Matthew A. Smith

Paclitaxel, Estramustine Phosphate, and Carboplatin in Patients With Advanced Prostate Cancer

PURPOSE To determine the safety and activity of weekly paclitaxel in combination with estramustine and carboplatin (TEC) in patients with advanced prostate cancer. PATIENTS AND METHODS In a dose-escalation study, patients with advanced prostate cancer were administered paclitaxel (weekly 1-hour infusions of 60 to 100 mg/m(2)), oral estramustine (10 mg/kg), and carboplatin (area under the curve, 6 mg/mL-min every 4 weeks). Paclitaxel levels were determined 0, 30, 60, 90, and 120 minutes and 18 hours after infusion, and a concentration-time curve was estimated. Once a safe dose was established, a multi-institutional phase II trial was conducted in patients with progressive androgen-independent disease. RESULTS Fifty-six patients with progressive androgen-independent disease were treated for a median of four cycles. The dose of paclitaxel was escalated from 60 to 100 mg/m(2) without the occurrence of DLT. Posttherapy decreases in serum prostate-specific antigen levels of 50%, 80%, and 90% were seen in 67%, 48%, and 39% (95% confidence interval, 55% to 79%, 35% to 61%, 26% to 52%) of the patients, respectively. Of the 33 patients with measurable disease, two (6%) had a complete response and 13 (39%) had a partial response. The overall median time to progression was 21 weeks, and the median survival time for all patients was 19.9 months. Major grade 3 or 4 adverse effects were thromboembolic disease (in 25% of patients), hyperglycemia (in 38%), and hypophosphatemia (in 42%). Significant leukopenia, thrombocytopenia, and peripheral neuropathy were not observed. CONCLUSION TEC has significant antitumor activity and is well tolerated in patients with progressive androgen-independent prostate cancer.

Preoperative therapy for esophageal cancer: a randomized comparison of chemotherapy versus radiation therapy.

Ninety-six patients with operable epidermoid cancer of the esophagus were entered into a phase III, random assignment study designed to compare the efficacy of two preoperative approaches (chemotherapy [CT] or radiation therapy [RT]). Major study end points were objective response rates, surgical outcome, and recurrence pattern. Patients were randomly assigned to receive either two cycles of cisplatin, vindesine, and bleomycin or 55 Gy of radiation before a planned surgical procedure. Postoperative crossover therapy (radiation to those receiving preoperative CT and vice versa) was given to patients with T3Nany or unresectable tumors. Objective response rates of the primary tumor to preoperative therapy were similar (RT 64%, CT 55%), as were operability rates (RT 77%, CT 75%), resection rates (RT 65%, CT 58%), and operative mortality (RT 13.5%, CT 11.1%). Significantly higher doses of CT could be administered when CT was given as initial therapy, rather than after RT/surgery. Local failure or persistence occurred in 33% of operable patients. The median survival for all patients was 11 months; 20% remain alive without disease (median follow-up, 34 months). Because of the crossover design, it was not possible to analyze survival according to the preoperative therapy arm alone. This study suggests that since CT is as effective in treating local tumor as RT, but can also potentially treat systemic disease, investigational programs using CT before surgery as part of initial treatment for localized esophageal cancer should continue. However, if a significant impact on overall survival is to be achieved, more effective chemotherapy regimens or schedules need to be identified. Outside of carefully designed clinical trials, surgery alone or radiation alone remain standard therapy.

