Identification of a novel form of caspase-independent cell death triggered by BH3-mimetics in diffuse large B-cell lymphoma cell lines.

BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.

Increased MCL1 dependency leads to new applications of BH3-mimetics in drug-resistant neuroblastoma.

BACKGROUND: Neuroblastoma is a paediatric cancer that is characterised by poor prognosis for chemoresistant disease, highlighting the need for better treatment options. Here, we asked whether BH3-mimetics inhibiting BCL2 proteins may eliminate chemoresistant neuroblastoma cells. METHODS: We utilised cisplatin-adapted neuroblastoma cell lines as well as patient tissues before and after relapse to study alterations of BCL2 proteins upon chemoresistance. RESULTS: In a direct comparison of cisplatin-resistant cells we identified a prominent loss of sensitivity to BCL2/BCL-XL inhibitors that is associated with an increase in MCL1 dependency and high expression of MCL1 in patient tumour tissues. Screening of FDA-approved anti-cancer drugs in chemoresistant cells identified therapeutics that may be beneficial in combination with the clinically tested BH3-mimetic ABT263, but no synergistic drug interactions with the selective MCL1 inhibitor S63845. Further exploration of potential treatment options for chemoresistant neuroblastoma identified immunotherapy based on NK cells as highly promising, since NK cells are able to efficiently kill both parental and chemoresistant cells. CONCLUSIONS: These data highlight that the application of BH3-mimetics may differ between first line treatment and relapsed disease. Combination of NK cell-based immunotherapy with BH3-mimetics may further increase killing of chemoresistant neuroblastoma, outlining a new treatment strategy for relapsed neuroblastoma.

Computational modeling of DLBCL predicts response to BH3-mimetics.

In healthy cells, pro- and anti-apoptotic BCL2 family and BH3-only proteins are expressed in a delicate equilibrium. In contrast, this homeostasis is frequently perturbed in cancer cells due to the overexpression of anti-apoptotic BCL2 family proteins. Variability in the expression and sequestration of these proteins in Diffuse Large B cell Lymphoma (DLBCL) likely contributes to variability in response to BH3-mimetics. Successful deployment of BH3-mimetics in DLBCL requires reliable predictions of which lymphoma cells will respond. Here we show that a computational systems biology approach enables accurate prediction of the sensitivity of DLBCL cells to BH3-mimetics. We found that fractional killing of DLBCL, can be explained by cell-to-cell variability in the molecular abundances of signaling proteins. Importantly, by combining protein interaction data with a knowledge of genetic lesions in DLBCL cells, our in silico models accurately predict in vitro response to BH3-mimetics. Furthermore, through virtual DLBCL cells we predict synergistic combinations of BH3-mimetics, which we then experimentally validated. These results show that computational systems biology models of apoptotic signaling, when constrained by experimental data, can facilitate the rational assignment of efficacious targeted inhibitors in B cell malignancies, paving the way for development of more personalized approaches to treatment.

Characterization of BV6-Induced Sensitization to the NK Cell Killing of Pediatric Rhabdomyosarcoma Spheroids.

Although the overall survival in pediatric rhabdomyosarcoma (RMS) has increased over the last decades, the most aggressive subtype of alveolar RMS is in dire need of novel treatment strategies. RMS cells evade cell death induction and immune control by increasing the expression of inhibitors of apoptosis proteins (IAPs), which can be exploited and targeted with stimulation with Smac mimetics. Here, we used the Smac mimetic BV6 to re-sensitize RMS spheroids to cell death, which increased killing induced by natural killer (NK) cells. Single BV6 treatment of RMS spheroids did not reduce spheroidal growth. However, we observed significant spheroidal decomposition upon BV6 pre-treatment combined with NK cell co-cultivation. Molecularly, IAPs s are rapidly degraded by BV6, which activates NF-κB signal transduction pathways in RMS spheroids. RNA sequencing analysis validated NF-κB activation and identified a plethora of BV6-regulated genes. Additionally, BV6 released caspases from IAP-mediated inhibition. Here, caspase-8 might play a major role, as knockdown experiments resulted in decreased NK cell-mediated attack. Taken together, we improved the understanding of the BV6 mechanism of RMS spheroid sensitization to cytotoxic immune cells, which could be suitable for the development of novel combinatory cellular immunotherapy with Smac mimetics.

