Massively parallel assessment of human variants with base editor screens.

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.

ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance.

Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response.

Anti-apoptotic MCL-1 promotes long-chain fatty acid oxidation through interaction with ACSL1.

MCL-1 is essential for promoting the survival of many normal cell lineages and confers survival and chemoresistance in cancer. Beyond apoptosis regulation, MCL-1 has been linked to modulating mitochondrial metabolism, but the mechanism(s) by which it does so are unclear. Here, we show in tissues and cells that MCL-1 supports essential steps in long-chain (but not short-chain) fatty acid ß-oxidation (FAO) through its binding to specific long-chain acyl-coenzyme A (CoA) synthetases of the ACSL family. ACSL1 binds to the BH3-binding hydrophobic groove of MCL-1 through a non-conventional BH3-domain. Perturbation of this interaction, via genetic loss of Mcl1, mutagenesis, or use of selective BH3-mimetic MCL-1 inhibitors, represses long-chain FAO in cells and in mouse livers and hearts. Our findings reveal how anti-apoptotic MCL-1 facilitates mitochondrial metabolism and indicate that disruption of this function may be associated with unanticipated cardiac toxicities of MCL-1 inhibitors in clinical trials.

Complementarity and redundancy of IL-22-producing innate lymphoid cells.

Intestinal T cells and group 3 innate lymphoid cells (ILC3 cells) control the composition of the microbiota and gut immune responses. Within the gut, ILC3 subsets coexist that either express or lack the natural cytoxicity receptor (NCR) NKp46. We identified here the transcriptional signature associated with the transcription factor T-bet-dependent differentiation of NCR(-) ILC3 cells into NCR(+) ILC3 cells. Contrary to the prevailing view, we found by conditional deletion of the key ILC3 genes Stat3, Il22, Tbx21 and Mcl1 that NCR(+) ILC3 cells were redundant for the control of mouse colonic infection with Citrobacter rodentium in the presence of T cells. However, NCR(+) ILC3 cells were essential for cecal homeostasis. Our data show that interplay between intestinal ILC3 cells and adaptive lymphocytes results in robust complementary failsafe mechanisms that ensure gut homeostasis.

Genes with differential expression across ancestries are enriched in ancestry-specific disease effects likely due to gene-by-environment interactions.

Multi-ancestry genome-wide association studies (GWASs) have highlighted the existence of variants with ancestry-specific effect sizes. Understanding where and why these ancestry-specific effects occur is fundamental to understanding the genetic basis of human diseases and complex traits. Here, we characterized genes differentially expressed across ancestries (ancDE genes) at the cell-type level by leveraging single-cell RNA-sequencing data in peripheral blood mononuclear cells for 21 individuals with East Asian (EAS) ancestry and 23 individuals with European (EUR) ancestry (172,385 cells); then, we tested whether variants surrounding those genes were enriched in disease variants with ancestry-specific effect sizes by leveraging ancestry-matched GWASs of 31 diseases and complex traits (average n ∼ 90,000 and ∼ 267,000 in EAS and EUR, respectively). We observed that ancDE genes tended to be cell-type specific and enriched in genes interacting with the environment and in variants with ancestry-specific disease effect sizes, which suggests cell-type-specific, gene-by-environment interactions shared between regulatory and disease architectures. Finally, we illustrated how different environments might have led to ancestry-specific myeloid cell leukemia 1 (MCL1) expression in B cells and ancestry-specific allele effect sizes in lymphocyte count GWASs for variants surrounding MCL1. Our results imply that large single-cell and GWAS datasets from diverse ancestries are required to improve our understanding of human diseases.

RVFV virulence factor NSs triggers the mitochondrial MCL-1-BAK axis to activate pathogenic NLRP3 pyroptosis.

