https://openworks.mdanderson.org/sumexp22/1118/thumbnail.jp

Abaya, Camille A

Kadara, Humam

Liu, Yuejiang

Rahal, Zahraa

Sinjab, Ansam

English

OpenWorks @ MD Anderson

Prevention of early stage Kras-mutant lung adenocarcinoma via targeted KrasG12D inhibitionCamille Abaya1, Yuejiang Liu2, Zahraa Rahal3, Ansam Sinjab3, Humam Kadara31Trinity University, San Antonio, TX ; 2 MD Anderson Cancer Center Graduate School of Biomedical Sciences, Houston, TX; 3 Department of Translational Molecular PathologyBackgroundResults• Lung adenocarcinoma (LUAD) represents the most common subtype of lung cancer1. Patients with KRAS-mutant LUADs (KM-LUADs) common to smokers display dismal clinical outcomes and resistance to targeted therapies3. • Our group recently identified alveolar intermediary cells (AICs) that arise early on post-tobacco carcinogen exposure in vivo and during AT2-mediated repair mechanisms. Notably, and prior to tumor onset, a subset of AICs harbored driver KrasG12D mutations which were later found in the resultant LUADs, and which comprise the same variants found in human smokers with KM-LUADs. These findings support a role for AICs as precursors for KM-LUAD pathogenesis (Fig 1a). Our ongoing studies are studying AICs at high resolution to identify targets for early prevention and/or interception of KM-LUAD pathogenesis, including specifically targeting KrasG12D mutation (Fig 1b).• Mutated KRAS has been an elusive target prior to the discovery of a conserved switch II binding pocket among all KRAS proteins. A structure-based drug design identified MRTX 1133 as a potent and selective KRASG12D inhibitor in both in vitro and in vivo models (Fig 2)2. While previous testing utilized patient-derived xenograft tumor models and orthotopic pancreatic cancer models2, we investigate the effectiveness of MRTX 1133 in murine lung cancer cells as a precursor to potential clinical prevention of KM-LUAD in vivo.Objectives• Confirm the ability of MRTX 1133 to specifically downregulate downstream products of KrasG12D in a murine LUAD cell line (mF471).• Study the pharmacokinetics of murine KrasG12D inhibition by MRTX 1133 to determine the onset of action and minimal inhibitory concentration (MIC).Methods• MTT Proliferation Assay: mF471 cells were plated on day 1, treated with MRTX 1133 (0nM, 1 nM, 10 nM, and 100nM) and Epidermal Growth Factor (50 ng/mL) on day 2, and MTT proliferation assay measured on day 4 (Fig 3a). Each well contains 1000 cells. • Western Blot: mF471 cells were plated on day 1, treated with MRTX 1133 (0 nM, 1nM, 10nM, and 100nM) and Epidermal Growth Factor (50 ng/mL) on day 3 for 1 and 3 hours before analysis (Fig 3b). 200 ug protein was loaded per lane.Figure 3. (a) MTT Proliferation Assay of mF471 cells treated with MRTX1133. * P <0.05, ** P <0.01, *** P <0.001. (b) Immunoblot protein western analyses of mF471 cells treated with (C) media alone for control; (E) 50 ng/mL EGF; (1) 50 ng/mL EGF + 1nM MRTX1133; (10) 50 ng/mL EGF + 10 nM MRTX1133; (100) 50 ng/ mL EGF + 100 nM MRTX 1133.Figure 1. (a) Trajectory analysis of KACs and malignant cells implicated in human KM-LUAD development. Adapted from Han G (2022).(b) Fraction of KrasG12D mutant cells (right) in KACs, malignant, AT1 and AT2 subsets. Absolute numbers of KrasG12D mutant cells are indicated on top of each bar. Figure and legend adapted from Han G, Sinjab A (2022).AFigure 2. Crystal structure of KRASG12D bound to MRTX 1133 via the switch II binding pocket. Figure adapted from Christensen J (2022). BConclusions & Future Directions• Increasing concentrations of MRTX 1133 result in statistically significant decreases in cell viability when compared to control. • MRTX1133 shows dose-dependent inhibition of Kras signaling at all time points tested. A significant reduction in key KRAS pathway signaling molecules were observed as early as 3 hours and with concentration as little as 1 nM.• These results suggest that MRTX 1133 is a potent KrasG12D inhibitor which has the potential to inhibit murine Krasmutant lung cancer cell lines. • Further experiments are warranted to assess the role of inhibiting KrasG12D during the early stages of LUAD pathogenesis in an in vivo model (Figure 4). Our group previously showed that mice with knockout of Gprc5a (Gprc5a-/-) develop pre-malignant lesions (PMLs) and LUADs which were markedly accelerated with exposure to nicotine-specific nitrosamine ketone (NNK), a tobacco carcinogen that is casually linked to lung cancer in humans. Hence, we will investigate the anti-tumor effects (including preventive) of MRTX 1133 in a mouse model of tobacco-associated KM-LUAD (Gprc5a-/-) and in 3D organoid models of tumor precursors from the same mice.AReferences & SupportThank you to Dr. Humam Kadara, Dr. ZahraaRahal, Dr. Ansam Sinjab, Yuejiang Liu for mentorship, and the directors and organizers of the ITERT Summer Experience, etc.Thank you to Mirati Therapeutics for the use of MRTX 1133.1) Han G, Sinjab A, et al. BioRxiv. 20222) Christensen J, et al. Research Square. 20223) Fujimoto J et al. Int J Cancer.2017;141(8):1589-1599.Figure 4. Experimental scheme to investigate the anti-tumor potential of MRTX 1133 in Gprc5a-/-mice.  Briefly, NNK will be given for 8 weeks. MRTX1133 (or control) will be administered at several stages of LUAD pathogenesis (AICs, PMLs, and invasive LUAD).B

Prevention of early stage Kras-mutant lung adenocarcinoma via targeted KrasG12D inhibition

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1118&context=sumexp22

Prevention of early stage Kras-mutant lung adenocarcinoma via targeted KrasG12D inhibition

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