Efficacy and immune modulation of KRAS G12C inhibitor sotor…

Efficacy and immune modulation of KRAS G12C inhibitor sotorasib in murine KRAS G12C mutant non-small cell lung cancers with major co-occurring genomic alterations

Teng Zhou, Minh Truong Do, Haniel Araujo, Richard Lee, Ferdinandos Skoulidis

Department of Thoracic and Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

Background KRAS is the most frequently mutated oncogene in lung cancers, and KRAS G12C is the most frequent mutant isoform in non-small cell lung cancers (NSCLCs). Sotorasib (AMG510) has been approved by FDA in 2021 as the first potent and selective KRAS G12C inhibitor. However, the clinical efficacy of inhibitor monotherapy is curtailed by molecular adaptation and characterized by broad heterogeneity in the depth and duration of individual responses. In addition to their tumor cell-autonomous effects, KRAS G12C inhibitor may also recondition the tumor immune microenvironment (TIME) and synergize with anti-PD-1 therapy to promote tumor regressions and T cell memory. The contributions of major co-occurring genomic alterations to KRAS G12C inhibitor-triggered efficacy and immune modulation are poorly understood. Here we established several murine KRAS G12C mutant lung cancer cell lines with co-alterations of STK11/LKB1 loss (K G12C L), TP53 R172H oncogenic mutant (K G12C P), and sgRNA-induced TP53 loss (K G12C sgP), investigated the efficacies and immune modulations of KRAS G12C inhibitor sotorasib on these major K G12C NSCLC subtypes. Methods We derived several murine KRAS G12C lung cancer cell lines by delivering Adeno-Cre to K G12C L ( Kras LSL- G12C/+ ; Stk11 flox/flox ), K G12C P ( Kras LSL-G12C/+ ; Trp53 LSL- R172H/+ ), or delivering Lenti-Cre-Cas9-sgRNA( Trp53 ) to K G12C ( Kras LSL-G12C/+ ) genetically engineered mouse models (GEMMs). We validated the LKB1 expression and p53 function by western blot, characterized the histological tumor types by H&E staining of subcutaneously implanted tumors, assessed the cell growth capability by CellTiterGlo luminescence assays. We further determined the dose-dependent sensitivity in response to sotorasib treatment in vitro by CellTiterGlo luminescence assays, and the efficacy of sotorasib in vivo on different co-mutation cell lines in syngeneic C57BL6 wild type mice. Finally, we investigated the immune modulation effect of sotorasib on K G12C L, K G12C P, andK G12C sgP tumors by implanting cells subcutaneously in syngeneic C57BL6 wild type mice, treating mice with sotorasib for one week, and then collecting tumors for FACS-based immune profiling assays.






Figure 1: Characterization of murine KRAS G12C lung cancer cell lines derived from genetically engineered mouse models (GEMMs). (A) Western blot determination of LKB1 and p53 expression in derived K G12C L, K G12C P, and K G12C sgP cell lines. (B) Western blot validation of loss of normal p53 downstream signaling function in K G12C P, and K G12C sgP cell lines. (C) Western blot validation of MAPK signaling inhibition in response to K G12C inhibitor sotorasib. (D) Histological H&E staining of subcutaneously implanted tumors in syngeneic C57BL6 wild typemice.



Conclusions Sotorasib has significant inhibitory effects on K G12C L, K G12C P, and K G12C sgP cell lines in vitro, with K G12C L be the most sensitive. Sotorasib shows significant initial inhibition of tumor growth in syngeneic models, while resistance and re-growth finally occur in all lines . K G12C L, K G12C P and K G12C sgP tumors have different compositions of infiltrated immune cells. Sotorasib triggers a significant immune sensitization on K G12C L andK G12C Pbut not K G12C sgP tumors. The characterization of immune microenvironment modulation induced by sotorasib may contribute to design of combination strategies. Figure 4: Immune modulation of sotorasib on K G12C L, K G12C P, andK G12C sgP tumors. Cells were implanted as in Figure 3 and treated with vehicle or sotorasib (100 mg/kg, q.d.) for one week before subjected to FACS-based immune profiling. To allow having enough sample, mice were treated with a starting tumor volume 300-500 mm 3 .

Figure 2: In vitro cell growth and sotorasib sensitivity assays. (A) Cell growth were recorded as CellTiterGlo luminescence and normalized relative to 24 h post cell seeding. (B) Cells were seeded in 96-well non-transparent plate, after 24 h, sotorasib or DMSO vehicle at different concentrations were added, and incubated with cells for another 72 h before subjected to CellTiterGlo luminescence determination.


Figure 3: In vivo efficacy study of sotorasib on K G12C L, K G12C P, andK G12C sgP cell lines. 2 × 10 6 cells of each indicated cell line was implanted subcutaneously in syngeneic C57BL6 wild type mice. For each mouse, consecutive vehicle or sotorasib treatment (100 mg/kg, q.d.) was started when the tumor volume reached 200-250 mm 3 . Tumor volume was measured every day until it reached endpoint. Tumor volume and time to tumor doubling curves were plotted to show the efficacies.

1. Sotorasib for Lung Cancers with KRAS p.G12C Mutation. N Engl J Med 2021; 384:2371-2381 2. Co-occurring genomic alterations in non-small-cell lung cancer biology and therapy. Nat Rev Cancer 2019; 19(9):495-509

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