Pediatric multicellular tumor spheroid models illustrate a therapeutic potential by combining BH3 mimetics with Natural Killer (NK) cell-based immunotherapy.

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

Unleashing the power of NK cells in anticancer immunotherapy.

Due to their physiological role in removing damaged cells, natural killer (NK) cells represent ideal candidates for cellular immunotherapy in the treatment of cancer. Thereby, the cytotoxicity of NK cells is regulated by signals on both, the NK cells as well as the targeted tumor cells, and the interplay and balance of these signals determine the killing capacity of NK cells. One promising avenue in cancer treatment is therefore the combination of NK cell therapy with agents that either help to increase the killing capacity of NK cells or sensitize tumor cells to an NK cell-mediated attack. In this mini-review, we present different strategies that can be explored to unleash the potential of NK cell immunotherapy. In particular, we summarize how modulation of apoptosis signaling within tumor cells can be exploited to sensitize tumor cells to NK cell-mediated cytotoxicity.

It's time to die: BH3 mimetics in solid tumors.

The removal of cells by apoptosis is an essential process regulating tissue homeostasis. Cancer cells acquire the ability to circumvent apoptosis and survive in an unphysiological tissue context. Thereby, the Bcl-2 protein family plays a key role in the initiation of apoptosis, and overexpression of the anti-apoptotic Bcl-2 proteins is one of the molecular mechanisms protecting cancer cells from apoptosis. Recently, small molecules targeting the anti-apoptotic Bcl-2 family proteins have been identified, and with venetoclax the first of these BH3 mimetics has been approved for the treatment of leukemia. In solid tumors the anti-apoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL are frequently overexpressed or genetically amplified. In this review, we summarize the role of Mcl-1 and Bcl-xL in solid tumors and compare the different BH3 mimetics targeting Mcl-1 or Bcl-xL.

MCL-1 inhibitors, fast-lane development of a new class of anti-cancer agents.

Cell death escape is one of the most prominent features of tumor cells and closely linked to the dysregulation of members of the Bcl-2 family of proteins. Among those, the anti-apoptotic family member myeloid cell leukemia-1 (MCL-1) acts as a master regulator of apoptosis in various human malignancies. Irrespective of its unfavorable structure profile, independent research efforts recently led to the generation of highly potent MCL-1 inhibitors that are currently evaluated in clinical trials. This offers new perspectives to target a so far undruggable cancer cell dependency. However, a detailed understanding about the tumor and tissue type specific implications of MCL-1 are a prerequisite for the optimal (i.e., precision medicine guided) use of this novel drug class. In this review, we summarize the major functions of MCL-1 with a special focus on cancer, provide insights into its different roles in solid vs. hematological tumors and give an update about the (pre)clinical development program of state-of-the-art MCL-1 targeting compounds. We aim to raise the awareness about the heterogeneous role of MCL-1 as drug target between, but also within tumor entities and to highlight the importance of rationale treatment decisions on a case by case basis.

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models.

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2's hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366's cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

DLBCL Cells with Acquired Resistance to Venetoclax Are Not Sensitized to BIRD-2 But Can Be Resensitized to Venetoclax through Bcl-XL Inhibition.

