Felicia L. Lenzo

Analytical Validation of a Next-Generation Sequencing Assay to Monitor Immune Responses in Solid Tumors

We have developed a next-generation sequencing assay to quantify biomarkers of the host immune response in formalin-fixed, paraffin-embedded (FFPE) tumor specimens. This assay aims to provide clinicians with a comprehensive characterization of the immunologic tumor microenvironment as a guide for therapeutic decisions on patients with solid tumors. The assay relies on RNA-sequencing (seq) to semiquantitatively measure the levels of 43 transcripts related to anticancer immune responses and 11 transcripts that reflect the relative abundance of tumor-infiltrating lymphocytes, as well as on DNA-seq to estimate mutational burden. The assay has a clinically relevant 5-day turnaround time and can be conducted on as little as 2.5 ng of RNA and 1.8 ng of genomic DNA extracted from three to five standard FFPE sections. The standardized next-generation sequencing workflow produced sequencing reads adequate for clinical testing of matched RNA and DNA from several samples in a single run. Assay performance for gene-specific sensitivity, linearity, dynamic range, and detection threshold was estimated across a wide range of actual and artificial FFPE samples selected or generated to address preanalytical variability linked to specimen features (eg, tumor-infiltrating lymphocyte abundance, percentage of necrosis), and analytical variability linked to assay features (eg, batch size, run, day, operator). Analytical precision studies demonstrated that the assay is highly reproducible and accurate compared with established orthogonal approaches.

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAKs critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein–protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTKHigh cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTKLow cells. Finally, HER2+ cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2+ cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028–39. ©2016 AACR.

PD-L2 amplification and durable disease stabilization in patient with urothelial carcinoma receiving pembrolizumab

ABSTRACT We report the immunological profile of a patient with upper-tract urothelial carcinoma experiencing stable disease on pembrolizumab for 20 months. The tumor exhibited extensive infiltration by CD8+ cytotoxic T lymphocytes, low-to-moderate mutational burden, no PD-L1 staining by commercially available immunohistochemical assays, but amplification of CD274 (coding for PD-L1) and/or PDCD1LG2 (encoding PD-L2) by fluorescence in situ hybridization. RNA-seq revealed multiple biomarkers of an ongoing immune response and compensatory immune evasion, including moderate PD-L1 levels coupled with robust PD-L2 expression. Pending validation in additional patients, these findings suggest that PD-L2 expression levels may constitute a biomarker of response to immune checkpoint blockade in urothelial carcinoma.

Abstract A64: Human epidermal growth factor receptor 2 (HER2) directly binds and activates focal adhesion kinase (FAK) to promote oncogenesis

Introduction: Human epidermal growth factor receptor 2 (HER2) is known to require focal adhesion kinase (FAK) for cellular transformation, tumor growth, invasion, and metastasis. FAK is overexpressed in nearly all types of human cancer, serving as a molecular scaffold protein to integrate oncogenic growth factor receptors with focal adhesion proteins. We have identified a novel direct protein-protein interaction between HER2 and FAK. In this study, we investigated whether the HER2-FAK direct interaction is required for HER2-dependent oncogenesis and its potential as a novel drug target. Methods: We have taken a multidisciplinary approach (biophysics, biochemistry, molecular modeling, structural biology, and molecular biology) to map the HER2-FAK binding interface and to study the functional aspects of the interaction. Results: Our data indicated that the N-lobe of the HER2 kinase domain directly interacts with the F1 lobe of the FAK FERM domain. In addition, molecular modeling studies have identified a putative HER2-FAK interface suitable for the binding of small molecule inhibitors. Site-directed mutagenesis studies validated this binding interface. Functional data indicated that HER2 directly transphosphorylated FAK at key tyrosine residues, Y397, Y861, and Y925, irrespective of FAK kinase activity. Additionally, kinome reprogramming analysis revealed upregulation of HER2 signaling and maintenance of FAK phosphorylation after FAK-kinase inhibitor treatment. Finally, cellular data suggested that loss of the HER2-FAK interaction, but not FAK-kinase activity, resulted in a defect in Heregulin β1-stimulated cell migration/invasion. Conclusion: We have identified a direct interaction between HER2 and FAK that is required for HER2-dependent oncogenic signaling and is amenable to targeting by small molecule therapeutics. In addition, this interaction mediated a novel drug resistance mechanism whereby HER2 rescued and maintained FAK activation under FAK-kinase inhibition. Citation Format: Timothy Marlowe, Sheila Figel, Felicia Lenzo, Vita Golubovskaya, Elena Kurenova, Alexander Tropsha, William Cance. Human epidermal growth factor receptor 2 (HER2) directly binds and activates focal adhesion kinase (FAK) to promote oncogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A64.

Abstract C77: Oncogenic drivers reactivate kinase-inhibited focal adhesion kinase (FAK) through phosphorylation of effector residues

Introduction: Focal Adhesion Kinase (FAK) is a critical signaling molecule showing elevated expression in nearly all tumor types. Phosphorylation of key tyrosine residues within FAK underlies its dual roles as both a kinase and a molecular scaffold that integrates signals from major oncogenes. Though FAK has emerged as a major drug target, inhibitors developed to date singularly target its kinase activity, leaving the scaffolding of oncogenic drivers intact. Our studies demonstrate that cells treated with FAK kinase inhibitors PF-573228, PF-562271, and defactinib retain phosphorylation of several key tyrosine residues downstream of oncogenic receptor tyrosine kinases (RTKs). Methods: We examined the phosphorylation status of key FAK scaffolding sites Y861 and Y925 in RTK expressing normal and cancer cells treated with FAK kinase inhibitors PF-573228, PF-562271, and defactinib. As Src kinase is known to directly phosphorylate Y861 and Y925, we also tested phosphorylation of these sites by RTKs in Src/Yes/Fyn (SYF) MEFs, which lack the major Src family kinases. Results: In cancer cells treated with FAK kinase inhibitors, multiple RTKs (HER2/3, PDGFR, and EGFR) activated FAK through the phosphorylation of effector tyrosines. Activation of HER2/3 in MDA-MB-453 breast cancer cells, PDGFR in U-87 glioblastoma cells, and EGFR in A549 lung cancer cells all stimulated Y861/Y925 phosphorylation in the presence of each of the three FAK kinase inhibitors. Moreover, Y861/Y925 phosphorylation occurred downstream of PDGFR and EGFR in Src-null SYF MEFs, demonstrating that RTK FAK targeting does not require Src. Finally, in vitro studies demonstrated that RTKs can directly phosphorylate these sites on FAK. Conclusion: We have identified a novel mechanism through which FAK retains signaling activity in the presence of kinase inhibitors. Continued FAK signaling in tumor cells which overexpress HER2, EGFR or PDGFR may therefore be a mode of clinical resistance to FAK kinase inhibitors. Citation Format: Sheila Figel, Felicia Lenzo, Timothy Marlowe, William Cance. Oncogenic drivers reactivate kinase-inhibited focal adhesion kinase (FAK) through phosphorylation of effector residues. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C77.