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Purpose: Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody–drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6–23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker. Experimental Design: A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6–23-ADC. The antitumor efficacy of CLDN6–23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6−) xenografts and patient-derived xenograft (PDX) models of human cancers. Results: CLDN6–23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6–23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target. Conclusions: We report the development of a novel ADC, CLDN6–23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6–23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.

Supplemental Figure S2: Amino acid sequence alignment of CLDN6 (P56747) and CLDN9 (O95484) with the extracellular loops highlighted

Methods for Supplemental Data

Purpose:Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody–drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6–23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker.Experimental Design:A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6–23-ADC. The antitumor efficacy of CLDN6–23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6−) xenografts and patient-derived xenograft (PDX) models of human cancers.Results:CLDN6–23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6–23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target.Conclusions:We report the development of a novel ADC, CLDN6–23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6–23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.

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Anticancer therapies have been limited by the emergence of mutations and other adaptations. In bacteria, antibiotics activate the SOS response, which mobilizes error-prone factors that allow for continuous replication at the cost of mutagenesis. We investigated whether the treatment of lung cancer with EGFR inhibitors (EGFRi) similarly engages hypermutators. In cycling drug-tolerant persister (DTP) cells and in EGFRi-treated patients presenting residual disease, we observed upregulation of GAS6, whereas ablation of GAS6's receptor, AXL, eradicated resistance. Reciprocally, AXL overexpression enhanced DTP survival and accelerated the emergence of T790M, an EGFR mutation typical to resistant cells. Mechanistically, AXL induces low-fidelity DNA polymerases and activates their organizer, RAD18, by promoting neddylation. Metabolomics uncovered another hypermutator, AXL-driven activation of MYC, and increased purine synthesis that is unbalanced by pyrimidines. Aligning anti-AXL combination treatments with the transition from DTPs to resistant cells cured patient-derived xenografts. Hence, similar to bacteria, tumors tolerate therapy by engaging pharmacologically targetable endogenous mutators.Significance:EGFR-mutant lung cancers treated with kinase inhibitors often evolve resistance due to secondary mutations. We report that in similarity to the bacterial SOS response stimulated by antibiotics, endogenous mutators are activated in drug-treated cells, and this heralds tolerance. Blocking the process prevented resistance in xenograft models, which offers new treatment strategies.This article is highlighted in the In This Issue feature, p. 2483

Supplementary Figure from AXL and Error-Prone DNA Replication Confer Drug Resistance and Offer Strategies to Treat EGFR-Mutant Lung Cancer

Supplementary Data from AXL and Error-Prone DNA Replication Confer Drug Resistance and Offer Strategies to Treat EGFR-Mutant Lung Cancer

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HER2 levels post-treatment.

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Supplementary Figure S3 describes the effects of distinct MEK inhibitors in combination with BCL-2/XL inhibition and contribution of BCL-2 vs. BCL-XL to apoptotic vulnerability of HGSOC PDX models

HER2 levels post-treatment.

Supplementary Movie Legends 1-12 from Ovarian Cancer Spheroids Use Myosin-Generated Force to Clear the Mesothelium

Supplementary Table 1 shows the p53 mutation in the PDX models

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ABT-737 enhances the effectiveness of T-DM1 in vivo.

p63 MCL-1 HER3 post-treatment.

Supplementary Figures 1-3 from Ovarian Cancer Spheroids Use Myosin-Generated Force to Clear the Mesothelium

Most patients with late-stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient–derived xenograft models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the proapoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/XL inhibitor (ABT-263) significantly reduced cell number, increased cell death, and displayed synergy in vitro in most models. In vivo, GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models—those with high BIM at baseline that was increased with MEK inhibition and those with low basal BIM and high pERK levels. Models with low BIM and low pERK were nonresponsive. Our findings demonstrate that combined MEK and BCL-2/XL inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for the clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.

Assessment of ERBB2 amplification.

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Comparison of continuous versus pulsatile combination treatments.

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Macroscopic comparison of tumors.

