Androgen deprivation-induced senescence confers sensitivity to a senolytic strategy in prostate cancer.

We have previously demonstrated that androgen-dependent prostate cancer (PCa) cell lines enter a state of senescence following exposure to androgen deprivation therapies (ADT). ADT-induced senescence was found to be transient, as senescent cells develop castration resistance and re-emerge into a proliferative state even under continuous androgen deprivation in vitro. Moreover, the BCL-XL/BCL-2 inhibitor, ABT-263 (navitoclax), an established senolytic agent, promoted apoptosis of senescent PCa cells, suppressing proliferative recovery and subsequent tumor cell outgrowth. As this strategy has not previously been validated in vivo, we used a clinically relevant, syngeneic murine model of PCa, where mice were either castrated or castrated followed by the administration of ABT-263. Our results largely confirm the outcomes previously reported in vitro; specifically, castration alone results in a transient tumor growth suppression with characteristics of senescence, which is prolonged by exposure to ABT-263. Most critically, mice that underwent castration followed by ABT-263 experienced a statistically significant prolongation in survival, with an increase of 14.5 days in median survival time (56 days castration alone vs. 70.5 days castration + ABT-263). However, as is often the case in studies combining the promotion of senescence with a senolytic (the "one-two" punch approach), the suppression of tumor growth by the inclusion of the senolytic agent was transient, allowing for tumor regrowth once the drug treatment was terminated. Nevertheless, the results of this work suggest that the "one-two" punch senolytic strategy in PCa may effectively interfere with, diminish, or delay the development of the lethal castration-resistant phenotype.

Bruton's Tyrosine Kinase Inhibitors with Distinct Binding Modes Reveal Differential Functional Impact on B-Cell Receptor Signaling.

Small molecule inhibitors of Bruton's tyrosine kinase (BTK) have been approved for the treatment of multiple B-cell malignancies and are being evaluated for autoimmune and inflammatory diseases. Various BTK inhibitors (BTKi) have distinct potencies, selectivity profiles, and binding modes within the ATP-binding site. On the basis of the latter feature, BTKis can be classified into those that occupy the back-pocket, H3 pocket, and the hinge region only. Hypothesizing that differing binding modes may have differential impact on the B-cell receptor (BCR) signaling pathway, we evaluated the activities of multiple BTKis in B-cell lymphoma models in vitro and in vivo. We demonstrated that, although all three types of BTKis potently inhibited BTK-Y223 autophosphorylation and phospholipase C gamma 2 (PLCγ2)-Y1217 transphosphorylation, hinge-only binders were defective in inhibiting BTK-mediated calcium mobilization upon BCR activation. In addition, PLCγ2 activation was effectively blocked by back-pocket and H3 pocket binders but not by hinge-only binders. Further investigation using TMD8 cells deficient in Rac family small GTPase 2 (RAC2) revealed that RAC2 functioned as a bypass mechanism, allowing for residual BCR signaling and PLCγ2 activation when BTK kinase activity was fully inhibited by the hinge-only binders. These data reveal a kinase activity-independent function of BTK, involving RAC2 in transducing BCR signaling events, and provide mechanistic rationale for the selection of clinical candidates for B-cell lymphoma indications.

Enabling, Decagram-Scale Synthesis of Macrocyclic MCL-1 Inhibitor ABBV-467.

ABBV-467 is a highly potent and selective MCL-1 inhibitor that was advanced to a phase I clinical trial for the treatment of multiple myeloma. Due to its large size and structural complexity, ABBV-467 is a challenging synthetic target. Herein, we describe the synthesis of ABBV-467 on a decagram scale, which enabled preclinical characterization. The strategy is convergent and stereoselective, featuring a hindered biaryl cross coupling, enantioselective hydrogenation, and conformationally preorganized macrocyclization by C-O bond formation as key steps.

Selective MCL-1 inhibitor ABBV-467 is efficacious in tumor models but is associated with cardiac troponin increases in patients.

BACKGROUND: MCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902). METHODS: Binding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy. RESULTS: Here we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings. CONCLUSIONS: The selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.

Apoptosis is a type of cell death that removes abnormal cells from the body. Cancer cells can have increased levels of MCL-1, a protein that helps cells survive and prevents apoptosis. ABBV-467 is a new drug that blocks the action of MCL-1 (an MCL-1 inhibitor) and could promote apoptosis. In animal models, ABBV-467 led to cancer cell death and delayed tumor growth. ABBV-467 was also studied in a clinical trial in 8 patients with multiple myeloma, a blood cancer. In 1 patient, ABBV-467 treatment prevented the cancer from getting any worse for 8 months. However, in 4 out of 8 patients ABBV-467 increased the levels of troponin, a protein associated with damage to the heart. This concerning side effect may impact the future development of MCL-1 inhibitors as anticancer drugs.

