ixazomib and Lung-Neoplasms

mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.

Topics: Animals; Antineoplastic Agents; Benzodioxoles; Boron Compounds; Cell Line, Tumor; Enhancer of Zeste Homolog 2 Protein; Gene Knockdown Techniques; Gene Knockout Techniques; Glycine; GTPase-Activating Proteins; HEK293 Cells; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Mice; Mutagenesis, Site-Directed; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase Inhibitors; Protein Stability; Proto-Oncogene Proteins p21(ras); Quinazolines; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

Topics: Amino Acids; Animals; Antineoplastic Agents; Boron Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Drug Resistance, Neoplasm; Fatty Acids; Glucose Transporter Type 4; Glycine; HCT116 Cells; Humans; Lung Neoplasms; Metabolome; Mice; Oxidation-Reduction; Proteasome Inhibitors; Proto-Oncogene Proteins p21(ras); Xenograft Model Antitumor Assays