Supplementary Table 1. Tumor IHC marker specifics; Supplementary Table 2. p-values for Figure 4 comparisons; Supplementary Table 3. p-values for Figure 5 comparisons

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Normal cells explore multiple states to survive stresses encountered during development and self-renewal as well as environmental stresses such as starvation, DNA damage, toxins or infection. Cancer cells co-opt normal stress mitigation pathways to survive stresses that accompany tumour initiation, progression, metastasis and immune evasion. Cancer therapies accentuate cancer cell stresses and invoke rapid non-genomic stress mitigation processes that maintain cell viability and thus represent key targetable resistance mechanisms. In this Review, we describe mechanisms by which tumour ecosystems, including cancer cells, immune cells and stroma, adapt to therapeutic stresses and describe three different approaches to exploit stress mitigation processes: (1) interdict stress mitigation to induce cell death; (2) increase stress to induce cellular catastrophe; and (3) exploit emergent vulnerabilities in cancer cells and cells of the tumour microenvironment. We review challenges associated with tumour heterogeneity, prioritizing actionable adaptive responses for optimal therapeutic outcomes, and development of an integrative framework to identify and target vulnerabilities that arise from adaptive responses and engagement of stress mitigation pathways. Finally, we discuss the need to monitor adaptive responses across multiple scales and translation of combination therapies designed to take advantage of adaptive responses and stress mitigation pathways to the clinic.

Both, p38/MAPK, and ERK/MEK pathway contribute to MYC and YAP up-regulation in resistant cell lines.

Sensitivity Analyses For the Median-Center and Proliferation Normalization of Microarray Data.

Description of additional methods and procedures used in the study.

Gene Expression Signatures

Aberrant activation of the PI3K/mTOR pathway is a common feature of many cancers and an attractive target for therapy, but resistance inevitably evolves as is the case for any cancer cell–targeted therapy. In animal tumor models, chronic inhibition of PI3K/mTOR initially inhibits tumor growth, but over time, tumor cells escape inhibition. In this study, we identified a context-dependent mechanism of escape whereby tumor cells upregulated the proto-oncogene transcriptional regulators c-MYC and YAP1. This mechanism was dependent on both constitutive ERK activity as well as inhibition of the stress kinase p38. Inhibition of p38 relieved proliferation arrest and allowed upregulation of MYC and YAP through stabilization of CREB. These data provide new insights into cellular signaling mechanisms that influence resistance to PI3K/mTOR inhibitors. Furthermore, they suggest that therapies that inactivate YAP or MYC or augment p38 activity could enhance the efficacy of PI3K/mTOR inhibitors. Cancer Res; 76(24); 7168–80. ©2016 AACR.

Table S5: Somatic Sequence Alterations in parental tumor and xenograft DF149

Legends for Supplementary Figures S1-S4.

Table S4: Somatic Sequence Alterations in parental tumor and xenograft DF101

Purpose: To provide further insight into the role of proliferation and other cellular processes in chemosensitivity and resistance, we evaluated the association of a diverse set of gene expression signatures with response to neoadjuvant chemotherapy (NAC) in breast cancer.Experimental Design: Expression data from primary breast cancer biopsies for 1,419 patients in 17 studies prior to NAC were identified and aggregated using common normalization procedures. Clinicopathologic characteristics, including response to NAC, were collected. Scores for 125 previously published breast cancer–related gene expression signatures were calculated for each tumor.Results: Within each receptor-based subgroup or PAM50 subtype, breast tumors with high proliferation signature scores were significantly more likely to achieve pathologic complete response to NAC. To distinguish “proliferation-associated” from “proliferation-independent” signatures, we used correlation and linear modeling approaches. Most signatures associated with response to NAC were proliferation associated: 90.5% (38/42) in ER+/HER2− and 63.3% (38/60) in triple-negative breast cancer (TNBC). Proliferation-independent signatures predictive of response to NAC in ER+/HER2− breast cancer were related to immune activity, while those in TNBC comprised a diverse set of signatures, including immune, DNA damage, signaling pathways (PI3K, AKT, Ras, and EGFR), and “stemness” phenotypes.Conclusions: Proliferation differences account for the vast majority of predictive capacity of gene expression signatures in neoadjuvant chemosensitivity for ER+/HER2− breast cancers and, to a lesser extent, TNBCs. Immune activation signatures are proliferation-independent predictors of pathologic complete response in ER+/HER2− breast cancers. In TNBCs, significant proliferation-independent signatures include gene sets that represent a diverse set of cellular processes. Clin Cancer Res; 22(24); 6039–50. ©2016 AACR.

