Evolved histone tail regulates 53BP1 recruitment at damaged chromatin.

The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.

Enhancer-activated RET confers protection against oxidative stress to KMT2A-rearranged acute myeloid leukemia.

Ectopic activation of rearranged during transfection (RET) has been reported to facilitate lineage differentiation and cell proliferation in different cytogenetic subtypes of acute myeloid leukemia (AML). Herein, we demonstrate that RET is significantly (p < 0.01) upregulated in AML subtypes containing rearrangements of the lysine methyltransferase 2A gene (KMT2A), commonly referred to as KMT2A-rearranged (KMT2A-r) AML. Integrating multi-epigenomics data, we show that the KMT2A-MLLT3 fusion induces the development of CCCTC-binding (CTCF)-guided de novo extrusion enhancer loop to upregulate RET expression in KMT2A-r AML. Based on the finding that RET expression is tightly correlated with the selective chromatin remodeler and mediator (MED) proteins, we used a small-molecule inhibitor having dual inhibition against RET and MED12-associated cyclin-dependent kinase 8 (CDK8) in KMT2A-r AML cells. Dual inhibition of RET and CDK8 restricted cell proliferation by producing multimodal oxidative stress responses in treated cells. Our data suggest that epigenetically enhanced RET protects KMT2A-r AML cells from oxidative stresses, which could be exploited as a potential therapeutic strategy.

Resveratrol induces major histocompatibility complex class I antigen presentation in a STING-dependent and independent manner in melanoma.

Immune checkpoint inhibitor therapy has drastically improved outcomes in treating cancer, particularly in melanoma. However, half of melanoma patients are resistant to treatment. One mechanism used by tumor cells to evade immune attack is to down-regulate major histocompatibility complex (MHC) class I molecules, which are required for cytotoxic CD8 T-cells to eliminate cancer cells. To increase immunotherapeutic efficacy, it is critical to identify how to restore MHC-I expression on cancer cells so that tumor antigens are presented. We found that resveratrol elevated MHC-I expression, so that tumor antigens are presented to cytotoxic CD8 T-cell killing. Through proteomic interrogation, we identified the STING pathway as a potential mechanism of action. Further studies indicated that resveratrol-mediated regulation of STING induced MHC-I expression potentially through both interferon-independent and dependent pathways. Our results have indicated the potential of STING to induce MHC-I expression independent of interferon signaling, broadening the potential of STING modulation as a tool to improve immune checkpoint blockade.

Chemical inhibition of DNA-PKcs impairs the activation and cytotoxicity of CD4+ helper and CD8+ effector T cells.

Modulation of T cell activity is an effective strategy for the treatment of autoimmune diseases, immune-related disorders and cancer. This highlights a critical need for the identification of proteins that regulate T cell function. The kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is emerging as a potent regulator of the immune system, spurring interest in its use as a therapeutic target. In murine models of immune-related diseases including asthma and rheumatoid arthritis, treatment with small-molecule DNA-PKcs inhibitors decreased the disease severity. Additionally, DNA-PKcs inhibitors reduced T cell-mediated graft rejection in a murine allogenic skin graft model. These in vivo studies suggest the use of DNA-PKcs inhibitors as immunotherapy for autoimmune and T cell-mediated disorders. In this study, we sought to characterize further the effects of DNA-PKcs inhibitors on T cells to better understand their clinical potential. We determined that inhibition of DNA-PKcs using inhibitor NU7441 and the inhibitors currently in clinical trials for cancer therapy, M3184 and AZD7648, abrogated the activation of murine and human CD4+ and CD8+ T cells as evidenced by the reduced expression of the activation markers CD69 and CD25. Furthermore, inhibition of DNA-PKcs impeded metabolic pathways and the proliferation of activated T cells. This reduced the ability of OTI-CD8+ T cells to kill cancer cells and the expression of IFNγ and cytotoxic genes. These results highlight a critical role for DNA-PKcs in T cells and validate future studies using DNA-PKcs inhibitors as immune modulation therapy for the treatment of immune-related diseases.

CRISPR/dCas9-KRAB-Mediated Suppression of S100b Restores p53-Mediated Apoptosis in Melanoma Cells.

Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of S100B is poorly correlated (R < 0.3; p > 0.05) to alterations in S100B copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in S100B upregulation in melanoma, we stably suppressed S100b (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Krüppel-associated box (KRAB). Selective combination of S100b-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of S100b in murine B16 melanoma cells without noticeable off-target effects. S100b suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to S100b suppression. S100b-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of S100b therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma.

CAR-T Therapies in Solid Tumors: Opportunities and Challenges.