Infection of Rift Valley fever virus (RVFV), a highly pathogenic mosquito-borne zoonotic virus, triggers severe inflammatory pathogenesis but the underlying mechanism of inflammation activation is currently unclear. Here, we report that the non-structural protein NSs of RVFV triggers mitochondrial damage to activate the NLRP3 inflammasome leading to viral pathogenesis in vivo. It is found that the host transcription inhibition effect of NSs causes rapid down-regulation of myeloid cell leukemia-1(MCL-1), a pro-survival member of the Bcl-2 (B-cell lymphoma protein 2) protein family. MCL-1 down-regulation led to BAK activation in the mitochondria, which triggered mtROS production and release of oxidized mitochondrial DNA (ox-mtDNA) into the cytosol. Cytosolic ox-mtDNA binds and activates the NLRP3 inflammasome triggering NLRP3-GSDMD pyroptosis in RVFV infected cells. A NSs mutant virus (RVFV-NSsRM) that is compromised in inducing transcription inhibition did not trigger MCL-1 down-regulation nor NLRP3-GSDMD pyroptosis. RVFV infection of the Nlrp3-/- mouse model demonstrated that the RVFV-triggered NLRP3 pyroptosis contributed to RVFV inflammatory pathogenesis and fatal infection in vivo. Infection with the RVFV-NSsRM mutant virus similarly showed alleviated inflammatory pathogenesis and reduced fatality rate. Taken together, these results revealed a mechanism by which a virulence factor activates the mitochondrial MCL-1-BAK axis through inducing host transcription inhibition to trigger NLRP3-dependent inflammatory pathogenesis.

A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics.

ABSTRACT: To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.

Dynamic Regulation of Long-Chain Fatty Acid Oxidation by a Noncanonical Interaction between the MCL-1 BH3 Helix and VLCAD.

MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid ß-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1Matrix) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1Matrix alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.

Novel markers of MCL1 inhibitor sensitivity in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer sub-type with limited treatment options and poor prognosis. Currently, standard treatments for TNBC include surgery, chemotherapy, and anti-PDL1 therapy. These therapies have limited efficacy in advanced stages. Myeloid-cell leukemia 1 (MCL1) is an anti-apoptotic BCL2 family protein. High expression of MCL1 contributes to chemotherapy resistance and is associated with a worse prognosis in TNBC. MCL1 inhibitors are in clinical trials for TNBC, but response rates to these inhibitors can vary and predictive markers are lacking. Currently, we identified a 4-member (AXL, ETS1, IL6, EFEMP1) gene signature (GS) that predicts MCL1 inhibitor sensitivity in TNBC cells. Factors encoded by these genes regulate signaling pathways to promote MCL1 inhibitor resistance. Small molecule inhibitors of the GS factors can overcome resistance and sensitize otherwise resistant TNBC cells to MCL1 inhibitor treatment. These findings offer insights into potential therapeutic strategies and tumor stratification for MCL1 inhibitor use in TNBC.

Genetic events associated with venetoclax resistance in CLL identified by whole-exome sequencing of patient samples.

Although BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported though remain poorly understood. Here, we analyze longitudinal tumor samples from 11 patients with disease progression while receiving venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at the posttreatment time point. We found the previously described acquired BCL2-G101V mutation in only 4 of 11 patients, with 2 patients showing a very low variant allele fraction (0.03%-4.68%). Whole-exome sequencing revealed acquired loss(8p) in 4 of 11 patients, of which 2 patients also had gain (1q21.2-21.3) in the same cells affecting the MCL1 gene. In vitro experiments showed that CLL cells from the 4 patients with loss(8p) were more resistant to venetoclax than cells from those without it, with the cells from 2 patients also carrying gain (1q21.2-21.3) showing increased sensitivity to MCL1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to the combination of MCL1 inhibitor and venetoclax. Differential gene expression analysis comparing bulk RNA sequencing data from pretreatment and progression time points of all patients showed upregulation of proliferation, B-cell receptor (BCR), and NF-κB gene sets including MAPK genes. Cells from progression time points demonstrated upregulation of surface immunoglobulin M and higher pERK levels compared with those from the preprogression time point, suggesting an upregulation of BCR signaling that activates the MAPK pathway. Overall, our data suggest several mechanisms of acquired resistance to venetoclax in CLL that could pave the way for rationally designed combination treatments for patients with venetoclax-resistant CLL.

Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia.

Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL-selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

TCEB3 initiates ovarian cancer apoptosis by mediating ubiquitination and degradation of MCL-1.

Platinum resistance remains a major contributor to the poor prognosis of ovarian cancer. Anti-apoptotic protein myeloid cell leukemia-1 (MCL-1) has emerged as a promising target for overcoming drug resistance, but different cancer cells utilize distinct protein degradation pathways to alter MCL-1 level. We systematically investigated E3 ligases to identify novel candidates that mediate platinum resistance in ovarian cancer. Transcription Elongation Factor B (TCEB3) has been identified as a novel E3 ligase recognition subunit that targets MCL-1 in the cytoplasm during platinum treatment other than its traditional function of targeting the Pol II in the nuclear compartment. TCEB3 expression is downregulated in platinum-resistant cell lines and this low expression is associated with poor prognosis. The ubiquitination of MCL-1 induced by TCEB3 leads to cell death in ovarian cancer. Moreover, platinum treatment increased the cytoplasm proportion of TCEB3, and the cytoplasm localization of TCEB3 is important for its targeting of MCL-1. This study emphasizes the dual function of TCEB3 in homeostasis maintenance and in cell fate determination under different conditions, and provides a new insight into drug resistance in ovarian cancer.

