Maria Gomez-Caraballo

Patient-derived models of acquired resistance can identify effective drug combinations for cancer

Targeted cancer therapies have produced substantial clinical responses, but most tumors develop resistance to these drugs. Here, we describe a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance. We established cell culture models derived from biopsy samples of lung cancer patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected these cells to genetic analyses and a pharmacological screen. Multiple effective drug combinations were identified. For example, the combination of ALK and MAPK kinase (MEK) inhibitors was active in an ALK-positive resistant tumor that had developed a MAP2K1 activating mutation, and the combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant resistant cancer with a mutation in FGFR3. Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, this strategy could help direct therapeutic choices for individual patients. Secondary chemotherapies can be developed by screening drug-resistant cells from individual cancer patients. Drug resistance, up close and personal Cancer therapies that target specific genetic mutations driving tumor growth have shown promising results in patients; however, the response is often short-lived because the tumors acquire new mutations that render them resistant to these therapies. Complicating matters, the mechanism of resistance can vary from patient to patient. To identify drugs most likely to be effective against resistant tumors, Crystal et al. established cell lines from the tumors of individual patients after resistance occurred and performed a drug screen and genetic analysis on the cultured cells. This strategy successfully identified drug combinations that halted the growth of resistant tumor cells both in culture and in mice. In the future, pharmacological profiling of patient-derived cells could be an efficient way to direct therapeutic choices for individual cancer patients. Science, this issue p. 1480

Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition

Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFRT790M gatekeeper mutation can occur either by selection of pre-existing EGFRT790M-positive clones or via genetic evolution of initially EGFRT790M-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFRT790M; treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor–resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic.

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer

Significance Small-cell lung cancer (SCLC) is an aggressive carcinoma with few effective treatment options beyond first-line chemotherapy. BH3 mimetics, such as ABT-263, promote apoptosis in SCLC cell lines, but early phase clinical trials demonstrated no significant clinical benefit. Here, we examine the sensitivity of a large panel of cancer cell lines, including SCLC, to ABT-263 and find that high Bcl2-interacting mediator of cell death (BIM) and low myeloid cell leukemia 1 (MCL-1) expression together predict sensitivity. SCLC cells relatively resistant to ABT-263 are sensitized by TORC1/2 inhibition via MCL-1 reduction. Combination of ABT-263 and TORC1/2 inhibition stabilizes or shrinks tumors in xenograft models, in autochthonous SCLC tumors in a genetically engineered mouse model, and in a patient-derived xenograft SCLC model. Collectively, these data support a compelling new therapeutic strategy for treating SCLC. BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

Programmed Cell Death Ligand 1 Expression in Resected Lung Adenocarcinomas: Association with Immune Microenvironment

Introduction Programmed cell death ligand 1 (PD‐L1) expression on tumor cells can be upregulated via activation of CD8+ cytotoxic T lymphocytes (CTLs) or the T helper cell (Th1) pathway, counterbalancing the CTL/Th1 microenvironment. However, PD‐L1 expression in association with subtypes of tumor‐associated lymphocytes and molecular alterations has not been well characterized in lung adenocarcinomas. Methods PD‐L1 expression was evaluated in 261 resected lung adenocarcinomas using tissue microarrays and various scoring systems, and was correlated with clinicopathologic/molecular features, including the extent/subtype of tumor‐associated lymphocytes (i.e., CD8, T‐bet [Th1 transcription factor], and GATA3 [Th2 transcription factor]), and patient outcomes. Results PD‐L1 expression was present in 129 (49%), 95 (36.5%), and 62 (24%) cases using cutoffs of ≥1%, ≥5%, and ≥50%, respectively, 98 (38%) by H score and 72 (28%) by immune score. PD‐L1 expression was associated with abundant CD8+ and/or T‐bet+ tumor‐infiltrating lymphocytes and EGFR wild‐type, significant smoking history, and aggressive pathologic features. In addition, concurrent PD‐L1 expression and abundant CD8+ tumor‐associated lymphocytes were seen in 25% of KRAS mutants or cases with no alterations by clinical molecular testing as opposed to only 7.4% of EGFR mutants. PD‐L1 expression was significantly associated with decreased progression‐free and overall survival rates by univariate analysis, but not by multivariate analysis. Conclusion PD‐L1 expression in resected lung adenocarcinomas is frequently observed in the presence of CTL/Th1 microenvironment, in particular in those with KRAS mutations or no common molecular alterations, suggesting that blockade of the PD‐1/PD‐L1 axis may be a promising treatment strategy to reinstitute active immune response for at least a subset of such patient populations.

