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PurposeLung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g. alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). We previously demonstrated a positive association of a TKI-induced interferon gamma (IFNγ) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used murine models of EML4-ALK lung cancer to test a role for host immunity in the therapeutic response to alectinib.Experimental DesignThree murine EML4-ALK cell lines (EA1, EA2, EA3) were implanted orthotopically into the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by μCT. Immune cell content was measured by flow cytometry, multispectral immunofluorescence and CyTOF. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA.ResultsAll cell lines were sensitive to alectinib in vitro. EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of treatment while EA2 tumors were eliminated by TKI treatment. Alectinib induced inflammatory transcriptional programs and multiple chemokines in all cell lines while untreated tumors exhibited distinct baseline chemokine expression patterns and content of CD8+ T cells and myeloid subsets. When propagated in immune-deficient mice, all three cell line-derived lung tumor models exhibited significant shrinkage followed by prompt progression despite continuous alectinib treatment.ConclusionsThe findings support an hypothesis that host and TKI-stimulated production of chemokines by tumor cells promotes functional engagement of adaptive immune cells within the tumor microenvironment that enhances the durability and depth of TKI response.Statement of Translational RelevancePatients with metastatic lung cancer harboring ALK fusions are treated with targeted tyrosine kinase inhibitors (TKI) in the first line setting. Despite bearing the same driver oncogene, patients experience a range of tumor burden reduction and variable amounts of residual disease. Residual disease burden associates with patient survival and contributes to the emergence of drug resistance yielding treatment failure. The factors mediating this differential response to TKI and residual disease are incompletely understood. Our group has developed a panel of murine ALK driven lung cancer cell lines that reproducibly show differences in the depth and duration of response when implanted into immunocompetent mice. Data using this model indicate that the presence of CD8+ T cells is a major contributor to the depth and duration of response. These models will be critical in developing rational combination therapies to augment the immune microenvironment engagement along with TKIs to improve outcomes for these patients.

One person dies from melanoma every hour worldwide. The American Cancer Society estimates that in 2013 the 5-year survival rate for stage IV melanoma will remain unchanged at about 15%, even with new therapies. Autophagy (self-eating pathway) is a cellular recycling pathway that has been shown to play a role in normal physiological processes including nutrient and/or stress responses, antigen presentation, and aging. In melanoma, basal levels of autophagy appear to vary across tumor types and higher levels of autophagy are associated with increased hypoxia and poor clinical prognosis. Understanding the relationships between autophagy, melanoma and oncogene status will elucidate the molecular mechanisms/pathways contributing to this disease. Our preliminary research indicates that cutaneous malignant melanoma (CMM) cell lines differ in their response to chloroquine (CLQ), a classic inhibitor of autophagy. This study was designed to determine how differences in the autophagy pathway are influenced by oncogene status and whether induction of autophagy is oncogene dependent. Using LC3-II puncta and western blot quantification we evaluated the differential rate of autophagy by oncogene status using CMM cell lines. The rate of autophagy was characterized in 6 melanoma cell lines (e.g. SK-Mel 2, 19, 29, 94,103, and 147) and primary melanocytes to determine the extent to which these cell lines responded to CLQ and determined any decrease in cellular viability caused by treatment. Two common cutaneous melanoma activating mutations, BRAF and NRAS, appear to have opposing roles on the autophagy pathway in our metastatic cell lines. Our data indicate that the targeting of autophagy using CLQ in the BRAF oncogenic cell lines increases apoptosis and leads a significant decrease in viability. In addition, BRAF cell lines have higher basal protein expression of ATG12-ATG5 and LC3-II and a higher number of basal LC3-II punctae. Finally, the induction of autophagy through serum starvation results in differential basal LC3-II kinetics as measured by number of punctae between the BRAF and NRAS cell lines. These results indicate that BRAF is more dependent on autophagy for survival than NRAS mutant melanoma and that oncogene status dictates the sensitivity of melanoma cells to CLQ by altering autophagic flux, subsequently increasing apoptosis. Ongoing experiments include the evaluation of autophagy in patient samples to quantify expression of known autophagy markers as well an evaluation of LC3-II expression correlated with oncogene status. These findings are immediately translatable for patients that would benefit from CLQ co-therapy, which is already approved for clinical use. Citation Format: Kirsten A. M. White, Salina M. Torres, Todd A. Thompson, Chien-An A. Hu, Natalia J. Gurule, Marianne Berwick. Differential inhibition of autophagic pathways in melanoma is oncogene-dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2635. doi:10.1158/1538-7445.AM2014-2635

Metastatic melanoma offers an ongoing clinical challenge. According to the American Cancer Society, the 5-year survival rate in 2012 for stage IV melanoma is only about 15% even with therapies. To improve outcomes, it is important to investigate new pathways involved in the regulation of metastasis as possible targets for prevention or treatment, such as autophagy. Autophagy (self-eating pathway) destroys a cell's damaged proteins and organelles through lysosomal degradation and has been shown to play roles in a wide variety of normal physiological processes including energy metabolism, nutrient or stress responses, growth regulation, and aging. The role of dysregulated autophagy in melanoma has not been well characterized. Preliminary research found cutaneous malignant melanoma cells display high levels of autophagy suggesting that this cellular process may provide an active metabolic state for invasive and metastatic melanoma. Chloroquine (CLQ), a classic antimalarial agent, is a potent inhibitor of autophagy and has been used safely in human patients for decades. This study is designed to dissect the role of autophagy in melanoma using melanoma cell lines (e.g. SK-Mel 19, 103, and 147), and primary melanocytes as controls to determine whether these cell types respond to CLQ as measured by cell viability . Similar to pancreatic and prostate cancer cell lines, our preliminary results indicate that metastatic melanoma cells require autophagy for growth and show high sensitivity to CLQ at doses as low as 20uM which resulted in greater than 60% decreases in cell viability. These findings suggest that metastatic melanoma cell lines rely on lysosomal degradation to a greater extent than what has been reported for other cell types. In addition, both N-ras mutation and androgen deprivation mitigate the effects of CLQ on melanoma cells. Ongoing experiments will confirm the expression of autophagy related genes, as well as imaging to determine the rate of autophagy in these cell lines. Finally, we will introduce stripped hormonal factors including vitamin D and estrogen, to evaluate whether this addition will progressively restore the original chloroquine sensitive phenotype. Findings from this work have the potential to be immediately translatable to patients as CLQ is already approved for therapeutic use in other human diseases. Citation Format: Kirsten A. White, Salina M. Torres, Natalia J. Gurule, Marianne Berwick, Chien-An A. Hu. Inhibition of autophagy in melanoma: using cell lines to model chloroquine 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 2041. doi:10.1158/1538-7445.AM2013-2041