Anti-apoptotic Bcl-2-family members are frequently dysregulated in both blood and solid cancers, contributing to their survival despite ongoing oncogenic stress. Yet, such cancer cells often are highly dependent on Bcl-2 for their survival, a feature that is exploited by so-called BH3-mimetic drugs. Venetoclax (ABT-199) is a selective BH3-mimetic Bcl-2 antagonist that is currently used in the clinic for treatment of chronic lymphocytic leukemia patients. Unfortunately, venetoclax resistance has already emerged in patients, limiting the therapeutic success. Here, we examined strategies to overcome venetoclax resistance. Therefore, we used two diffuse large B-cell lymphoma (DLBCL) cell lines, Riva WT and venetoclax-resistant Riva (VR). The latter was obtained by prolonged culturing in the presence of venetoclax. We report that Riva VR cells did not become more sensitive to BIRD-2, a peptide targeting the Bcl-2 BH4 domain, and established cross-resistance towards BDA-366, a putative BH4-domain antagonist of Bcl-2. However, we found that Bcl-XL, another Bcl-2-family protein, is upregulated in Riva VR, while Mcl-1 expression levels are not different in comparison with Riva WT, hinting towards an increased dependence of Riva VR cells to Bcl-XL. Indeed, Riva VR cells could be resensitized to venetoclax by A-1155463, a selective BH3 mimetic Bcl-XL inhibitor. This is underpinned by siRNA experiments, demonstrating that lowering Bcl-XL-expression levels also augmented the sensitivity of Riva VR cells to venetoclax. Overall, this work demonstrates that Bcl-XL upregulation contributes to acquired resistance of DLBCL cancer cells towards venetoclax and that antagonizing Bcl-XL can resensitize such cells towards venetoclax.

A direct comparison of selective BH3-mimetics reveals BCL-XL, BCL-2 and MCL-1 as promising therapeutic targets in neuroblastoma.

BACKGROUND: Despite advances in the treatment of neuroblastoma, patients with high-risk disease still have dismal survival prognosis. Neuroblastoma cells display elevated expression of the antiapoptotic BCL-2 proteins, suggesting that BH3-mimetics may be a promising treatment option. Here, we investigated the role of BCL-2, BCL-XL and MCL-1 in neuroblastoma. METHODS: A panel of neuroblastoma cell lines and primary patient-derived cells were exposed to BH3-mimetics targeting BCL-2 (ABT-199), BCL-XL (A1331852) or MCL-1 (S63845). In addition, protein expression and interaction patterns were analysed using Western blotting and immunoprecipitation. RESULTS: All tested BH3-mimetics were able to induce apoptosis in neuroblastoma cell lines, indicating that not only BCL-2 but also BCL-XL and MCL-1 may be promising therapeutic targets. Primary patient-derived cells displayed highest sensitivity to A1331852, highlighting the important role of BCL-XL in neuroblastoma. Further analysis into the molecular mechanisms of apoptosis revealed that A1331852 and S63845 displaced proapoptotic proteins like BIM and BAK from their antiapoptotic targets, subsequently leading to the activation of BAX and BAK and caspase-dependent apoptosis. CONCLUSIONS: By using selective BH3-mimetics, this study demonstrates that BCL-2, BCL-XL, and MCL-1 are all relevant therapeutic targets in neuroblastoma. A1331852 and S63845 induce rapid apoptosis that is initiated following a displacement of BAK from BCL-XL or MCL-1, respectively.

Proteasome inhibitors and Smac mimetics cooperate to induce cell death in diffuse large B-cell lymphoma by stabilizing NOXA and triggering mitochondrial apoptosis.

Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Second mitochondria-derived activator of caspases (Smac) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pretreatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZ-induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies.

Targeting BCL-2 proteins in pediatric cancer: Dual inhibition of BCL-XL and MCL-1 leads to rapid induction of intrinsic apoptosis.

With the development of potent and selective inhibitors of MCL-1 (S63845) and BCL-XL (A-1331852) novel cancer treatment options have emerged. BCL-2 family proteins are important regulators of apoptosis in pediatric solid tumors. In the current study, we discover that rhabdomyosarcoma, Ewing sarcoma, osteosarcoma and neuroblastoma cell lines are co-dependent on BCL-XL and MCL-1 for survival. A-1331852/S63845 co-treatment, but not combinations of either inhibitor with ABT-199, synergistically induces rapid intrinsic apoptosis in vitro and demonstrates efficiency in an in vivo embryonic chicken model of rhabdomyosarcoma. Interestingly, A-1331852/S63845-induced apoptosis is BAX/BAK-dependent and mediated by displacement of BAK from BCL-XL and MCL-1, respectively. Moreover, BAK interacts with BAX to build a pore-forming complex in the outer mitochondrial membrane, leading to loss of mitochondrial outer membrane potential and caspase activation. Furthermore, in RD cells A-1331852/S63845 co-treatment disrupts BIM and NOXA in their interactions with BCL-XL and MCL-1, respectively, thereby contributing to apoptosis. Altogether, this study is the first to demonstrate the potency of A-1331852/S63845 in pediatric solid tumor cells and to describe the molecular mechanisms of A-1331852/S63845 co-treatment underlining the potential of BCL-XL and MCL-1 inhibition as treatment regime.