Clitocine enhances the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis.

Chemotherapy resistance to colon cancer is an unavoidable obstacle in the clinical management of the disease. Clitocine, an adenosine analog, played a significant role in the chemosensitivity of human colon cancer cells by promoting myeloid cell leukemia 1 (MCL-1) protein degradation. However, the detailed mechanism remains to be further elucidated. We found that clitocine upregulates the expression of F-box and WD repeat domain containing 7 (FBXW7), a ubiquitin ligase involved in the MCL-1 degradation. Transcriptome sequencing analysis revealed that clitocine significantly inhibits the cyclic adenosine monophosphate (cAMP) and extracellular regulated protein kinases (ERK) downstream signaling pathways in colon cancer cells, thereby enhancing FBXW7 expression and subsequently promoting the ubiquitination degradation of MCL-1 protein. We verified that clitocine regulated intracellular cAMP levels by competitive binding with the adenosine receptor A2B. A molecular docking assay also verified the binding relationship. By decreasing intracellular cAMP levels, clitocine blocks the activation of downstream signaling pathways, which ultimately enhances the drug sensitivity of colon cancer cells through increased FBXW7 expression due to the inhibition of its promoter DNA methylation. Both knockout of the adenosine receptor A2B and Br-cAMP treatment can effectively attenuate the function of clitocine in vitro and in vivo. This study clarified that clitocine enhanced the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis, providing further knowledge of the clinical application for clitocine.NEW & NOTEWORTHY Our study found that clitocine enhances the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis.

Polypyrimidine tract binding protein 1 (PTBP1) contains a novel regulatory sequence, the rBH3, that binds the prosurvival protein MCL1.

The maturation of RNA from its nascent transcription to ultimate utilization (e.g., translation, miR-mediated RNA silencing, etc.) involves an intricately coordinated series of biochemical reactions regulated by RNA-binding proteins (RBPs). Over the past several decades, there has been extensive effort to elucidate the biological factors that control specificity and selectivity of RNA target binding and downstream function. Polypyrimidine tract binding protein 1 (PTBP1) is an RBP that is involved in all steps of RNA maturation and serves as a key regulator of alternative splicing, and therefore, understanding its regulation is of critical biologic importance. While several mechanisms of RBP specificity have been proposed (e.g., cell-specific expression of RBPs and secondary structure of target RNA), recently, protein-protein interactions with individual domains of RBPs have been suggested to be important determinants of downstream function. Here, we demonstrate a novel binding interaction between the first RNA recognition motif 1 (RRM1) of PTBP1 and the prosurvival protein myeloid cell leukemia-1 (MCL1). Using both in silico and in vitro analyses, we demonstrate that MCL1 binds a novel regulatory sequence on RRM1. NMR spectroscopy reveals that this interaction allosterically perturbs key residues in the RNA-binding interface of RRM1 and negatively impacts RRM1 association with target RNA. Furthermore, pulldown of MCL1 by endogenous PTBP1 verifies that these proteins interact in an endogenous cellular environment, establishing the biological relevance of this binding event. Overall, our findings suggest a novel mechanism of regulation of PTBP1 in which a protein-protein interaction with a single RRM can impact RNA association.

Initial AXL and MCL-1 inhibition contributes to abolishing lazertinib tolerance in EGFR-mutant lung cancer cells.

Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells.

Synergistic cytotoxicity of decitabine and YM155 in leukemia cells through upregulation of SLC35F2 and suppression of MCL1 and survivin expression.

