Autophagy as a Target Pathway in Multiple Myeloma: A Forward Chemical Genetic Approach.

Autophagy is an evolutionarily conserved process by which cellular structures may be degraded to support ongoing biogenesis. Recent evidence has identified the induction of autophagy following growth factor pathway inhibition, rapid tumor expansion and proteasome inhibition. Thus, autophagy is a highly desirable pathway for the development of targeted therapeutics in cancer, in particular in tumors characterized by misfolded protein stress such as multiple myeloma (MM). Unfortunately, there are only several known classes of autophagy inhibitors and the emerging biology of autophagy has identified few validated targets. We therefore performed a forward chemical genetic study of autophagy using novel techniques in high-content imaging, high-throughput screening and multidimensional data analysis. Using LN229 glioblastoma cells stably transfected with an LC3-EGFP construct, we created a robust screening platform capable of quantifying the number, size and fluorescence intensity of autophagosomes. Assay validation was performed with the known autophagy inhibitor chloroquine, which induces a characteristic shift from diffuse to punctate fluorescence. Initially, we profiled a library of approximately 3,500 structurally diverse bioactive small molecules to leverage possible downstream mechanistic insights. Assay positives and controls were re-tested in the adherent H1299 adenocarcinoma cell line likewise stably expressing LC3-EGFP. Known autophagy inhibitors were enriched with this approach: nocodazole, bafilomycin A1, colchicine and monensin. Structure-activity relationship analysis of the primary screening data and 34 novel autophagy modulators revealed a compelling activity of bis-indolyl maleimides. We confirmed sub-micromolar inhibition of autophagy by three such compounds using a quantitative, radiolabeled protein degradation assay (tritiated tyrosine pulse-chase), LC3-II immunoblotting and quantitative measurement of autophagosomes across three cell lines. From these studies we selected a lead compound, K252a, for further characterization and tested twenty-two closely related analogues. Detailed SAR analysis has identified features of the molecule required for inhibition of autophagy and sites permissive for further functionalization in ongoing target identification studies. Using MM as a translational model system of misfolded protein stress, we found dose-dependent, selective cytotoxicity in MM of K252a compared to analogues lacking autophagy inhibitory activity. We report here the results of a forward chemical genetic study of autophagy which identifies and characterizes novel small molecule inhibitors of autophagy and establishes a rationale for clinical studies of autophagy inhibitors in the treatment of MM.