Specific interactions of BCL-2 family proteins mediate sensitivity to BH3-mimetics in diffuse large B-cell lymphoma.

The BCL-2-specific inhibitor, ABT-199 (venetoclax) has exhibited remarkable clinical activity in nearly all cases of chronic lymphocytic leukemia. In contrast, responses are usually much less in diffuse large B-cell lymphoma (DLBCL), despite high level expression of BCL-2 in over 40% of cases, indicating that co-expression of related anti-apoptotic BCL-2 family proteins may limit the activity of ABT-199. We have investigated the roles of BCL-2 proteins in DLBCL cells using a panel of specific BCL-2 homology 3 (BH3)-mimetics and identified subgroups of these cells that exhibited marked and specific dependency on either BCL-2, BCL-XL or MCL-1 for survival. Dependency was associated with selective sequestration of the pro-apoptotic proteins BIM, BAX and BAK by the specific anti-apoptotic BCL-2 protein which was important for cellular survival. Sensitivity to BH3-mimetics was independent of genetic alterations involving the BCL-2 family and only partially correlated with protein expression levels. Treatment with ABT-199 displaced BAX and BIM from BCL-2, subsequently leading to BAK activation and apoptosis. In contrast, apoptosis induced by inhibiting BCL-XL with A1331852 was associated with a displacement of both BAX and BAK from BCL-XL and occurred independently of BIM. Finally, the MCL-1 inhibitor S63845 induced mainly BAX-dependent apoptosis mediated by a displacement of BAK, BIM and NOXA from MCL-1. In conclusion, our study indicates that in DLBCL, the heterogeneous response to BH3-mimetics is mediated by selective interactions between BAX, BAK and anti-apoptotic BCL-2 proteins.

Next-generation hypomethylating agent SGI-110 primes acute myeloid leukemia cells to IAP antagonist by activating extrinsic and intrinsic apoptosis pathways.

Therapeutic efficacy of first-generation hypomethylating agents (HMAs) is limited in elderly acute myeloid leukemia (AML) patients. Therefore, combination strategies with targeted therapies are urgently needed. Here, we discover that priming with SGI-110 (guadecitabine), a next-generation HMA, sensitizes AML cells to ASTX660, a novel antagonist of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2) and X-linked IAP (XIAP). Importantly, SGI-110 and ASTX660 synergistically induced cell death in a panel of AML cell lines as well as in primary AML samples while largely sparing normal CD34+ human progenitor cells, underlining the translational relevance of this combination. Unbiased transcriptome analysis revealed that SGI-110 alone or in combination with ASTX660 upregulated the expression of key regulators of both extrinsic and intrinsic apoptosis signaling pathways such as TNFRSF10B (DR5), FAS, and BAX. Individual knockdown of the death receptors TNFR1, DR5, and FAS significantly reduced SGI-110/ASTX660-mediated cell death, whereas blocking antibodies for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or FAS ligand (FASLG) failed to provide protection. Also, TNFα-blocking antibody Enbrel had little protective effect on SGI-110/ASTX660-induced cell death. Further, SGI-110 and ASTX660 acted in concert to promote cleavage of caspase-8 and BID, thereby providing a link between extrinsic and intrinsic apoptotic pathways. Consistently, sequential treatment with SGI-110 and ASTX660-triggered loss of mitochondrial membrane potential (MMP) and BAX activation which contributes to cell death, as BAX silencing significantly protected from SGI-110/ASTX660-mediated apoptosis. Together, these events culminated in the activation of caspases-3/-7, nuclear fragmentation, and cell death. In conclusion, SGI-110 and ASTX660 cooperatively induced apoptosis in AML cells by engaging extrinsic and intrinsic apoptosis pathways, highlighting the therapeutic potential of this combination for AML.

Side-by-side comparison of BH3-mimetics identifies MCL-1 as a key therapeutic target in AML.