Antitumor efficacy of the anti-interleukin-6 (IL-6) antibody siltuximab in mouse xenograft models of lung cancer

Introduction: Interleukin-6 (IL-6) can activate downstream signaling pathways in lung cancer cells, such as the STAT3 pathway, and is reported to be produced by tumor cells with activating EGFR mutations. We examined IL-6/STAT3 in lung cancer tumor tissues and the effects of siltuximab, a neutralizing antibody to human IL-6, in mouse models of lung cancer. Methods: IL-6 and STAT3 activation levels were compared with tumor histology and presence of KRAS mutations in snap-frozen, non–small-cell lung cancer tumors. The effects of siltuximab alone or in combination with erlotinib were examined in mouse xenograft models constructed using three cell line xenograft models and one primary explant mouse model. We examined the influence of cancer-associated fibroblasts (CAFs) on tumor growth and siltuximab effects. Results: IL-6 levels were higher in tumors of squamous cell versus adenocarcinoma histology and were not associated with presence of KRAS mutations. Tyrosine phosphorylation status of STAT3 did not correlate with tumor IL-6 levels. Serine phosphorylation of STAT3 was correlated with KRAS mutation status. Both tumor and stromal cells contributed to total IL-6 within tumors. Siltuximab had minimal effect as a single agent in xenografts with tumor cells alone; however, in models coadministered with CAFs, siltuximab had more potent effects on tumor inhibition. We observed no effects of combined erlotinib and siltuximab. Conclusions: IL-6 is elevated in subsets of human NSCLCs, especially with squamous cell histology. Tumors supported by stromal production of IL-6 seem to be the most vulnerable to tumor growth inhibition by siltuximab.

Signaling Control by Epidermal Growth Factor Receptor and MET: Rationale for Cotargeting Strategies in Lung Cancer

In a report accompanying our article, Spigel et al present their findings on combining erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), with onartuzumab (MetMAb; Genentech, South San Francisco, CA), a humanized monovalent monoclonal antibody that blocks ligand activation of MET receptor tyrosine kinase (RTK). MET-positive patients (defined by immunohistochemistry) treated with erlotinib and onartuzumab demonstrated improved progression-free and overall survival, whereas MET-negative patients had worse outcomes. This article provides an overview of signaling control by EGFR and MET and the biologic rationale for cotargeting strategies. EGFR and MET are membrane-bound RTKs consisting of extracellular domains that bind to growth factors and intracellular domains that contain a kinase domain (Fig 1). MET is activated by its cognate ligand, hepatocyte growth factor (HGF), which is produced by stromal cells, also in an autocrine fashion. EGFR is activated by several ligands, including EGF, transforming growth factor alpha (TGF ), and heparin-binding EGF. On receptor binding of the ligands, the kinase domain becomes activated, leading to transautophosphorylation of tyrosine residues on the cytoplasmic tail. Phosphorylation of these tyrosine residues creates intramolecular docking sites for proteins containing Src homology 2 domains that serve as readers for tyrosine phosphorylation. This includes adaptor proteins such as growth factor receptor–bound protein 2 (GRB2), GAB1 (GRB2-associated binding protein 1), and Src homology 2–containing transforming protein 1 (SHC1). These proteins serve as scaffolds to support signal transduction to the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), phospholipase C (PLC), and signal transducer and activator of transcription (STAT) pathways. EGFR and MET use a highly overlapping repertoire of signaling adaptors and downstream effector pathways, highlighting their combined ability to codrive oncogenic signaling. Three general approaches are being employed in clinic to target EGFR and MET. The first uses small-molecule TKIs, such as erlotinib for EGFR and crizotinib for MET, that bind the ATP-binding pocket in the kinase domain and inhibit the kinase function. The second approach uses antibody therapy directed against the extracellular domain of the MET receptor (onartuzumab) or the HGF ligand (ficlatuzumab). The third approach targets downstream proteins involved in signaling, such as inhibitors of PI3K, or molecules regulating receptor trafficking and degradation, such as heat shock proteins. The EGFR pathway can be activated in lung cancer through genomic events, such as activating EGFR mutations or gene amplification, or through tumor environmental events, such as autocrine production of EGFR-activating ligands. Likewise, MET activation occurs through HGF produced in the tumor microenvironment and through MET gene amplification. MET amplification by fluorescent in situ hybridization occurs in approximately 5% to 15% of lung adenocarcinomas. Increased MET is associated with advanced stage of disease, poor outcome, brain metastasis, and high levels of EGFR. Coactivation of EGFR and MET occurs in subsets of lung cancer cell lines and tumor tissues and is associated with aggressive disease. In cells lacking EGFR mutation or high levels of MET, activation of either receptor can produce extensive signaling crosstalk to the opposing RTK and downstream substrates. EGFR activation can induce accumulation of activated MET protein through a c-SRC– dependent mechanism, and dual MET/EGFR inhibition has shown increased activity in xenograft models. Phosphoproteomic analysis of lung tumors and cell lines can identify tumors with concomitant EGFR and MET activation. EGFR, either through mutation or ligand-mediated activation, can also regulate MET levels through hypoxia-inducible factor-1 . Importantly, MET signaling is one actionable mechanism driving resistance to EGFR TKIs. MET activation can drive intrinsic as well as acquired resistance to EGFR through genomic and tumor microenvironment–mediated mechanisms. Primary resistance to anti-EGFR antibody therapy has been associated with MET activation in primary explant models of lung cancer. Approximately 5% of EGFR mutation–positive tumors with acquired resistance to EGFR TKIs are found to have MET gene amplification. Mechanistically, amplified MET co-opts use of ERBB3 to maintain the PI3K pathway activation. Combining MET and EGFR TKIs or EGFR/HSP90 inhibition has been effective in mouse models of lung cancer with T790M EGFR and MET amplification. High levels of HGF are associated with intrinsic or acquired resistance to EGFR TKIs in patients with lung cancer with activating EGFR mutations and in those with other solid tumors. HGF production by stromal fibroblasts leads to fibroblast-induced JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 31 NUMBER 32 NOVEMBER 1

