Dual inhibition of HERs and PD-1 counteract resistance in KRAS G12C -mutant head and neck cancer

Basket clinical trials targeting the KRAS -mutation in solid tumors have shown initial promise, including in orphan KRAS head and neck cancer (HNC). However, development of resistance to KRAS -mutant-specific inhibitors (KRAS i) remains a major obstacle. Here, we investigated the intrinsic (tumor-cell autonomus) and tumor-microenvironment (TME) mechanisms of resistance to the KRAS i-MRTX849 and AMG510 in a unique syngenic murine KRAS -mutated HNC cell line. Western-blotting was used for protein abundance and activation, overexpression, and ligand activation studies to verify the intrinsic mechanism of resistance to KRAS i in KRAS -mutated HNC cell line, 4NQO-L. In vitro KRAS -acquired-resistant cells were developed from 4NQO-L (4NQO-L-AcR). MRTX849/lapatinib combination efficacy, and CD8 T-cells depletion, were assessed in C57BL/6 J mice and supplementation of anti-PD-1 (αPD-1) to MRTX849/lapatinib was also performed in 4NQO-L- KRAS i-senisitve and 4NQO-L-AcR tumors. Immunohistochemistry (IHC) and Immunoflourescence (IF) analyses were performed to profile the TME and programmed death-ligand 1 (PD-L1) expression in tumors. Activation and upregulation of EGFR and HER2/3 (pan-HERs) are the intrinsic mechanism of resistance to KRAS i in 4NQO-L cells, and blocking pan-HERs signaling with lapatinib enhanced MRTX849 efficacy in vitro by inhibiting the MAPK and AKT/mTOR pathways. 4NQO-L-AcR upregulated the expression of pan-HERs, and lapatinib treatment re-sensitized 4NQO-L-AcR to MRTX849. In mice, MRTX849 showed a slight anti-tumor effect, but in combination with lapatinib a significant tumor growth delay was observed, but all tumors progressed over time. Histopathology analysis of the TME revealed infiltration of CD8 T-cells after treatment combination, and these CD8 T-cells play a key role in MRTX849/lapatinib efficacy. MRTX849/lapatinib treatment upregulated PD-L1 overexpression in both stromal and tumor cells, which presumably suppressed CD8 T-cells and enabled immune escape and tumor progression. Supplementation of αPD-1 prolonged the progression-free survival of 4NQO-L-bearing mice treated with MRTX849/lapatinib. MRTX849/lapatinib treatment delayed tumor growth of 4NQO-L-AcR in mice; however, the percentages of CD8 T-cells in 4NQO-L-AcR were low, and supplementation of MRTX849/lapatinib with αPD-1 did not improve the outcome. Our study highlights the critical need for blocking both intrinsic and extrinsic mechanisms of resistance for the prolonged response