Resistance remains a key issue limiting the clinical benefit from RAS-targeting therapeutic agents and necessitates combination approaches. In this study, we identified persistent mTORC1 activity in preclinical KRAS-mutant non-small cell lung cancer (NSCLC) and colorectal cancer models as a frequent, nongenetic driver of inherent and adaptive resistance to RAS inhibition. This vulnerability was targetable with the farnesyl transferase inhibitor darlifarnib (KO-2806), which blocks mTORC1 activation via RHEB while sparing mTORC2 to limit associated toxicities. The addition of KO-2806 to NSCLC or colorectal cancer tumors progressing on mutant-selective RAS inhibitors led to rapid and durable tumor regression. In contrast, switching from mutant-selective to pan-RAS inhibitor monotherapy resulted in only stasis of NSCLC tumors and had no effect on colorectal cancer tumor progression. Furthermore, the addition of KO-2806 rescued sensitivity of progressing tumors to the pan-RAS inhibitor RMC-6236. These results establish mTORC1 as an important mediator of escape from RAS inhibition and highlight KO-2806 as a promising RAS companion inhibitor in patients with prior RAS inhibitor exposure.Significance: KO-2806 salvages RAS inhibitor activity by controlling parallel mTORC1 in RAS inhibitor-resistant tumors in which vertical inhibition of MAPK is insufficient to restore sensitivity, providing a combination strategy for resistant patients.

Identifying ideal candidates for cancer therapies is challenging, especially with multiple oncogenic variants involved. In head and neck squamous cell carcinoma, the PI3Kα-AKT-mTOR and HRAS-MAPK pathways are frequently dysregulated. This study demonstrates how a quantitative systems pharmacology (QSP) model can help optimize clinical trial design by guiding patient selection and dosing strategies based on oncogenic genotypes. A QSP model was developed to capture the experimentally observed dynamics of the HRAS and PI3K pathways across five molecularly defined patient cohorts in the KURRENT-HN Phase I/II trial (NCT04997902). The model assessed which genotypes would benefit the most from combination therapy with tipifarnib (farnesyl transferase inhibitor) and alpelisib (PIK3CA inhibitor). Simulation results identified the PIK3CA gain of function (GOF) as the genotype most likely to benefit. Virtual population analysis of PIK3CA GOF with dose escalation to 600 mg b.i.d. tipifarnib and 250 mg q.d. alpelisib suggested that a higher tipifarnib dose could enhance tumor response, potentially due to significant dependency of the PIK3CA-mutant cells on mTORC1 signaling. These simulations were consistent with the clinical data. This key dependency can be targeted by tipifarnib by blocking farnsylation of RHEB, an essential activator of mTORC1. Vpop responders showed that reduced intracellular mTOR activity in simulations increased the likelihood of tumor volume reduction. Global sensitivity analysis identified compensatory feedback, tumor proliferation rate, and PI3K-mTOR crosstalk as key determinants of tumor response. This novel QSP application exemplifies an innovative bottom-up modeling approach to support patient selection and dosing strategies for future clinical studies.

Supplementary figures related to figures 1-5.

