Severine Kako

Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV-, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.

Abstract Oncogenic kinase fusions of ALK, ROS1, RET, and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1+ lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naïve cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion–positive lung cancer cells, mouse models, and human clinical specimens before the onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to cotarget EGFR to reduce the risks of developing drug resistance. Cancer Res; 77(13); 3551–63. ©2017 AACR.

8023 Background: The identification and therapeutic targeting of oncogenic drivers in lung adenocarcinoma has led to significant clinical improvements for patients with EGFR mutations or ALK fusions. However, many lung cancer patients do not yet have an identified oncogenic driver and the discovery of new actionable oncogenic drivers is thus an active area of investigation. Methods: Tumor samples from 36 ‘pan-negative’ (EGFR, KRAS, ALK, and ROS1) lung adenocarcinoma patients were analyzed using a next generation sequencing (NGS) test performed in a CLIA-certified lab (Foundation Medicine, Cambridge, MA). Fluorescence in situ hybridization (FISH) screening using a novel NTRK1 break-apart assay was performed on an additional 61 pan-negative samples. Cells expressing the novel NTRK1 fusions were assayed for transformation and pharmacologic inhibition. Results: Two tumor samples were identified with gene fusions containing the kinase domain of TrkA, encoded by NTRK1, including one each with an MPRIP-NTRK1 (M21;N14) and CD74-NTRK1 (C8;N12) fusion. RT-PCR confirmed mRNA expression and identity of the fusion partner and FISH analysis detected split 5’/3’ signals corresponding to the NTRK1 gene. A third sample was identified by FISH analysis. Cloning and expression of MPRIP- and CD74-NTRK1 into NIH3T3 and Ba/F3 cells show constitutive activation of the TrkA kinase domain and transformation. Treatment of cells expressing NTRK1 fusions with several candidate pan-Trk inhibitors (ARRY-772, -523, and -470) as well as CEP-701 and crizotinib demonstrate decreased phosphorylation of the fusion oncoprotein and inhibition of cell proliferation. Treatment of the index patient harboring the MPRIP-NTRK1fusion with crizotinib led to minor transient tumor shrinkage. Conclusions: We identified a novel class of oncogenes, NTRK1 fusions, in lung adenocarcinomas that can be detected by NGS or FISH. Additional studies to determine the frequency and characteristics of NTRK1 fusions in lung cancer are ongoing. Our findings suggest prospective clinical trials of Trk inhibitors in NTRK1 fusion positive patients may be warranted. Support: CO Bioscience Discovery and Evaluation Grant and CO Clinical and Translational Sciences Institute Grant.