MET–GRB2 Signaling-Associated Complexes Correlate with Oncogenic MET Signaling and Sensitivity to MET Kinase Inhibitors

Purpose: Targeting MET in cancer is hampered by lack of diagnostics that accurately reflect high MET signaling and dependence. We hypothesized that assays reflecting MET signaling associated protein complexes could redefine tumors dependent on MET and could add additional precision beyond genomic assessments. Experimental Design: We used biochemical approaches, cellular viability studies, and proximity ligation assays to assess MET dependence. We examined MET signaling complexes in lung cancer patient specimens (N = 406) and patient-derived xenograft (PDX) models of solid tumors (N = 308). We evaluated response to crizotinib in a MET-amplified cohort of PDX models of lung cancer (N = 6) and provide a case report of a lung cancer patient harboring a Δexon14 MET splice variant. Results: We found the interaction of MET with the adaptor protein GRB2 is necessary for oncogenic survival signaling by MET. MET-GRB2 complexes were identified only within MET-amplified PDX models and patient specimens but exhibit substantial variability. Lack of MET-GRB2 complexes was associated with lack of response to MET TKI in cell lines and PDX models. Presence of MET-GRB2 complexes can further subtype tumors with Δexon14 MET splice variants. Presence of these complexes correlated with response to crizotinib in one patient with Δexon14 MET lacking MET gene amplification. Conclusions: Proximity assays measuring MET-GRB2 signaling complexes provide novel insights into MET-mediated signaling and could complement current clinical genomics-based assay platforms. Clin Cancer Res; 23(22); 7084–96. ©2017 AACR.