Purpose: Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, with high rates of recurrence and eventual resistance to cytotoxic chemotherapy. Model systems that allow for accurate and reproducible target discovery and validation are needed to support further drug development in this disease.Experimental Design: Clinically annotated patient-derived xenograft (PDX) models were generated from tumor cells isolated from the ascites or pleural fluid of patients undergoing clinical procedures. Models were characterized by IHC and by molecular analyses. Each PDX was luciferized to allow for reproducible in vivo assessment of intraperitoneal tumor burden by bioluminescence imaging (BLI). Plasma assays for CA125 and human LINE-1 were developed as secondary tests of in vivo disease burden.Results: Fourteen clinically annotated and molecularly characterized luciferized ovarian PDX models were generated. Luciferized PDX models retain fidelity to both the nonluciferized PDX and the original patient tumor, as demonstrated by IHC, array CGH, and targeted and whole-exome sequencing analyses. Models demonstrated diversity in specific genetic alterations and activation of PI3K signaling pathway members. Response of luciferized PDX models to standard-of-care therapy could be reproducibly monitored by BLI or plasma markers.Conclusions: We describe the establishment of a collection of 14 clinically annotated and molecularly characterized luciferized ovarian PDX models in which orthotopic tumor burden in the intraperitoneal space can be followed by standard and reproducible methods. This collection is well suited as a platform for proof-of-concept efficacy and biomarker studies and for validation of novel therapeutic strategies in ovarian cancer. Clin Cancer Res; 23(5); 1263–73. ©2016 AACR.

Table S2: Array CGH characterization of PDX models

Supplementary Figure 1 from Fibroblast Growth Factor Receptor 1–Transformed Mammary Epithelial Cells Are Dependent on RSK Activity for Growth and Survival

Supplementary Figure 2 from Fibroblast Growth Factor Receptor 1–Transformed Mammary Epithelial Cells Are Dependent on RSK Activity for Growth and Survival

Supplementary Figure 4 from Fibroblast Growth Factor Receptor 1–Transformed Mammary Epithelial Cells Are Dependent on RSK Activity for Growth and Survival

Supplementary Figure 3 from Fibroblast Growth Factor Receptor 1–Transformed Mammary Epithelial Cells Are Dependent on RSK Activity for Growth and Survival

Supplementary Figure 5 from Fibroblast Growth Factor Receptor 1–Transformed Mammary Epithelial Cells Are Dependent on RSK Activity for Growth and Survival

Fibroblast growth factor receptor 1 (FGFR1) is frequently amplified and highly expressed in lobular carcinomas of the breast. In this report, we evaluated the biological activity of FGFR1 in a wide range of in vitro assays. Conditional activation of FGFR1 in the nontransformed MCF10A human mammary cell line, MCF10A, resulted in cellular transformation marked by epidermal growth factor–independent cell growth, anchorage-independent cell proliferation and survival, loss of cell polarity, and epithelial-to-mesenchymal transition. Interestingly, small-molecule or small interfering RNA inhibition of ribosomal S6 kinase (RSK) activity induced death of the FGFR1-transformed cells, but not of the parental MCF10A cell line. The dependence of FGFR1-transformed cells on RSK activity was further confirmed in cell lines derived from mouse and human lobular carcinomas that possess high FGFR1 activity. Taken together, these results show the transforming activity of FGFR1 in mammary epithelial cells and identify RSK as a critical component of FGFR1 signaling in lobular carcinomas, thus implicating RSK as a candidate therapeutic target in FGFR1-expressing tumors. [Cancer Res 2009;69(6):2244–51]

Intratumoral heterogeneity has been described for various tumor types and models of human cancer, and can have profound effects on tumor progression and drug resistance. This study describes an in-depth analysis of molecular and functional heterogeneity among subclonal populations (SCPs) derived from a single triple-negative breast cancer cell line, including copy number analysis, whole-exome and RNA sequencing, proteome analysis, and barcode analysis of clonal dynamics, as well as functional assays. The SCPs were found to have multiple unique genetic alterations and displayed significant variation in anchorage independent growth and tumor forming ability. Analyses of clonal dynamics in SCP mixtures using DNA barcode technology revealed selection for distinct clonal populations in different in vitro and in vivo environmental contexts, demonstrating that in vitro propagation of cancer cell lines using different culture conditions can contribute to the establishment of unique strains. These analyses also revealed strong enrichment of a single SCP during the development of xenograft tumors in immune-compromised mice. This SCP displayed attenuated interferon signaling in vivo and reduced sensitivity to the antiproliferative effects of type I interferons. Reduction in interferon signaling was found to provide a selective advantage within the xenograft microenvironment specifically. In concordance with the previously described role of interferon signaling as tumor suppressor, these findings suggest that similar selective pressures may be operative in human cancer and patient-derived xenograft models.