Antitumor Activity of Tipifarnib and PI3K Pathway Inhibitor in HRAS-associated HNSCC

Purpose/Objective(s) HRAS-MAPK and PI3K-AKT-mTOR are important oncogenic pathways in squamous cell carcinomas (SCCs) including those of the head and neck (HNSCC). Although HRAS mutations occur at a rate of ∼5% in HNSCCs, HRAS overexpression is present in up to 30% of HNSCC tumors, raising the possibility that some HRAS wild-type (WT) HNSCCs may also display a degree of HRAS dependence. PI3Kα (the catalytic subunit of PI3K), another prominent driver in HNSCC, is activated by gain-of-function mutations or PIK3CA gene amplification in about 30% of HNSCC patients. Multiple reports indicate that the HRAS and PI3K pathways cooperate and crosstalk to drive tumor progression and resistance to targeted therapies in SCCs. In this study, we explored whether combined inhibition of HRAS (via tipifarnib) and PI3K signaling (via PI3Ka inhibitor alpelisib) would have greater anti-tumor activity in cell line and patient-derived xenograft (PDX) models of HRAS-associated SCCs relative to monotherapy approaches. Materials/Methods HNSCC PDX combination studies were performed in Balb/c nu/nu or SCID mice. PDX were allowed to establish to 250-300mm3 after which the animals were randomized and treated orally with vehicle or tipifarnib (60-80 mg/kg BID) alone or in combination with alpelisib (40mg/kg QD) for 25-30 days. Results In a panel of HNSCC cell lines harboring HRAS and/or PIK3CA alterations (mutation, overexpression or amplification), tipifarnib reduced cell growth and, in combination with PI3K-a inhibitor alpelisib, induced cytotoxicity. Consistent with in vitro findings, regressions with the tipifarnib-PI3Ka inhibitor doublet were observed in tumors bearing HRAS mutation or overexpression, PIK3CA mutation or gene amplification, as well as tumors harboring alterations in both genetic drivers, suggesting that concomitant inhibition of HRAS/PI3Ka may have surprisingly broad and potent anti-tumor activity in HNSCC. In dose-scheduling experiments in PDX models, simultaneous blockade of both targets was superior to split intermittent dosing of the two drugs, underlining the extent of pathway cooperativity in these models. Mechanistically, the tipifarnib/alpelisib combination inhibited mTOR activity more effectively than single-agent treatment. Tipifarnib blocked the compensatory mTOR reactivation induced by alpelisib alone, resulting in durable inhibition of mTOR signaling and cell growth. Conclusion Tipifarnib muted mTOR reactivation induced by alpelisib explaining cytotoxic cellular response to the combination in HRAS/PIk3CA dysregulated HNSCC models. We contend that combination of tipifarnib with a PI3Ka inhibitor holds therapeutic potential for the treatment of R/M HNSCCs harboring alterations in HRAS and/or PIK3CA which will be evaluated in the recently initiated KURRENT clinical trial (NCT04997902). HRAS-MAPK and PI3K-AKT-mTOR are important oncogenic pathways in squamous cell carcinomas (SCCs) including those of the head and neck (HNSCC). Although HRAS mutations occur at a rate of ∼5% in HNSCCs, HRAS overexpression is present in up to 30% of HNSCC tumors, raising the possibility that some HRAS wild-type (WT) HNSCCs may also display a degree of HRAS dependence. PI3Kα (the catalytic subunit of PI3K), another prominent driver in HNSCC, is activated by gain-of-function mutations or PIK3CA gene amplification in about 30% of HNSCC patients. Multiple reports indicate that the HRAS and PI3K pathways cooperate and crosstalk to drive tumor progression and resistance to targeted therapies in SCCs. In this study, we explored whether combined inhibition of HRAS (via tipifarnib) and PI3K signaling (via PI3Ka inhibitor alpelisib) would have greater anti-tumor activity in cell line and patient-derived xenograft (PDX) models of HRAS-associated SCCs relative to monotherapy approaches. HNSCC PDX combination studies were performed in Balb/c nu/nu or SCID mice. PDX were allowed to establish to 250-300mm3 after which the animals were randomized and treated orally with vehicle or tipifarnib (60-80 mg/kg BID) alone or in combination with alpelisib (40mg/kg QD) for 25-30 days. In a panel of HNSCC cell lines harboring HRAS and/or PIK3CA alterations (mutation, overexpression or amplification), tipifarnib reduced cell growth and, in combination with PI3K-a inhibitor alpelisib, induced cytotoxicity. Consistent with in vitro findings, regressions with the tipifarnib-PI3Ka inhibitor doublet were observed in tumors bearing HRAS mutation or overexpression, PIK3CA mutation or gene amplification, as well as tumors harboring alterations in both genetic drivers, suggesting that concomitant inhibition of HRAS/PI3Ka may have surprisingly broad and potent anti-tumor activity in HNSCC. In dose-scheduling experiments in PDX models, simultaneous blockade of both targets was superior to split intermittent dosing of the two drugs, underlining the extent of pathway cooperativity in these models. Mechanistically, the tipifarnib/alpelisib combination inhibited mTOR activity more effectively than single-agent treatment. Tipifarnib blocked the compensatory mTOR reactivation induced by alpelisib alone, resulting in durable inhibition of mTOR signaling and cell growth. Tipifarnib muted mTOR reactivation induced by alpelisib explaining cytotoxic cellular response to the combination in HRAS/PIk3CA dysregulated HNSCC models. We contend that combination of tipifarnib with a PI3Ka inhibitor holds therapeutic potential for the treatment of R/M HNSCCs harboring alterations in HRAS and/or PIK3CA which will be evaluated in the recently initiated KURRENT clinical trial (NCT04997902).