Cell signaling heterogeneity is modulated by both cell-intrinsic and -extrinsic mechanisms: An integrated approach to understanding targeted therapy

During the last decade, our understanding of cancer cell signaling networks has significantly improved, leading to the development of various targeted therapies that have elicited profound but, unfortunately, short-lived responses. This is, in part, due to the fact that these targeted therapies ignore context and average out heterogeneity. Here, we present a mathematical framework that addresses the impact of signaling heterogeneity on targeted therapy outcomes. We employ a simplified oncogenic rat sarcoma (RAS)-driven mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) signaling pathway in lung cancer as an experimental model system and develop a network model of the pathway. We measure how inhibition of the pathway modulates protein phosphorylation as well as cell viability under different microenvironmental conditions. Training the model on this data using Monte Carlo simulation results in a suite of in silico cells whose relative protein activities and cell viability match experimental observation. The calibrated model predicts distributional responses to kinase inhibitors and suggests drug resistance mechanisms that can be exploited in drug combination strategies. The suggested combination strategies are validated using in vitro experimental data. The validated in silico cells are further interrogated through an unsupervised clustering analysis and then integrated into a mathematical model of tumor growth in a homogeneous and resource-limited microenvironment. We assess posttreatment heterogeneity and predict vast differences across treatments with similar efficacy, further emphasizing that heterogeneity should modulate treatment strategies. The signaling model is also integrated into a hybrid cellular automata (HCA) model of tumor growth in a spatially heterogeneous microenvironment. As a proof of concept, we simulate tumor responses to targeted therapies in a spatially segregated tissue structure containing tumor and stroma (derived from patient tissue) and predict complex cell signaling responses that suggest a novel combination treatment strategy.

Abstract 311: Novel role for MIG-6 in mediating TKI resistance in ALK-rearranged lung cancer

INTRODUCTION: Patients with ALK-rearranged non-small cell lung cancer (NSCLC) derive significant anti-tumor responses when treated with ALK tyrosine kinase inhibitors (TKIs), such as crizotinib, the first-in-class ALK TKI. However despite the high response rates to these agents, acquired resistance to ALK TKI therapy remains a significant barrier to overcome in order to maximize therapeutic responses in patients with ALK+ lung cancer. In crizotinib-resistant tumors, EGFR activation has been demonstrated to mediate acquired resistance in several independent studies. The tumor suppressor gene product MIG-6 (encoded by ERFFI1) acts as a natural inhibitor of signaling through EGFR (ErbB1) and other ErbB family members. However, a role for MIG-6 in ALK+ lung cancer has not yet been elucidated. Here, we investigated the regulation of human MIG-6 in ALK+ lung cancer cells. DESIGN: MIG-6 expression, protein interaction and phosphorylation status were evaluated in ALK+ lung cancer cell lines and human tumor samples. The impact of MIG-6 loss and gain of function on ErbB receptor activity and on cell proliferation/survival were determined in several models of TKI-sensitive and TKI-resistant ALK+ lung cancer. RESULTS: MIG-6 protein level is regulated by EML4-ALK fusion protein at both transcriptional and post-translational levels. Genetic or pharmacologic knock-down of ALK significantly reduced MIG-6 protein levels in ALK+/TKI sensitive cells. We established a novel interaction between MIG-6 and ALK fusion proteins (both EML4-ALK and other fusion partners), and this association was dependent on ALK kinase activity. MIG-6 can also be tyrosine-phosphorylated by EML4-ALK and be threonine-phosphorylated through EML4-ALK-dependent MAPK activation. The reduced MIG-6 protein level was accompanied by increased total and phosphorylated ErbB family members (EGFR, HER2, and HER3) in ALK+/TKI sensitive lung cancer cells. Consistent with this observation, MIG-6 protein level is also attenuated in ALK+/TKI resistant cells, following the decreased EML4-ALK activity, while EGFR signaling activity is remarkably up-regulated in these resistant cells. Crizotinib-resistant cells were re-sensitized upon exposure to EGFR/HER2 inhibitors in combination with crizotinib. In addition, MIG-6 reconstitution impeded the development of early adaptive resistance in crizotinib-sensitive cells and was also able to inhibit the proliferation of crizotinib-resistant cells. CONCLUSION: Our study presents an in-depth mechanistic understanding of how ErbB family members, such as EGFR, are up-regulated at the time of crizotinib resistance and provides insights into the early escape mechanisms tumor may have to evade ALK TKI therapy. Citation Format: Huan Qiao, Yingjun Yan, Matthew A. Smith, Eric B. Haura, Marc Ladanyi, Christine M. Lovly. Novel role for MIG-6 in mediating TKI resistance in ALK -rearranged lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 311.