The hypomethylation agent decitabine (DAC), in combination with other apoptosis inducers, is considered a potential modality for cancer treatment. We investigated the mechanism underlying the combined cytotoxicity of DAC and YM155 in acute myeloid leukemia (AML) cells because of increasing evidence that YM155 induces apoptosis in cancer cells. Co-administration of DAC and YM155 resulted in synergistic cytotoxicity in AML U937 cells, which was characterized by the induction of apoptosis, NOXA-dependent degradation of MCL1 and survivin, and depolarization of mitochondria. Restoration of MCL1 or survivin expression attenuated DAC/YM155-induced U937 cell death. DAC initiated AKT and p38 MAPK phosphorylation in a Ca2+/ROS-dependent manner, thereby promoting autophagy-mediated degradation of ß-TrCP mRNA, leading to increased Sp1 expression. DAC-induced Sp1 expression associated with Ten-eleven-translocation (TET) dioxygenases and p300 was used to upregulate the expression of SLC35F2. Simultaneously, the activation of p38 MAPK induced by DAC, promoted CREB-mediated NOXA expression, resulting in survivin and MCL1 degradation. The synergistic cytotoxicity of DAC and YM155 in U937 cells was dependent on elevated SLC35F2 expression. Additionally, YM155 facilitated DAC-induced degradation of MCL1 and survivin. A similar mechanism explained DAC/YM155-mediated cytotoxicity in AML HL-60 cells. Our data demonstrated that the synergistic cytotoxicity of DAC and YM155 in AML cell lines U937 and HL-60 is dependent on AKT- and p38 MAPK-mediated upregulation of SLC35F2 and p38 MAPK-mediated degradation of survivin and MCL1. This indicates that a treatment regimen that amalgamates YM155 and DAC may be beneficial for AML.

Regulatory axis of circular RNA DTNB, microRNA-485-5p, and myeloid cell leukemia 1 attenuates inflammation and apoptosis in caerulein-treated AR42J cells.

Acute pancreatitis (AP) is an inflammatory disease of the pancreas and the main cause of hospital admissions for gastrointestinal diseases. Here, the work studied the circular RNA DTNB/microRNA-485-5p/MCL1 axis in AP and hoped to unravel the related mechanism. Caerulein exposure replicated an AP model in AR42J cells, and caerulein-mediated expression of circDTNB, miR-485-5p, and MCL1 was recorded. After exposure, cells were intervened with transfection plasmids and tested for LDH release, apoptosis, and inflammation. To determine the interwork of circDTNB, miR-485-5p, and MCL1, prediction results and verification experiments were conducted. Caerulein exposure reduced circDTNB and MCL1, while elevated miR-485-5p levels in AR42J cells. Upregulating circDTNB protected AR42J cells from caerulein-induced LDH cytotoxicity, apoptosis, and inflammation, but circDTNB upregulation-induced protections could be muffled by inhibiting MCL1. On the contrary, downregulating circDTNB further damaged AR42J cells under caerulein exposure, however, this phenomenon could be partially rescued after silencing miR-485-5p. miR-485-5p was mechanistically verified to be a target of circDTNB to mediate MCL1. Overall, the circDTNB/miR-485-5p/MCL1 axis protects inflammatory response and apoptosis in caerulein-exposed AR42J cells, promisingly identifying circDTNB as a novel molecule for AP treatment.

Venetoclax synergizes with gilteritinib in FLT3 wild-type high-risk acute myeloid leukemia by suppressing MCL-1.

BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML.

Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Electron transport chain and mTOR inhibition synergistically decrease CD40 signaling and counteract venetoclax resistance in chronic lymphocytic leukemia.

CD40 signaling upregulates BCL-XL and MCL-1 expression in the chronic lymphocytic leukemia (CLL) lymph node microenvironment, affording resistance to the BCL-2 inhibitor, venetoclax. Venetoclax resistance in the therapeutic setting and after long-term laboratory selection has been linked to metabolic alterations, but the underlying mechanism(s) are unknown. We aimed here to discover how CD40 stimulation as a model for tumor microenvironment-mediated metabolic changes, affects venetoclax sensitivity/resistance. CD40 stimulation increased oxidative phosphorylation and glycolysis, but only inhibition of oxidative phosphorylation countered venetoclax resistance. Furthermore, blocking mitochondrial import of pyruvate, glutamine or fatty acids affected CLL metabolism, but did not prevent CD40-mediated resistance to venetoclax. In contrast, inhibition of the electron transport chain (ETC) at complex I, III or V attenuated CLL activation and ATP production, and downregulated MCL-1 and BCL-XL, correlating with reduced CD40 surface expression. Moreover, ETC inhibition equaled mTOR1/2 but not mTOR1 inhibition alone for venetoclax resistance, and all three pathways were linked to control of general protein translation. In line with this, ETC plus mTOR inhibition synergistically counteracted venetoclax resistance. These findings link oxidative CLL metabolism to CD40 expression and cellular signaling, and may hold clinical potential.