Pharmaceutical Development Challenges in a Beyond Rule of Five Prodrug: Case Study of ABBV-167, Phosphate Prodrug of Venetoclax.

ABBV-167, a phosphate prodrug of BCL-2 inhibitor venetoclax, was recently progressed into the clinic as an alternative means of reducing pill burden for patients in high-dose indications. The dramatically enhanced aqueous solubility of ABBV-167 allowed for high drug loading within a crystalline tablet and, when administered in phase I clinical study, conferred venetoclax exposure commensurate with the equivalent dose administered as an amorphous solid dispersion. In enabling the progression into the clinic, we performed a comprehensive evaluation of the CMC development aspects of this beyond the rule of five (bRo5) prodrug. Adding a phosphate moiety resulted in excessively complex chemical speciation and solid form landscapes with significant physical-chemical stability liabilities. A combination of experimental and computational methods including microelectron diffraction (MicroED), total scattering, tablet colorimetry, finite element, and molecular dynamics modeling were used to understand CMC developability across drug substance and product manufacture and storage. The prodrug's chemical structural characteristics and loose crystal packing were found to be responsible for the loss of crystallinity during its manufacturing, which in turn led to high solid-state chemical reactivity and poor shelf life stability. The ABBV-167 case exemplifies key CMC development challenges for complex chemical matter such as bRo5 phosphate prodrugs with significant ramifications during drug substance and drug product manufacturing and storage.

New era for myelofibrosis treatment with novel agents beyond Janus kinase-inhibitor monotherapy: Focus on clinical development of BCL-XL /BCL-2 inhibition with navitoclax.

Myelofibrosis is a heterogeneous myeloproliferative neoplasm characterized by chronic inflammation, progressive bone marrow failure, and hepatosplenic extramedullary hematopoiesis. Treatments like Janus kinase inhibitor monotherapy (e.g., ruxolitinib) provide significant spleen and symptom relief but demonstrate limited ability to lead to a durable disease modification. There is an urgent unmet medical need for treatments with a novel mechanism of action that can modify the underlying pathophysiology and affect the disease course of myelofibrosis. This review highlights the role of B-cell lymphoma (BCL) protein BCL-extra large (BCL-XL ) in disease pathogenesis and the potential role that navitoclax, a BCL-extra large/BCL-2 inhibitor, may have in myelofibrosis treatment.

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma.

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein-protein interactions are critical in MCL1's nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53-/- CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53-/- CRC via exploitation of this unique MCL1-based chemoresistance mechanism.

Senolytic-Mediated Elimination of Head and Neck Tumor Cells Induced Into Senescence by Cisplatin.

Therapeutic outcomes achieved in head and neck squamous cell carcinoma (HNSCC) patients by concurrent cisplatin-based chemoradiotherapy initially reflect both tumor regression and tumor stasis. However, local and distant metastasis and disease relapse are common in HNSCC patients. In the current work, we demonstrate that cisplatin treatment induces senescence in both p53 wild-type HN30 and p53 mutant HN12 head and neck cancer models. We also show that tumor cells can escape from senescence both in vitro and in vivo. We further establish the effectiveness of the senolytic, ABT-263 (Navitoclax), in elimination of senescent tumor cells after cisplatin treatment. Navitoclax increased apoptosis by 3.3-fold (P ≤ 0.05) at day 7 compared with monotherapy by cisplatin. Additionally, we show that ABT-263 interferes with the interaction between B-cell lymphoma-x large (BCL-XL) and BAX, anti- and pro-apoptotic proteins, respectively, followed by BAX activation, suggesting that ABT-263-induced apoptotic cell death is mediated through BAX. Our in vivo studies also confirm senescence induction in tumor cells by cisplatin, and the promotion of apoptosis coupled with a significant delay of tumor growth after sequential treatment with ABT-263. Sequential treatment with cisplatin followed by ABT-263 extended the humane endpoint to ∼130 days compared with cisplatin alone, where mice survived ∼75 days. These results support the premise that senolytic agents could be used to eliminate residual senescent tumor cells after chemotherapy and thereby potentially delay disease recurrence in head and neck cancer patients. SIGNIFICANCE STATEMENT: Disease recurrence is the most common cause of death in head and neck cancer patients. B-cell lymphoma-x large inhibitors such as ABT-263 (Navitoclax) have the capacity to be used in combination with cisplatin in head and neck cancer patients to eliminate senescent cells and possibly prevent disease relapse.

