Abstract PR001: Mechanisms by which Mito-LND inhibits KRAS mutant lung cancer
Authors: Weh Katherine M. , Howard Connor L. , Zhang Yun , Riethoven Jean-Jack , Clarke Jennifer L. , Kalyanaraman Balaraman , You Ming , Kresty Laura A. 
Published: January 2023

Abstract
Lung cancer remains the most frequently diagnosed cancer and leading cause of cancer mortality in the United States and worldwide. Efficacious targeted options are urgently needed for lung cancer prevention and treatment. Approximately 30% of non-small cell lung cancer cases have KRAS mutations, yet pharmacological targeting has eluded the scientific community due to structural and biochemical properties of the KRAS protein. We recently synthesized Mito-lonidamine (Mito-LND), a mitochondrial targeted analog of lonidamine which inhibits aerobic glycolysis in cancer cells. We identified that Mito-LND induces autophagy, specifically mitophagy, preferentially in KRAS mutant lung adenocarcinoma cells and inhibits lung tumor development and brain metastasis in vivo. The cancer inhibitory mechanisms by which Mito-LND induces mitophagy, including requisite receptors and effects on immune signaling cascades remains unknown. We evaluated FDA-approved cell death inhibitors for autophagy (chloroquine; CQ), mitophagy (cyclosporin A; CsA) and necrosis (ponatinib) in KRAS (H2030 and H2030BrM3) mutant cells using cellular viability assays in combination with Mito-LND. siRNA was utilized to knockdown autophagy/mitophagy receptors (P62, PINK1, NDP52, OPTN and BECN1) in KRAS mutant cells, followed by Mito-LND treatment and viability assessments. Last, KRAS mutant cells were treated with Mito-LND to evaluate transcriptomic changes by RNA sequencing and deconvolution analysis performed to interrogate alterations in immune cell populations. CQ and CsA significantly inhibited Mito-LND induced cell death in H2030BrM3 cells. Interestingly, necrosis inhibition with ponatinib synergistically increased Mito-LND induced cell death in both cell lines. Of the receptors tested (P62, PINK1, OPTN and NDP52), NDP52 in H2030BrM3 cells appears requisite for Mito-LND induced cell death. Following transcriptomic sequencing, we conducted pathway analysis in Metacore on the significantly dysregulated genes (P and FDR&lt;0.05). Mito-LND significantly upregulated heat shock protein signaling, HIF1A transcriptional targets, ER stress and the immune response. Downregulation of cell adhesion was noted in both cell lines, with changes in EMT and WNT signaling noted only in H2030 cells. Gene set enrichment analysis revealed TNFα signaling via NF-κB as the only hallmark significantly enriched by Mito-LND. Deconvolution analysis discerned alterations to T and B cell populations, highlighting a potential role for Mito-LND in modulating adaptive immunity. These results begin to define the cancer inhibitory mechanisms by which Mito-LND induces cancer cell death, including requisite receptors, signaling pathways and immune cell populations in KRAS mutant cells. Future directions include evaluating potential synergy of Mito-LND in combination with additional cell death and KRAS-targeting drugs as well as characterizing autophagy/mitophagy receptors in vivo utilizing chemically induced and genetically driven mouse models of lung carcinogenesis.
Citation Format: Katherine M. Weh, Connor L. Howard, Yun Zhang, Jean-Jack Riethoven, Jennifer L. Clarke, Balaraman Kalyanaraman, Ming You, Laura A. Kresty. Mechanisms by which Mito-LND inhibits KRAS mutant lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR001.