Abstract B54: Characterizing pathway vulnerabilities and tumor heterogeneity through signaling-associated complexes

Signaling pathways controlling cellular proliferation are major targets of cancer therapeutics, however, accurate assessment of activation status of targetable signaling cascades remains a challenge. We have developed proximity ligation assay (PLA)-based methodologies to detect signaling-associated complexes for a variety of receptor tyrosine kinases, intracellular adaptors and phosphorylation events in formalin-fixed, paraffin-embedded tissues. By assessing the degree of functional association of oncogenic kinases (such as EGFR and ALK) with intracellular adaptor molecules, it is possible to characterize the activation status of targetable kinase activity and monitor variations across tissue types, within tissues and in response to perturbations. Here, we present data for a panel of PLAs using cell lines, patient specimens and patient-derived xenografts models. We show that EGFR-GRB2 PLA reflects erlotinib pharmacodynamics and has predictive capacity for response to both erlotinib and cetuximab. We demonstrate that PLA can identify alterations in multiple bypass signaling cascades associated with therapeutic escape using both in vitro models of acquired resistance and patient specimens. We observe striking intratumoral variation in abundance of signaling-associated complexes, likely reflecting influences from the tumor microenvironment and phenotypic heterogeneity. We are currently expanding our PLA panel to include emerging therapeutic targets (MET and FGFR1) and evaluating performance of these assays to characterize signaling complexes in circulating tumor cells obtained from advanced stage non-small cell lung cancer patients. Collectively, our data suggests that PLA-based methodologies can be harnessed to explore the cancer interactome and can be important tools to enable precision medicine. Citation Format: Matthew A. Smith, Leah Clark, Richard Hall, Kate Fisher, Takeshi Yoshida, Vincent Vuaroqueaux, Heinz-Herbert Fiebig, Ann Chen, Eric B. Haura. Characterizing pathway vulnerabilities and tumor heterogeneity through signaling-associated complexes. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B54.

Abstract B27: Proximity ligation assays as biomarkers for receptor tyrosine kinase activity in lung tumors.