Androgen-deprivation induced senescence in prostate cancer cells is permissive for the development of castration-resistance but susceptible to senolytic therapy.

Prostate cancer (PCa) is one of the leading causes of cancer-related deaths in men. Although androgen deprivation therapies (ADT) and antiandrogens confer increased survival rates, most patients eventually develop castration resistant disease (CRPC). Previous studies have shown that these treatments have limited cytotoxicity, and instead, promote tumor cell growth arrest. We show here that PCa cells grown in either charcoal-stripped serum or exposed to the antiandrogen, bicalutamide, undergo a senescent growth arrest marked by morphological changes, upregulated senescence-associated-ß-galactosidase (SA-ß-Gal), cathepsin D accumulation, and expression of the senescence-associated secretory phenotype (SASP). The senescent growth arrest is, however, transient, as cells can resume proliferation upon restoration of normo-androgenic conditions. Intriguingly, enrichment for senescent cells confirmed that ADT-induced senescent cells recover their proliferative capacity, even under prolonged androgen deprivation, and form androgen-independent outgrowths. Transplantation of the enriched senescent population into castrated, syngeneic mice confirmed that senescent cells escape the growth arrest and form castration-resistant tumors in vivo. Outgrowth from senescence was associated with increased expression of constitutively active androgen receptor splice variants, a common mechanism of resistance to ADT. Finally, the selective elimination of senescent PCa cells following ADT in vitro by the senolytic navitoclax (ABT-263) interfered with the development of androgen-independent outgrowth. Taken together, these data support the premise that ADT-induced senescence is a transient cell state from which CRPC populations can emerge, identifying senescence as a potential driver of disease progression. Furthermore, it is feasible that senolytic therapy to eliminate senescent PCa cells could delay disease recurrence and/or progression to androgen independence.

Balancing Properties with Carboxylates: A Lead Optimization Campaign for Selective and Orally Active CDK9 Inhibitors.

Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.

Unmasking BCL-2 Addiction in Synovial Sarcoma by Overcoming Low NOXA.

Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.

Structure-Based Design of A-1293102, a Potent and Selective BCL-XL Inhibitor.

BCL-XL, an antiapoptotic member of the BCL-2 family of proteins, drives tumor survival and maintenance and thus represents a key target for cancer treatment. Herein we report the rational design of a novel series of selective BCL-XL inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-XL inhibitor A-1155463 and the dual BCL-XL/BCL-2 inhibitors ABT-737 and navitoclax, while representing a distinct pharmacophore as assessed by an objective cheminformatic evaluation. A-1293102 exhibited picomolar binding affinity to BCL-XL and both efficiently and selectively killed BCL-XL-dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-XL, while the bent-back moiety achieved efficient occupancy of the P4 pocket in a manner similar to that of navitoclax. A-1293102 represents one of the few distinct structural series of selective BCL-XL inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization.

Venetoclax-based Rational Combinations are Effective in Models of MYCN-amplified Neuroblastoma.

Venetoclax is a small molecule inhibitor of the prosurvival protein BCL-2 that has gained market approval in BCL-2-dependent hematologic cancers including chronic lymphocytic leukemia and acute myeloid leukemia. Neuroblastoma is a heterogenous pediatric cancer with a 5-year survival rate of less than 50% for high-risk patients, which includes nearly all cases with amplified MYCN We previously demonstrated that venetoclax is active in MYCN-amplified neuroblastoma but has limited single-agent activity in most models, presumably the result of other pro-survival BCL-2 family protein expression or insufficient prodeath protein mobilization. As the relative tolerability of venetoclax makes it amenable to combining with other therapies, we evaluated the sensitivity of MYCN-amplified neuroblastoma models to rational combinations of venetoclax with agents that have both mechanistic complementarity and active clinical programs. First, the MDM2 inhibitor NVP-CGM097 increases the prodeath BH3-only protein NOXA to sensitize p53-wild-type, MYCN-amplified neuroblastomas to venetoclax. Second, the MCL-1 inhibitor S63845 sensitizes MYCN-amplified neuroblastoma through neutralization of MCL-1, inducing synergistic cell killing when combined with venetoclax. Finally, the standard-of-care drug cocktail cyclophosphamide and topotecan reduces the apoptotic threshold of neuroblastoma, thus setting the stage for robust combination efficacy with venetoclax. In all cases, these rational combinations translated to in vivo tumor regressions in MYCN-amplified patient-derived xenograft models. Venetoclax is currently being evaluated in pediatric patients in the clinic, including neuroblastoma (NCT03236857). Although establishment of safety is still ongoing, the data disclosed herein indicate rational and clinically actionable combination strategies that could potentiate the activity of venetoclax in patients with amplified MYCN with neuroblastoma.