Despite advances in the treatment of acute myeloid leukemia (AML), prognosis of AML patients is still dismal and better treatment options are required. B-cell Lymphoma 2 (BCL-2) homology domain 3 (BH3)-mimetics are emerging as a novel class of apoptosis-inducing agents that are currently being tested for the treatment of different hematological malignancies including AML. Particularly, the selective BCL-2 inhibitor ABT-199/Venetoclax is demonstrating clinical responses and has recently been approved in combination for the treatment of AML. Compounds targeting the related protein MCL-1 have recently entered clinical trials, highlighting the urgency to compare the different BH3-mimetics and identify the most promising antiapoptotic target in AML. We performed a side-by-side comparison of different highly selective and potent BH3-mimetics targeting BCL-2 (ABT-199), MCL-1 (S63845) or BCL-xL (A1331852) in a panel of AML cell lines and primary patient cells. Gene knockdown using siRNAs was utilized to investigate the functional relevance of BCL-2 proteins. Western blotting and immunoprecipitations were used to explore the influence of BH3-mimetics on interactions between pro- and antiapoptotic BCL-2 proteins. A1331852 induced apoptosis only in selected cases, indicating that BCL-xL is not a very promising therapeutic target in AML. However, S63845 displayed higher potency than ABT-199, with more cell lines and primary cells responding to S63845 than to ABT-199. MCL-1 dependency in AML cells was confirmed by siRNA-mediated knockdown of MCL-1, which was sufficient to induce apoptosis. S63845-induced cell death was accompanied by a displacement of the BH3-only protein BIM as well as BAK, resulting in BAK-dependent apoptosis. In contrast, ABT-199-induced cell death was mediated by BAX rather than BAK, indicating distinct non-redundant molecular functions of BCL-2 and MCL-1 in AML. Our study reveals that MCL-1 may be a more prevalent therapeutic target than BCL-2 in AML and identifies BIM and BAK as important mediators of S63845-induced apoptosis in AML.

Selective BH3-mimetics targeting BCL-2, BCL-XL or MCL-1 induce severe mitochondrial perturbations.

Induction of apoptosis by selective BH3-mimetics is currently investigated as a novel strategy for cancer treatment. Here, we report that selective BH3-mimetics induce apoptosis in a variety of hematological malignancies. Apoptosis is accompanied by severe mitochondrial toxicities upstream of caspase activation. Specifically, the selective BH3-mimetics ABT-199, A-1331852 and S63845, which target BCL-2, BCL-XL and MCL-1, respectively, induce comparable ultrastructural changes including mitochondrial swelling, a decrease of mitochondrial matrix density and severe loss of cristae structure. These shared effects on mitochondrial morphology indicate a similar function of these anti-apoptotic BCL-2 proteins in maintaining mitochondrial integrity and function.

Targeting intermediary metabolism enhances the efficacy of BH3 mimetic therapy in hematologic malignancies.

BH3 mimetics are novel targeted drugs with remarkable specificity, potency and enormous potential to improve cancer therapy. However, acquired resistance is an emerging problem. We report the rapid development of resistance in chronic lymphocytic leukemia cells isolated from patients exposed to increasing doses of navitoclax (ABT-263), a BH3 mimetic. To mimic such rapid development of chemoresistance, we developed simple resistance models to three different BH3 mimetics, targeting BCL-2 (ABT-199), BCL-XL (A-1331852) or MCL-1 (A-1210477), in relevant hematologic cancer cell lines. In these models, resistance could not be attributed to either consistent changes in expression levels of the anti-apoptotic proteins or interactions among different pro- and anti-apoptotic BCL-2 family members. Using genetic silencing, pharmacological inhibition and metabolic supplementation, we found that targeting glutamine uptake and its downstream signaling pathways, namely glutaminolysis, reductive carboxylation, lipogenesis, cholesterogenesis and mammalian target of rapamycin signaling resulted in marked sensitization of the chemoresistant cells to BH3 mimetic-mediated apoptosis. Furthermore, our findings highlight the possibility of repurposing widely used drugs, such as statins, to target intermediary metabolism and improve the efficacy of BH3 mimetic therapy.