Anti-PD1 prolongs the response of PI3K and farnesyl transferase inhibition in HRAS- and PIK3CA-mutant head and neck cancers

Abstract

Background: Tipifarnib, a farnesyl transferase inhibitor, has shown promising response in the treatment of HRAS-mutant HNSCC in the clinic, and in combination with a PI3K inhibitor in PIK3CA-mutant mouse models; however, the involvement of antitumor immunity in the efficacy of tipifarnib has not yet been investigated. This study aimed to evaluate the involvement of antitumor immunity in the efficacy of tipifarnib in HRAS- or PIK3CA-mutant HPV-positive and HPV-negative head and neck cancer murine models.

Methods: To investigate the role of antitumor immunity, we compared the efficacy of tipifarnib in immune-intact C57BL/6 mice and immunodeficient NSG mice. Histopathological analyses were conducted to evaluate PD-L1 expression and the activation of key signaling pathways. Additionally, the synergistic potential of tipifarnib with the PI3Kα inhibitor alpelisib (BYL719) was assessed in vitro and in vivo. Immunohistochemical analysis was performed to examine the infiltration of CD8⁺T cells, and anti-PD1 treatment was tested to evaluate its potential to prolong progression-free survival.

Results: In the HPV-positive HRAS-mutant HNSCC model, the antitumor efficacy of tipifarnib was primarily dependent on CD8⁺T cell activity, whereas in HPV-negative cancers, the contribution of antitumor immunity was less pronounced. Tipifarnib treatment upregulated PD-L1 expression, potentially inhibiting T cell antitumor activity and inducing hyperactivation of the AKT pathway, which mitigated MAPK inhibition and promoted cell proliferation. Blocking the PI3K pathway with alpelisib demonstrated synergistic antitumor effects in all models. The combination of tipifarnib and alpelisib exhibited greater efficacy in immune-intact mice than in immunodeficient mice, and was accompanied by increased CD8⁺T cell infiltration. Adding anti-PD1 treatment to the tipifarnib/alpelisib combination further prolonged progression-free survival in tumor-bearing mice.

Conclusion: These findings underscore the critical role of antitumor immunity, particularly CD8⁺T cell activity, in the efficacy of tipifarnib alone and in combination with alpelisib. The triple combination of tipifarnib, alpelisib, and anti-PD1 treatment showed superior antitumor activity and extended survival in preclinical models, suggesting its potential as a therapeutic strategy for HNSCC patients with HRAS- and PIK3CA-mutation.

Keywords: Alpelisib; Anti-PD1; HRAS; Head and neck cancer; PI3K; Tipifarnib.

Effect of combination therapy of tipifarnib, alpelisib and anti-PD1 on HRAS and PIK3CA mutant HNSCC tumors.

Fig. 1

Tipifarnib efficacy is enhanced by antitumor immunity. (A) Schematic representation of the in vitro viability assay using tipifarnib (created using www.biorender.com). (B) Viability of HPV-positive HRAS mutant (mEERL), HPV-negative HRAS-mutant (FT1, HRAS^V12 shp53 EpT), and PIK3CA-mutant (S26-117) cell lines treated with increasing doses of tipifarnib treatment for four days and the IC₅₀ values are shown. (C) Schematic representation of the pulsative treatment regimen (tipifarnib 60 mg/kg by oral administration, twice daily for three days) and the comparison of WT vs. NSG mice (created using www.biorender.com). (D) Tumor volume of the mEERL, FT1, HRAS^V12 shp53 EpT, and S26-117 syngeneic murine models in WT and NSG mice; 3  ×  10⁶ cells were injected orthotopically, mice were randomized into two groups. (n = 5) treated with vehicle or tipifarnib.

Fig. 2

Tipifarnib induces AKT activation in tumor cells and promotes immune escape via PD-L1 upregulation in HPV-negative HRAS mutant HNSCC, whereas tipifarnib efficacy depends on CD8⁺T cell infiltration in HPV-negative HRAS mutant HNSCC. (A) Representative immunohistochemical staining images showing the expression of cleaved caspase-3, Ki67, pMAPK, pAKT, pS6, CD45, CD8, and PD-L1 in mEERL tumors treated with vehicle or tipifarnib (60 mg/kg) for three days (scale bars: 100 μm; insets 20 μm). Quantification of positive cells (n = 3 tumors and n = 15 analysis fields). Error bars indicate SEM. Statistical significance was calculated using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); ns denotes not significant). (B) IHC images showing the expression of cleaved caspace-3, Ki67, pMAPK, pAKT, pS6, CD45, CD8, and PD-L1 in FT1 tumors treated with vehicle or tipifarnib (60 mg/kg) for three days (scale bars: 100 μm; insets 20 μm). Quantification of positive cells (n = 3 tumors and n = 15 analysis fields). Error bars indicate SEM. Statistical significance was calculated using a one-way analysis of variance (ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001; ns denotes not significant). (C) Growth of mEERL tumors in WT mice: 3 × 10⁶ cells were injected orthotopically, and mice were randomized into six treatment arms (n = 6) with tipifarnib, with or without depletion of CD8⁺T cells and CD20⁺B cells. Right: Fold change in volumes of mEERL tumors treated with tipifarnib with or without depletion of CD8⁺T cells and CD20⁺B cells.