Expanding the Repertoire for "Large Small Molecules": Prodrug ABBV-167 Efficiently Converts to Venetoclax with Reduced Food Effect in Healthy Volunteers.

Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. Although a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug (3, ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing.

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

Clearance of therapy-induced senescent tumor cells by the senolytic ABT-263 via interference with BCL-XL -BAX interaction.

Tumor cells undergo senescence in response to both conventional and targeted cancer therapies. The induction of senescence in response to cancer therapy can contribute to unfavorable patient outcomes, potentially including disease relapse. This possibiliy is supported by our findings that tumor cells induced into senescence by doxorubicin or etoposide can give rise to viable tumors in vivo. We further demonstrate sensitivity of these senescent tumor cells to the senolytic ABT-263 (navitoclax), therefore providing a "two-hit" approach to eliminate senescent tumor cells that persist after exposure to chemotherapy or radiation. The sequential combination of therapy-induced senescence and ABT-263 could shift the response to therapy toward apoptosis by interfering with the interaction between BCL-XL and BAX. The administration of ABT-263 after either etoposide or doxorubicin also resulted in marked, prolonged tumor suppression in tumor-bearing animals. These findings support the premise that senolytic therapy following conventional cancer therapy may improve therapeutic outcomes and delay disease recurrence.

Inhibition of cyclin-dependent kinase 9 synergistically enhances venetoclax activity in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive and largely incurable subtype of non-Hodgkin's lymphoma. Venetoclax has demonstrated efficacy in MCL patients with relapsed or refractory disease, however response is variable and less durable than CLL. This may be the result of co-expression of other anti-apoptotic proteins such as MCL-1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. One strategy for neutralizing MCL-1 and other short-lived survival factors is to inhibit CDK9, which plays a key role in transcription. Here, we report the response of MCL cell lines and primary patient samples to the combination of venetoclax and novel CDK9 inhibitors. Primary samples represented de novo patients and relapsed disease, including relapse after ibrutinib failure. Despite the diverse responses to each single agent, possibly due to variable expression of the BCL-2 family members, venetoclax plus CDK9 inhibitors synergistically induced apoptosis in MCL cells. The synergistic effect was also confirmed via venetoclax plus a direct MCL-1 inhibitor. Murine xenograft studies demonstrated potent in vivo efficacy of venetoclax plus CDK9 inhibitor that was superior to each agent alone. Together, this study supports clinical investigation of this combination in MCL, including in patients who have progressed on ibrutinib.

A novel CDK9 inhibitor increases the efficacy of venetoclax (ABT-199) in multiple models of hematologic malignancies.

MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.

The Ewing Family of Tumors Relies on BCL-2 and BCL-XL to Escape PARP Inhibitor Toxicity.

PURPOSE: It was recently demonstrated that the EWSR1-FLI1 t(11;22)(q24;12) translocation contributes to the hypersensitivity of Ewing sarcoma to PARP inhibitors, prompting clinical evaluation of olaparib in a cohort of heavily pretreated Ewing sarcoma tumors. Unfortunately, olaparib activity was disappointing, suggesting an underappreciated resistance mechanism to PARP inhibition in patients with Ewing sarcoma. We sought to elucidate the resistance factors to PARP inhibitor therapy in Ewing sarcoma and identify a rational drug combination capable of rescuing PARP inhibitor activity. EXPERIMENTAL DESIGN: We employed a pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenograft (PDX) and cell line xenograft models. RESULTS: We found olaparib sensitivity was diminished following chemotherapy. The matched cell line pair revealed increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apoptotic resistance to olaparib. Resistance to olaparib was maintained in this chemotherapy-resistant model in vivo, whereas the addition of the BCL-2/XL inhibitor navitoclax led to tumor growth inhibition. In 2 PDXs, olaparib and navitoclax were minimally effective as monotherapy, yet induced dramatic tumor growth inhibition when dosed in combination. We found that EWS-FLI1 increases BCL-2 expression; however, inhibition of BCL-2 alone by venetoclax is insufficient to sensitize Ewing sarcoma cells to olaparib, revealing a dual necessity for BCL-2 and BCL-XL in Ewing sarcoma survival. CONCLUSIONS: These data reveal BCL-2 and BCL-XL act together to drive olaparib resistance in Ewing sarcoma and reveal a novel, rational combination therapy that may be put forward for clinical trial testing.