FGFRs are commonly altered in non-small cell lung cancer (NSCLC). FGFRs activate multiple pathways including RAS/RAF/MAPK, PI3K/AKT, and STAT, which may play a role in the cellular response to radiation. We investigated the effects of combining the selective FGFR 1-3 tyrosine kinase inhibitor AZD4547 with radiation in cell line and xenograft models of NSCLC. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, autophagy, cell cycle, and DNA damage signaling and repair assays. In vivo xenografts and IHC were used to confirm in vitro results. NSCLC cell lines demonstrated varying degrees of FGFR protein and mRNA expression. In vitro clonogenic survival assays showed radiosensitization with AZD4547 in two NSCLC cell lines. In these two cell lines, an increase in apoptosis and autophagy was observed with combined radiation and AZD4547. The addition of AZD4547 to radiation did not significantly affect gH2AX foci formation. Enhanced xenograft tumor growth delay was observed with the combination of radiation and AZD4547 compared with radiation or drug alone. IHC results revealed inhibition of pMAPK and pS6 and demonstrated an increase in apoptosis in the radiation plus AZD4547 group. This study demonstrates that FGFR inhibition by AZD4547 enhances the response of radiation in FGFR-expressing NSCLC in vitro and in vivo model systems. These results support further investigation of combining FGFR inhibition with radiation as a clinical therapeutic strategy.

M6620, a selective ATP-competitive inhibitor of the ATM and RAD3-related (ATR) kinase, is currently under investigation with radiation in patients with non–small cell lung cancer (NSCLC) brain metastases. We evaluated the DNA damage response (DDR) pathway profile of NSCLC and assessed the radiosensitizing effects of M6620 in a preclinical NSCLC brain metastasis model. Mutation analysis and transcriptome profiling of DDR genes and pathways was performed on NSCLC patient samples. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, cell cycle, and DNA damage signaling and repair assays. NSCLC brain metastasis patient-derived xenograft models were used to assess intracranial response and overall survival. In vivo IHC was performed to confirm in vitro results. A significant portion of NSCLC patient tumors demonstrated enrichment of DDR pathways. DDR pathways correlated with lung squamous cell histology; and mutations in ATR, ATM, BRCA1, BRCA2, CHEK1, and CHEK2 correlated with enrichment of DDR pathways in lung adenocarcinomas. M6620 reduced colony formation after radiotherapy and resulted in inhibition of DNA DSB repair, abrogation of the radiation-induced G2 cell checkpoint, and formation of dysfunctional micronuclei, leading to enhanced radiation-induced mitotic death. The combination of M6620 and radiation resulted in improved overall survival in mice compared with radiation alone. In vivo IHC revealed inhibition of pChk1 in the radiation plus M6620 group. M6620 enhances the effect of radiation in our preclinical NSCLC brain metastasis models, supporting the ongoing clinical trial (NCT02589522) evaluating M6620 in combination with whole brain irradiation in patients with NSCLC brain metastases.

Quantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevents accurate quantification. We report a high-throughput, inexpensive, reliable and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.

<p>Supplementary Table 1. Short Tandem Repeat analysis of cell lines used in this study. Supplementary Table 2. Antibodies used in this study. Supplemental Table 3: Dose Enhancement factors at survival fraction of 0.1 (DEF0.1) and Pvalues for radiation clonogenic curves. Western blot showing FGFR1, FGFR2 and FGFR3 protein levels in HTE and BeasB2 cell lines. Supplementary figure 2. Normalized intensity quantification of pMAPK and pAKT proteins determined by western blot after treatment with AZD4547 in three NSCLC cell lines. Supplementary figure 3. Combined radiation and AZD4547 induces autophagy.</p>

<p>Supplementary Table 1. Short Tandem Repeat analysis of cell lines used in this study. Supplementary Table 2. Antibodies used in this study. Supplemental Table 3: Dose Enhancement factors at survival fraction of 0.1 (DEF0.1) and Pvalues for radiation clonogenic curves. Western blot showing FGFR1, FGFR2 and FGFR3 protein levels in HTE and BeasB2 cell lines. Supplementary figure 2. Normalized intensity quantification of pMAPK and pAKT proteins determined by western blot after treatment with AZD4547 in three NSCLC cell lines. Supplementary figure 3. Combined radiation and AZD4547 induces autophagy.</p>