Fig. 3

Enhanced efficacy of tipifarnib/alpelisib combination in both HRAS and PIK3CA mutant cells in vitro. (A) Colony formation assay with tipifarnib (1 μm), alpelisib (2 μm), and tipifarnib/alpelisib treatment in mEERL, FT1, S26-117, and S24-658 cell lines. (B) Four days cell proliferation assay was performed with tipifarnib (1μM), alpelisib (2 μm), and tipifarnib/alpelisib treatment in mEERL, FT1, S26-117, and S24-658 cell lines. (C) Synergy score and heat map calculated using SynergyFinder+ for the combination of tipifarnib and alpelisib in mEERL, FT1, S26-117, and S24-658 HNSCC cell lines. (D) Western blot analysis of the indicated proteins after 2 and 24 h of tipifarnib (1 μm), alpelisib (2 μm), and combination (tipifarnib/alpelisib) treatment in FT1 and S26-117 cell lines. Numbers indicate the fold-change in protein levels normalized to βactin. Data represent representative experiments from three independent experiments. Statistical significance was calculated using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 4

Tipifarnib/alpelisib displayed improved efficacy in HRAS and PIK3CA mutant in vivo models. (A) Tumor volume of FT1 syngeneic murine models of WT and NSG mice. 5  ×  10⁶ cells were injected subcutaneously, and mice were randomized into two groups. (WT, n = 14) and (NSG, n = 6) and treated with vehicle or tipifarnib/alpelisib [alpelisib (25 mg/kg daily once) and tipifarnib (60 mg/kg, twice daily]. (B) Representative immunohistochemical staining images of FT1 tumors harvested from WT mice at two different time points - 8^th and 16^th day, showing the expression of cleaved caspase-3, Ki67, pMAPK, pAKT, pS6, CD45, CD8, and PD-L1 in FT1 tumors treated with vehicle or tipifarnib/alpelisib (scale bars: 100 μm; insets: 20 μm). Error bars indicate SEM. Statistical significance was calculated using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns denotes not significant).

Fig. 5

A triple combination of tipifarnib, alpelisib, and anti-PD1 promoted tumor regression in HPV-negative HRAS- or PIK3CA-mutant tumors. Tumor volumes of (A) FT1 syngeneic murine models in C57BL/6 J (WT) (n = 15, 5  ×  10⁶ cells were injected orthotopically into the lip), (B) S26-117, and (C) S24-658 syngeneic murine models in C57BL/6 J (WT) (n = 18, 5  ×  10⁶ cells were injected subcutaneously), and mice were randomized into three groups. and treated with vehicle, tipifarnib/alpelisib + IgG, or tipifarnib/alpelisib + anti-PD1 (n = 5-6). IgG and anti-PD1 were administered via I.P injection once every five days. Right: Fold changes in tumor volumes of FT1, S26-117, and S24-658 tumors treated with vehicle, tipifarnib/alpelisib + IgG, or tipifarnib/alpelisib + anti-PD1. Error bars indicate SEM. Statistical significance was calculated using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns denotes not significant).

Fig. 6

Diagrammatic illustration of the intrinsic and extrinsic responses of tumors with HRAS/PIK3CA mutations to various treatments: tipifarnib, alpelisib, the combination of tipifarnib and alpelisib, and triple therapy with tipifarnib, alpelisib, and anti-PD1. (A) Resistance to tipifarnib treatment in HRAS-driven HNSCC. Tipifarnib monotherapy in HRAS-mutant tumors resulted in AKT overactivation due to a compensatory mechanism involving the PI3K-AKT-mTOR signaling cascade, leading to tumor proliferation and growth. (B) Resistance to alpelisib treatment in PIK3CA-driven HNSCC. In tumors with PIK3CA mutations, treatment with alplelisib triggers hyperactivation of the RAS-MAPK pathway, serving as a compensatory mechanism to counteract the inhibition of the PI3K pathway, ultimately promoting tumor survival. (C) Immune escape following tipifarnib/alpelisib treatment. The combined administration of tipifarnib and alpelisib effectively inhibited the compensatory pathway activation. However, this dual treatment also created an immunosuppressive environment by increasing PD-L1 expression. Consequently, the activity of infiltrating CD8⁺T cells is impaired, resulting in immune evasion and tumor progression. (D) Mechanism of the tipifarnib/alpelisib/anti-PD1 triple combination: the synergistic use of tipifarnib, alpelisib, and anti-PD1 effectively inhibited the compensatory resistance pathway and PD-L1, thus enhancing antitumor immunity. This was achieved by boosting the activity of infiltrating CD8⁺T cells, which resulted in delayed tumor growth and subsequent tumor elimination (Created using www.biorender.com).