PURPOSE OF REVIEW: This review will discuss the challenges facing chimeric antigen receptor (CAR)-T cell application for solid tumors and opportunities to overcome these obstacles. In addition, this review will examine therapies that are in development for pediatric solid tumors. RECENT FINDINGS: The similar success of CAR-T cell treatment for hematological malignancies has not been observed in solid tumors because of the hostile tumor microenvironment and tumor heterogeneity. Most strategies developed to combat these limitations emphasize combinatorial techniques that still require further testing. Preliminary results of multiple clinical trials, including GD2- and HER2-CAR-T cells, are encouraging but must be reproduced and validated on a larger scale. CAR-T cell application in solid tumors remains challenging, and most research is in development. Several clinical trials are ongoing for pediatric solid tumors. Early results are promising but demonstrate the need for CAR-T cell modification to prevent tumor recurrence.

Epigenetic Control of Cdkn2a.Arf Protects Tumor-Infiltrating Lymphocytes from Metabolic Exhaustion.

T-cell exhaustion in cancer is linked to poor clinical outcomes, where evidence suggests T-cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Environmental stress produces epigenome remodeling events within TIL resulting from loss of the histone methyltransferase EZH2. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TIL. A multiomics approach revealed a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo. Our data suggest that manipulation of T-cell EZH2 within the context of cellular therapies may yield lymphocytes that are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults. SIGNIFICANCE: These findings demonstrate that manipulation of T-cell EZH2 in cellular therapies may yield cellular products able to withstand solid tumor metabolic-deficient environments. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4707/F1.large.jpg.

Delivery of phosphatidylethanolamine blunts stress in hepatoma cells exposed to elevated palmitate by targeting the endoplasmic reticulum.

Genetic obesity increases in liver phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio, inducing endoplasmic reticulum (ER) stress without concomitant increase of ER chaperones. Here, it is found that exposing mice to a palm oil-based high fat (HF) diet induced obesity, loss of liver PE, and loss of the ER chaperone Grp78/BiP in pericentral hepatocytes. In Hepa1-6 cells treated with elevated concentration of palmitate to model lipid stress, Grp78/BiP mRNA was increased, indicating onset of stress-induced Unfolded Protein Response (UPR), but Grp78/BiP protein abundance was nevertheless decreased. Exposure to elevated palmitate also induced in hepatoma cells decreased membrane glycosylation, nuclear translocation of pro-apoptotic C/EBP-homologous-protein-10 (CHOP), expansion of ER-derived quality control compartment (ERQC), loss of mitochondrial membrane potential (MMP), and decreased oxidative phosphorylation. When PE was delivered to Hepa1-6 cells exposed to elevated palmitate, effects by elevated palmitate to decrease Grp78/BiP protein abundance and suppress membrane glycosylation were blunted. Delivery of PE to Hepa1-6 cells treated with elevated palmitate also blunted expansion of ERQC, decreased nuclear translocation of CHOP and lowered abundance of reactive oxygen species (ROS). Instead, delivery of the chemical chaperone 4-phenyl-butyrate (PBA) to Hepa1-6 cells treated with elevated palmitate, while increasing abundance of Grp78/BiP protein and restoring membrane glycosylation, also increased ERQC, expression and nuclear translocation of CHOP, non-mitochondrial oxygen consumption, and generation of ROS. Data indicate that delivery of PE to hepatoma cells under lipid stress recovers cell function by targeting the secretory pathway and by blunting pro-apoptotic branches of the UPR.

Histone Modifications as Biomarkers for Immunotherapy.

Recent advances in immunotherapy have revolutionized the treatment of certain cancers. Some patients show a durable response to these immunotherapies, while others show little benefit or develop resistance. Identification of biomarkers to predict responsiveness will be helpful for informing treatment strategies; and would furthermore lead to the identification of molecular pathways dysregulated in nonresponding patients that could be targeted for therapeutic development. Pathways of epigenetic modification, such as histone posttranslational modifications (PTMs), have been shown to be dysregulated in certain cancer and immune cells. Histones are abundant cellular proteins readily assayed with high-throughput technologies, making them attractive targets as biomarkers. We explore promising advancements for using histone PTMs as immunotherapy responsiveness biomarkers in both cancer and immune cells, and provide a methodological workflow for assaying histone PTMs in relevant samples.

PHF19 promotes multiple myeloma tumorigenicity through PRC2 activation and broad H3K27me3 domain formation.

Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an "onco"-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.

Effect of Sulforaphane and 5-Aza-2'-Deoxycytidine on Melanoma Cell Growth.

