Abstract 1978: The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer
Authors: Castel Myra E. , Conlon Neil T. , Walsh Naomi , Bischof Georg F. , Crown John P. , Collins Denis M. 
Published: March 2024

Abstract
Introduction: Constitutive activation of KRAS through gain-of-function mutations, such as KRASG12C, is thought to reduce sensitivity to HER-targeted TKIs like neratinib (N), lapatinib (L) and tucatinib (T). Sotorasib (S) is a KRASG12C specific inhibitor clinically approved for the treatment of advanced KRASG12C-mutated non-small cell lung cancer. One identified resistance mechanism to sotorasib is the increase of multiple phosphorylated receptor tyrosine kinases, including EGFR and HER2. The objective of this study was to assess the in vitro efficacy of the single agents N, L, T and S, and the combination of N and S in HER2-amplified, KRASG12C-mutated cancer cell lines.
Methods: The antiproliferative effects of N, L, T and S were assessed in the HER2-amplified and KRASG12C-mutated KYSE-410 (esophageal) and NCI-H2030 (lung) cancer cell lines, and KRASG12C-mutated MiaPaca-2 (pancreatic) cell line by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between N and S, matrix assays were performed and analyzed using Combenefit software. To further determine the efficacy of the drugs, changes in signaling pathways were assessed by Western blotting.
Results: N displayed nanomolar IC50 values (&lt; 100 nM) in all three cell lines, while nanomolar IC50 values with L and T were only achieved in KYSE-410 (IC50 = 426.84 nM and IC50 = 363.38 nM respectively). S was most potent in MiaPaca-2 (IC50 = 6.04 nM), followed by NCI-H2030 (IC50 = 270.60 nM) and KYSE-410 (IC50 = 5.46 µM). Matrix assays showed that NS was broadly additive in MiaPaca-2 cells, and synergistic in both NCI-H2030 and KYSE-410 cell lines. N and NS numerically decreased HER2 levels compared to control in NCI-H2030 after 24h of treatment (6.4-fold, p = 0.58 and 2.5-fold, p = 0.79 respectively) but not in KYSE-410. KRAS levels were significantly increased (1.89-fold, p ≤ 0.05) after treatment with NS in NCI-H2030. No significant effects were observed on the activation of HER2 and EGFR in either cell line at 24h. In NCI-H2030, NS had a significantly stronger inhibitory effect on pERK compared to N (p ≤ 0.0001) and S (p = 0.012) alone. In KYSE-410, pERK levels were also slightly decreased with NS, but not significantly different from N (p = 0.12). However, pAkt levels in KYSE-410 were significantly reduced by N (1.3-fold, p = 0.009) and NS (1.7-fold, p ≤ 0.0001) compared to control, with NS showing a significantly greater decrease than N (p = 0.026). It thus appears that the synergy between N and S occurs through a stronger inhibition of ERK activity in NCI-H2030, while it occurs through a stronger inhibition of Akt activity in KYSE-410.
Conclusion: NS is additive or synergistic in the three KRASG12C-mutated cancer cell lines examined. Further work is planned to investigate the basis of the synergy observed.
Citation Format: Myra E. Castel, Neil T. Conlon, Naomi Walsh, Georg F. Bischof, John P. Crown, Denis M. Collins. The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1978.