Background: In the curative setting for head and neck cancer (HNC) a common treatment is radiation combined with cetuximab, an antibody therapeutic targeting EGFR. Despite decades of research into improved treatments, therapeutic resistance remains a major challenge for this malignancy, with roughly 40% of patients developing recurrent disease. Recent evidence has suggested that autophagy, a cellular stress response, may be an additional contributor to therapy resistance, by protecting HNC cells from the cytotoxic effects of radiotherapy and the growth inhibitory effects of anti-EGFR treatment. The mechanism of radiation-induced autophagy is under current investigation. Methods: Cell lines were source from commercial sources, cultured under recommended conditions, and identity confirmed by short tandem repeat testing. Induction of autophagy was detected by immunoblot flux assays for LC3 and p62, immunofluorescent staining of autophagic vesicles, LC3 reporter flux assay, and flow cytometry using acridine orange. The effect of autophagy inhibition was tested using clonogenic survival assays. Induction of apoptosis was analyzed by immunoblot against cleaved caspase and PARP and via AnnexinV staining. Results: We evaluated a panel of both human papillomavirus (HPV) positive and negative HNC cell lines for autophagic response to both cetuximab (CTX) treatment and ionizing radiation (XRT). Flux assays revealed that both CTX and XRT treatment induced autophagy in a time- and dose-dependent manner. Immunofluorescent staining of LC3 to identify autophagic vesicles showed that a relatively small fraction of the total cell population is able to induce this response. Flow cytometry analysis demonstrated that autophagic cells were largely non-apoptoic. For example, in the UM-SCC47 cell line treated with CTX for 48 h, flow cytometry for autophagy (20.8%), apoptosis (13.7%) or dual staining (5.4%) suggests a cytoprotective role for autophagy. The addition of the ULK1 inhibitor, SBI-0206965) to CTX and XRT induced apoptosis as shown by caspase activity and AnnexinV staining and reduced clonogenic cell survival. Conclusions: These preclinical studies have established the proof of concept for the cytoprotective effect of autophagy in response to anticancer treatments including EGFR inhibition and radiotherapy in HNC. Further, we have identified the addition of specific autophagy inhibitors to standard treatments as a potential strategy to overcome this mechanism of resistance. Citation Format: Jaimee Eckers, Justin Skiba, Gopika Senthilkumar, Kwang P. Nickel, Adam D. Swick, Randall J. Kimple. Autophagy contributes to therapeutic resistance in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1344.

No abstract

Background: Radiation and EGFR-targeted therapies are commonly used in the treatment of head and neck squamous cell carcinoma (HNSCC). These treatments fail to control a significant number of cancers resulting in a 5-year survival rate that remains around 40-50%. We have shown that both cetuximab and radiation induce autophagy, a pro-survival cellular stress response, in head and neck cancer. In this study, we examine the consequence of autophagy inhibition and investigate the molecular mechanism underlying therapy-induced autophagy. Methods: Autophagy was assessed using a nano-Luc LC3 reporter (Promega), immunofluorescence for LC3, p62, and acridine orange in HNSCC cell lines. RNAi knockdown of EGFR and LAPTM4B were used to test the involved signaling molecules. Radiation was delivered using a RS225 cabinet irradiator at a dose rate of approximately 3 Gy/min with dose validation by TLD using custom phantoms. Cetuximab was delivered via intraperitoneal injection. Vps34 inhibitor SAR405 and ULK1 inhibitor SBI-0206965 were used to determine whether inhibition of autophagy reduces cell survival or represses cancer cell growth in the clonogenic assay. A flank xenograft model using A253 cells was used to test the combination of autophagy inhibitors and current therapies in vivo. Results: As previously shown, both cetuximab and radiation induced autophagy by two times. Knockdown of EGFR and LAPTM4B decreased autophagy (62.5% and 65%, respectively) when assessed using the nano-Luc reporter assay. Similar results were seen using IF. Using a clonogenic survival assay, the combination of SAR405 and radiation resulted in complete loss of cell survival suggesting a radiosensitizing effect. In vivo, SAR405 treatment improved tumor control when combined with radiation or cetuximab when compared to either treatment alone. Conclusions: Therapy induced autophagy is dependent upon expression of both EGFR and LAPTM4B. Inhibition of autophagy resulted decreased cell survival in vitro and resulted in decreased in vivo tumor growth. These results suggest that inhibition of autophagy may be a viable approach to sensitize HNSCC to anti-cancer treatments. Citation Format: Yong-Syu Lee, Justin Skiba, Jaimee Kubatzke, Kwangok P. Nickel, Randall Kimple. Inhibition of autophagy increases HNSCC sensitivity to cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4273.