Background: UV exposure-induced oxidative stress is implicated as a driving mechanism for melanoma. Increased oxidative stress results in DNA damage and epigenetic dysregulation. Accordingly, we explored whether a low dose of the antioxidant sulforaphane (SFN) in combination with the epigenetic drug 5-aza-2'-deoxycytidine (DAC) could slow melanoma cell growth. SFN is a natural bioactivated product of the cruciferous family, while DAC is a DNA methyltransferase inhibitor. Methods: Melanoma cell growth characteristics, gene transcription profiles, and histone epigenetic modifications were measured after single and combination treatments with SFN and DAC. Results: We detected melanoma cell growth inhibition and specific changes in gene expression profiles upon combinational treatments with SFN and DAC, while no significant alterations in histone epigenetic modifications were observed. Dysregulated gene transcription of a key immunoregulator cytokine-C-C motif ligand 5 (CCL-5)-was validated. Conclusions: These results indicate a potential combinatorial effect of a dietary antioxidant and an FDA-approved epigenetic drug in controlling melanoma cell growth.

Discovery of Potent Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors That Demonstrate in Vivo Activity in Mouse Xenograft Models of Human Cancer.

Overexpression of myeloid cell leukemia-1 (Mcl-1) in cancers correlates with high tumor grade and poor survival. Additionally, Mcl-1 drives intrinsic and acquired resistance to many cancer therapeutics, including B cell lymphoma 2 family inhibitors, proteasome inhibitors, and antitubulins. Therefore, Mcl-1 inhibition could serve as a strategy to target cancers that require Mcl-1 to evade apoptosis. Herein, we describe the use of structure-based design to discover a novel compound (42) that robustly and specifically inhibits Mcl-1 in cell culture and animal xenograft models. Compound 42 binds to Mcl-1 with picomolar affinity and inhibited growth of Mcl-1-dependent tumor cell lines in the nanomolar range. Compound 42 also inhibited the growth of hematological and triple negative breast cancer xenografts at well-tolerated doses. These findings highlight the use of structure-based design to identify small molecule Mcl-1 inhibitors and support the use of 42 as a potential treatment strategy to block Mcl-1 activity and induce apoptosis in Mcl-1-dependent cancers.

Loss of E-Cadherin Inhibits CD103 Antitumor Activity and Reduces Checkpoint Blockade Responsiveness in Melanoma.

Identifying controlling features of responsiveness to checkpoint blockade therapies is an urgent goal in oncology research. Our group and others have previously shown melanoma tumors resistant to checkpoint blockade display features of mesenchymal transition, including E-cadherin loss. Here, we present the first in vivo evidence that E-cadherin from tumor cells facilitate immune attack, using a B16F10 melanoma mouse model in which E-cadherin is exogenously expressed (B16.Ecad). We find, compared with vector control, B16.Ecad exhibits delayed tumor growth, reduced metastatic potential, and increased overall survival in vivo. Transplantation of B16.Ecad into Rag1-/- and CD103-/- mice abrogated the tumor growth delay. This indicates the anti-melanoma response against B16.Ecad is both immune and CD103+ mediated. Moreover, B16.Ecad showed increased responsiveness to combination immune checkpoint blockade (ICB) compared with vector control. This work establishes a rationale for ICB responses observed in high E-cadherin-expressing tumors and suggests therapeutic advancement through amplifying CD103+ immune cell subsets.Significance: These findings identify the mechanism behind checkpoint blockade resistance observed in melanoma that has undergone mesenchymal transition and suggest activation of CD103+ immune cells as a therapeutic strategy against other E-cadherin-expressing malignancies.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/6/1113/F1.large.jpg.

Indicators of responsiveness to immune checkpoint inhibitors.

Modulation of the immune system can produce anti-tumor responses in various cancer types, including melanoma. Recently, immune checkpoint inhibitors (ICI), in single agent and combination regimens, have produced durable and long-lasting clinical responses in a subset of metastatic melanoma patients. These monoclonal antibodies, developed against CTLA-4 and PD-1, block immune-inhibitory receptors on activated T-cells, amplifying the immune response. However, even when using anti-CTLA-4 and anti-PD-1 in combination, approximately half of patients exhibit innate resistance and suffer from disease progression. Currently, it is impossible to predict therapeutic response. Here, we report the first proteomic and histone epigenetic analysis of patient metastatic melanoma tumors taken prior to checkpoint blockade, which revealed biological signatures that can stratify patients as responders or non-responders. Furthermore, our findings provide evidence of mesenchymal transition, a known mechanism of immune-escape, in non-responding melanoma tumors. We identified elevated histone H3 lysine (27) trimethylation (H3K27me3), decreased E-cadherin, and other protein features indicating a more mesenchymal phenotype in non-responding tumors. Our results have implications for checkpoint inhibitor therapy as patient specific responsiveness can be predicted through readily assayable proteins and histone epigenetic marks, and pathways activated in non-responders have been identified for therapeutic development to enhance responsiveness.