In the era of precision medicine, matching patients to targeted therapies is a critical goal and novel biomarker strategies must be implemented as new therapeutic options are introduced into the clinic. However, most traditional biomarkers fail to capture the actual signaling activity occurring within a patients tumor. Proximity ligation assays (PLA) represent a novel approach to quantify receptor tyrosine kinase activity in clinical specimens, yielding a fluorescent readout of in situ signaling complexes. We have developed PLA assays for multiple receptor tyrosine kinase-adaptor pairs, including EGFR in complex with its major adaptor protein, GRB2. Here, we demonstrate robust performance of these assays in both cell lines and FFPE tissue specimens and provide evidence of predictive biomarker capacity for tyrosine kinase inhibitor response in lung cancer patients. For EGFR-GRB2, we demonstrate PLA is highly sensitive and specific, reflecting kinase activity and not merely expression levels of EGFR or GRB2. In non-small cell lung cancer (NSCLC) cell lines, high PLA signal is associated with EGFR mutation status and levels of phospho-EGFR. PLA also correlates with results from co-immunoprecipitation experiments and is abrogated by erlotinib in a dose-dependent manner. In multiple cell line xenograft models, PLA signal is observed in cytokeratin-staining regions and reveals dissociation of EGFR-GRB2 signaling complexes in response to erlotinib. In patient-derived lung adenocarcinoma xenografts, significantly higher PLA signal is observed in EGFR mutant xenografts relative to wildtype xenografts and is strongly reduced with erlotinib treatment. We have analyzed three cohorts of mixed histology NSCLC tumor specimens (N =354 patients) and scored these on a 0-3+ scale, finding good agreement of incidence of ≥ 2 PLA score across the three cohorts. EGFR protein expression levels as determined by Automated Quantitative Analysis (AQUA) correlate with EGFR-GRB2 PLA signal, but clear instances are defined where PLA signals are discordant, indicating “value-added” over EGFR expression analysis. High PLA score was not limited to adenocarcinomas and was observed in multiple histologies, with significant enrichment in metastatic brain lesions (P<.0001). PLA signal was enriched in tumors with known EGFR mutation status (P<0.01 by x2), but we also identified high signal in 24.5% (13 of 53) of KRAS mutant lung cancers and in 34% (26 of 77) of lung cancers lacking either EGFR or KRAS mutations (WT/WT). Among erlotinib-treated patients with follow-up data (N=91), we identified a strong trend of higher EGFR-GRB2 PLA signal associated with improved overall survival (P = 0.127) and a near doubling in median survival (10 months vs. 20 months) which was not observed when EGFR expression was evaluated by AQUA. Furthermore, PLA assays can be used on archival specimens in which phospho-IHC fails and can also be conducted using brightfield microscopy detection. Collectively, our data indicate that PLA assays measuring EGFR:GRB2 signaling complexes can serve as biomarkers for druggable kinase activity. We believe this novel proteome-level approach has the potential to be applied in a predictive capacity in multiple malignancies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B27. Citation Format: Matthew A. Smith, Richard Hall, Kate Fisher, Ann Chen, Eric B. Haura. Proximity ligation assays as biomarkers for receptor tyrosine kinase activity in lung tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B27.

Abstract 3502: Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity.

Predictive biomarkers are critical to optimize the use of receptor tyrosine kinase (RTK) inhibitors for cancer therapy. Because multi-protein complexes are critical to maintain oncogenic signaling, we hypothesized that assays that measure protein-protein interactions could identify patients with RTK activity and predict RTK inhibitor response. We developed in situ proximity ligation assays (PLA) to assess the degree of active EGFR signaling in both non-small cell lung cancer (NSCLC) cell lines and formalin-fixed paraffin-embedded (FFPE) patient specimens with the goal of establishing PLA assays as biomarkers of erlotinib sensitivity. Using in situ PLA, we evaluated EGFR association with its signaling adaptors, GRB2 and SHC1. By confocal microscopy we observed increased PLA signals in EGFR mutant NSCLC cell lines (PC9, H1650, HCC4006, HCC827) relative to EGFR wildtype (H23, H1299, H322, A549), indicative of constitutively active signaling complexes in mutant cell lines. PLA signal intensity was independent of EGFR, GRB2 and SHC1 protein expression levels, but did correlate with co-IP and pEGFR immunoblot results. Erlotinib dose-dependently reduced PLA signals in PC9 cells. H1650 xenografts exhibited high PLA signals that were reduced by erlotinib, corresponding with tumor shrinkage. Conversely, low PLA signals were observed in xenografts from EGFR wildtype cell lines (H322, H157). In human xenografts, PLA signals were significantly higher in EGFR mutant xenografts and reduced by erlotinib. In human FFPE specimens, PLA signals were absent in normal non-epithelial tissues and low in epithelial portions. FFPE lung tumor specimens (N = 221) revealed a wide range of PLA signal intensities (scored as 0+ to 3+), which localized to cytokeratin-positive regions. High PLA signals were found in approximately 25% of specimens across multiple histological classifications, including adenocarcinoma, squamous and large cell carcinoma. Furthermore, specimens from metastatic brain lesions were highly enriched for high PLA signal (p 2+) in specimens with relatively low EGFR protein expression and low PLA signal ( Citation Format: Matthew A. Smith, Richard Hall, Scott Haake, Jiannong Li, Eric B. Haura. Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3502. doi:10.1158/1538-7445.AM2013-3502