Abstract B084: Sotorasib/SHP2 inhibitors combo, KRASG12C(ON)I and RASMULTI(ON)I effectively target KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification
Authors: Solanki Hitendra S , Imbody Denis , Desai Bina , Kato Ryoji , Stewart Paul A , Stern Yaakov , Majumder Anurima , Bridenstine Liznair , Sarca Bhaswati , Miroshnychenko Daria , Aronchik Ida , Marusyk Andriy , Haura Eric B 
Published: December 2023

Abstract
Background: Amplification of KRASG12C mutant allele has been reported as one of the resistance mechanisms in tumors progressed to Sotorasib, the FDA approved KRASG12C(OFF) inhibitor. An identical mechanism was observed in one of our H358 xenograft tumors relapsed to Sotorasib. The cell line generated from relapsed tumor represents an appropriate in vitro model to study therapeutic opportunities in KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification.
Methods: We generated five Sotorasib resistant cell lines using relapsed tumors of Sotorasib-treated H358 xenografts (MR1-MR5) and two vehicle-treated tumors (MV1 and MV2). Whole exome sequencing (WES), Taqman copy number analysis and digital PCR to study mutant allelic fractions were performed to investigate acquired mechanisms of resistance. We have also generated a Dox-inducible system for H358 cells to overexpress KRASG12C protein and mimic KRAS amplification.
Results: We observed loss of sensitivity to Sotorasib in models MR1-MR5, when compared to MV1 and MV2. Western blot analysis show EMT-associated signatures in all resistant lines except MR2. The WES of resistant lines shows higher allelic frequency of KRASG12C mutation in MR2, which is homozygous (through loss of heterozygosity). The dPCR analysis further confirmed higher mutant allelic frequency in MR2. Copy-number analysis revealed the presence of more than 10 copies of KRAS in MR2. MR2 model represents the best model to investigate therapeutic options in KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification. Both RAS-RAF Proximity Ligation and Raf-RBD pull down assays confirm enhanced RAS signaling in MR2 cells. The combination of KRASG12C(OFF) and SHP2 inhibitors, or single agent KRASG12C(ON) or RASMULTI(ON) inhibitors suppressed enhanced MAPK signaling in MR2 cells and drove tumor suppressions in MR2 mice xenografts.
Conclusions: The stronger inhibition of KRAS pathway is necessary to suppress enhanced MAPK signaling in KRASG12C mutated lung cancer that developed secondary resistance to Sotorasib treatment through KRASG12C amplification. These preclinical data suggest that KRASG12C(OFF)/SHP2 inhibitors combo, or single agent KRASG12C(ON)I or RASMULTI(ON)I represent potential therapeutic strategies for these relapsed tumors.
Citation Format: Hitendra S Solanki, Denis Imbody, Bina Desai, Ryoji Kato, Paul A Stewart, Yaakov Stern, Anurima Majumder, Liznair Bridenstine, Bhaswati Sarca, Daria Miroshnychenko, Ida Aronchik, Andriy Marusyk, Eric B Haura. Sotorasib/SHP2 inhibitors combo, KRASG12C(ON)I and RASMULTI(ON)I effectively target KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B084.