Discovery and biological characterization of potent myeloid cell leukemia-1 inhibitors.

Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl-1 inhibitors. PDB ID CODES: Comp. 2: 5IEZ; Comp. 5: 5IF4.

Defining specificity and on-target activity of BH3-mimetics using engineered B-ALL cell lines.

One of the hallmarks of cancer is a resistance to the induction of programmed cell death that is mediated by selection of cells with elevated expression of anti-apoptotic members of the BCL-2 family. To counter this resistance, new therapeutic agents known as BH3-mimetic small molecules are in development with the goal of antagonizing the function of anti-apoptotic molecules and promoting the induction of apoptosis. To facilitate the testing and modeling of BH3-mimetic agents, we have developed a powerful system for evaluation and screening of agents both in culture and in immune competent animal models by engineering mouse leukemic cells and re-programming them to be dependent on exogenously expressed human anti-apoptotic BCL-2 family members. Here we demonstrate that this panel of cell lines can determine the specificity of BH3-mimetics to individual anti-apoptotic BCL-2 family members (BCL-2, BCL-XL, BCL-W, BFL-1, and MCL-1), demonstrate whether cell death is due to the induction of apoptosis (BAX and BAK-dependent), and faithfully assess the efficacy of BH3-mimetic small molecules in pre-clinical mouse models. These cells represent a robust and valuable pre-clinical screening tool for validating the efficacy, selectivity, and on-target action of BH3-mimetic agents.

Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways.

Nonresolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for antitumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor-cell subset, but not the granulocytic subset, requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, whereas their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival, and function of myeloid suppressor cells.

Requirement for antiapoptotic MCL-1 in the survival of BCR-ABL B-lineage acute lymphoblastic leukemia.

The response of Philadelphia chromosome (Ph(+)) acute lymphoblastic leukemia (ALL) to treatment by BCR-ABL tyrosine kinase inhibitors (TKIs) has been disappointing, often resulting in short remissions typified by rapid outgrowth of drug-resistant clones. Therefore, new treatments are needed to improve outcomes for Ph(+) ALL patients. In a mouse model of Ph(+) B-lineage ALL, MCL-1 expression is dysregulated by the BCR-ABL oncofusion protein, and TKI treatment results in loss of MCL-1 expression prior to the induction of apoptosis, suggesting that MCL-1 may be an essential prosurvival molecule. To test this hypothesis, we developed a mouse model in which conditional allele(s) of Mcl-1 can be deleted either during leukemia transformation or later after the establishment of leukemia. We report that endogenous MCL-1's antiapoptotic activity promotes survival during BCR-ABL transformation and in established BCR-ABL(+) leukemia. This requirement for MCL-1 can be overcome by overexpression of other antiapoptotic molecules. We further demonstrate that strategies to inhibit MCL-1 expression potentiate the proapoptotic action of BCL-2 inhibitors in both mouse and human BCR-ABL(+) leukemia cell lines. Thus, strategies focused on antagonizing MCL-1 function and expression would be predicted to be effective therapeutic strategies.

Deletion of MCL-1 causes lethal cardiac failure and mitochondrial dysfunction.

MCL-1 is an essential BCL-2 family member that promotes the survival of multiple cellular lineages, but its role in cardiac muscle has remained unclear. Here, we report that cardiac-specific ablation of Mcl-1 results in a rapidly fatal, dilated cardiomyopathy manifested by a loss of cardiac contractility, abnormal mitochondria ultrastructure, and defective mitochondrial respiration. Strikingly, genetic ablation of both proapoptotic effectors (Bax and Bak) could largely rescue the lethality and impaired cardiac function induced by Mcl-1 deletion. However, while the overt consequences of Mcl-1 loss were obviated by combining with the loss of Bax and Bak, mitochondria from the Mcl-1-, Bax-, and Bak-deficient hearts still revealed mitochondrial ultrastructural abnormalities and displayed deficient mitochondrial respiration. Together, these data indicate that merely blocking cell death is insufficient to completely overcome the need for MCL-1 function in cardiomyocytes and suggest that in cardiac muscle, MCL-1 also facilitates normal mitochondrial function. These findings are important, as specific MCL-1-inhibiting therapeutics are being proposed to treat cancer cells and may result in unexpected cardiac toxicity.

A competitive stapled peptide screen identifies a selective small molecule that overcomes MCL-1-dependent leukemia cell survival.

Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochemically by an antiapoptotic surface groove that neutralizes the proapoptotic BH3 α helix of death proteins. Antiapoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Although targeting the BCL-2 antiapoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of molecules tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor molecule that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochemical lock-hold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence.