table S5 shows cellular pathways enriched in our study.</p>
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