Programmed Cell Death Ligand (PD-L1) Expression in Stage II and III Lung Adenocarcinomas and Nodal Metastases

Introduction: Programmed death ligand 1 (PD‐L1) expression determined by immunohistochemistry (IHC) may serve as a predictive biomarker for anti–PD‐1/PD‐L1 therapies; however, little is known about intertumoral heterogeneity of PD‐L1 expression determined by IHC in lung adenocarcinomas (ADCs), and there have been conflicting results on the prognostic role of PD‐L1 expression in ADCs. Methods: PD‐L1 expression was evaluated in resected stage II and III ADCs by using various cutoffs and correlated with clinicopathologic parameters and survival. PD‐L1 expression was also compared between the primary tumor and lymph node metastases. Results: There were 109 study cases. PD‐L1 expression was seen in 56 (51%), 43 (39%), and 19 (17%) when cutoffs of at least 1%, at least 5%, and at least 50%, respectively, were used. Abundant intratumoral CD8‐positive T cells were a significant predictor of the expression in the primary tumor, with cutoffs of 1% and 5% (p < 0.001 for both) by multivariate analysis, whereas they were a nonsignificant predictor of the expression with a 50% cutoff (p = 0.076). PD‐L1 expression was concordant between the primary tumor and nodal metastasis in most of the cases, but it was discrepant in up to 38%. The discrepancy was attributed in part to different predominant histologic patterns between the primary and metastatic tumors. In the entire cohort, PD‐L1 expression with all cutoffs had no bearing on 5‐year recurrence‐free survival. Conclusions: PD‐L1 expression is associated with abundant intratumoral CD8‐positive T cells in resected ADCs, suggesting a predictive role of PD‐L1 expression in anti–PD‐1/PD‐L1 therapies; however, the possible intertumoral heterogeneity of PD‐L1 expression raises a concern about selecting the most appropriate sample for PD‐L1 IHC.

Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts

Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patients disease. Collectively, this work highlights the translational potential of this strategy.Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600-15. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Epithelial-to-Mesenchymal Transition Antagonizes Response to Targeted Therapies in Lung Cancer by Suppressing BIM

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes. Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers

There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers (NSCLC) and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) and SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and colorectal cancers.

Abstract 2163: Combined targeting of MEK and MCL-1 induces apoptosis and tumor regression of KRAS mutant NSCLC

There are currently no effective targeted therapeutic strategies for KRAS mutant non-small cell lung cancer (NSCLC). Single agent MEK inhibitors have demonstrated showed disappointing clinical activity, partly due to inability to induce a robust apoptotic response. Combining MEK inhibitors with BCL-XL/BCL-2 inhibitors may be effective for a subset of KRAS mutant cancers that are dependent on BCL-XL for survival, however this combination is unlikely to be an effective strategy for cancers dependent on MCL-1. We investigated the effect of combining the MEK inhibitor trametinib with a novel MCL-1 inhibitor (compound A), which possesses potent and selective anti-MCL-1 activity in vitro and in vivo, on KRAS mutant cancers. In contrast to colorectal cancer models, which are largely sensitive to combined MEK + BCL-XL inhibition, a subset of cell line and patient-derived mouse xenograft (PDX) KRAS mutant NSCLC models were significantly more sensitive to MEK + MCL-1 inhibition compared to MEK + BCL-XL. To model potential clinical strategies, we tested intermittent dosing regimens and unexpectedly discovered a method strategy for dramatically sensitizing KRAS mutant NSCLC cells to the MEK + MCL-1 combination. These studies provide rationale for the clinical evaluation of combined MEK + MCL-1 inhibitors for KRAS mutant NSCLC. Citation Format: Aaron N. Hata, Faria M. Siddiqui, Maria Gomez-Caraballo, Samantha J. Bilton, Daria Timonina, Varuna Nangia, Angela Coxon, Sean Caenepeel, Paul Hughes. Combined targeting of MEK and MCL-1 induces apoptosis and tumor regression of KRAS mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2163. doi:10.1158/1538-7445.AM2017-2163