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The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression.SignificanceMutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis.

Background: Basket clinical trials targeting the KRASG12C-mutation in solid tumors have shown initial promise, including in orphan KRASG12C head and neck cancer (HNC). However, development of resistance to KRASG12C-mutant-specific inhibitors (KRASG12Ci) remains a major obstacle. Here, we investigated the intrinsic (tumor-cell autonomus) and tumor-microenvironment (TME) mechanisms of resistance to the KRASG12Ci—MRTX849 and AMG510 in a unique syngenic murine KRASG12C-mutated HNC cell line. Methods: Western-blotting was used for protein abundance and activation, overexpression, and ligand activation studies to verify the intrinsic mechanism of resistance to KRASG12Ci in KRASG12C-mutated HNC cell line, 4NQO-L. In vitro KRASG12C-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– KRASG12Ci-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. Results: Activation and upregulation of EGFR and HER2/3 (pan-HERs) are the intrinsic mechanism of resistance to KRASG12Ci 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. Conclusions: Our study highlights the critical need for blocking both intrinsic and extrinsic mechanisms of resistance for the prolonged response and shows that such treatment is ineffective in KRASG12Ci-AcR tumors. [ABSTRACT FROM AUTHOR]