Several mutant-selective PI3Kα inhibitors have recently entered clinical development. Although these agents may have improved therapeutic indices compared to isoform-selective predecessors, feedback reactivation of PI3K-AKT-mTORC1 or compensatory parallel pathways may hinder their efficacy as monotherapies and necessitate combination strategies to improve clinical outcomes. We have previously demonstrated farnesyl transferase inhibitors (FTIs) enhance the antitumor activity of multiple targeted therapies, including the α-selective PI3K inhibitor alpelisib, by blocking RHEB-mediated mTORC1 activation. We hypothesized feedback reactivation of mTORC1 would remain a factor limiting the efficacy of mutant-selective PI3Kα inhibitors, and FTI KO-2806 could potentiate this efficacy by sustained inhibition of mTORC1 signaling. In this study, we assessed the potential therapeutic utility of combining KO-2806 with mutant-selective PI3Kα inhibitors STX-478 or RLY-2608 in a panel of in vitro cell line and in vivo xenograft models spanning the breadth of solid tumor indications with the highest frequency of PIK3CA mutation. We first evaluated the effect of these combinations on cell viability. PIK3CA-mutant breast, endometrial, ovarian, bladder, and colorectal carcinoma cell lines were cultured in monolayer or as 3D tumor spheroids and exposed to KO-2806 and STX-478 or RLY-2608 in dose-response matrices. Although the sensitivity of individual models to single-agent PI3Kα inhibition varied, KO-2806 deepened the antiproliferative effects of either PI3K inhibitor across the cell line panel. To examine the mechanism underlying this synergy, we probed cell signaling kinetics following PI3Kα inhibitor exposure in the presence or absence of KO-2806. Single-agent STX-478 or RLY-2608 rapidly inhibited AKT and mTORC1 activity, as evidenced by dephosphorylation of AKT/PRAS40 and S6K/S6/4EBP-1 at 1 hr, but phosphorylation partially or completely rebounded by 24-48 hrs. In contrast, when cells were simultaneously exposed to KO-2806 and a PIK3α inhibitor from the outset, initial mTORC1 inhibition was deeper, and rebound of activity was blocked. This correlated with apoptosis (PARP cleavage) and cell cycle arrest (inhibition of Rb phosphorylation), consistent with our cell viability results. Given promising in vitro findings, we asked whether this combination activity translated to in vivo models. In PIK3CA-mutant ER+ breast, triple-negative breast, gynecological, colorectal, and bladder xenograft models, KO-2806 enhanced the antitumor effects of STX-478 or RLY-2608, with responses ranging from tumor stasis to deep regressions. In the ER+ model MCF7, combination of KO-2806 and RLY-2608 was comparable to combination of fulvestrant and RLY-2608, resulting in tumor regressions, while the triplet further deepened tumor regression. These data suggest that, due to its ability to constrain mTORC1 signaling, KO-2806 holds promise as a combination agent for mutant-selective PI3Kα inhibitors across a broad range of PIK3CA-mutant solid tumor indications. Alison E. Smith, Ayuna Jombik, Linda Kessler, Francis Burrows, Shivani Malik, Alison Smith. The farnesyl transferase inhibitor KO-2806 constrains mTORC1 activity to enhance the antitumor efficacy of mutant-selective PI3Kα inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C084.

Anti-angiogenic tyrosine kinase inhibitors (TKIs) targeting VEGFR remain the backbone of therapy in advanced renal cell carcinoma (RCC). However, durability of responses is limited, and resistance typically arises. The TKI cabozantinib demonstrated modest activity in patients with prior anti-VEGFR exposure, with a 21% overall response rate and 7.4 months median progression-free survival (NCT01865747). Thus, there is an urgent need for therapeutic agents that enhance responses to cabozantinib. We have previously shown that farnesyl transferase inhibitors (FTIs) improve upfront responses of preclinical clear cell RCC (ccRCC) models to anti-VEGFR therapies. However, given that most ccRCC patients are likely to receive anti-VEGFR agents prior to cabozantinib, we modeled tumor progression on TKIs in ccRCC xenograft models and asked whether KO-2806, a next-generation FTI, could enhance cabozantinib activity. To probe the mechanism of action, we assessed changes in angiogenic and cell signaling proteins in vitro and in vivo. The combination of KO-2806 and cabozantinib led to deeper and more consistent tumor growth inhibition than cabozantinib alone in tumor models that had progressed on axitinib or lenvatinib, two TKIs used to treat RCC. Addition of KO-2806 to tumors progressing on cabozantinib also resulted in tumor regression. Assessment of the vascular markers CD31 and NG2 revealed significantly reduced expression in tumors treated with the combination compared to either single agent, suggesting that the combination has greater antiangiogenic activity. Primary human endothelial cells treated with cabozantinib showed partial inhibition of mTORC1 activity (as assessed by phosphorylation of substrates S6 and 4-EBP1), while the combination with KO-2806 led to near complete loss of mTORC1 signaling due to inhibition of RHEB farnesylation. Tumor transcriptomic and cell signaling studies suggest that KO-2806 also negatively affects the cell cycle, a well-documented effect of farnesyl transferase inhibition. Based on these findings, we posit that the enhanced antiangiogenic activity is a more prominent mechanism of KO-2806 in RCC and that the combined effects on the tumor and the vasculature lead to greater tumor growth inhibition for the combination over cabozantinib alone. Taken together, our findings demonstrate that KO-2806 augments the antitumor activity of cabozantinib, even after prior exposure to anti-VEGFR treatment, largely by enhancing anti-angiogenesis via mTORC1 signaling inhibition in endothelial cells. These data strongly support our clinical evaluation of KO-2806 as a combination partner with cabozantinib in ccRCC as part of the ongoing Phase 1 FIT-001 trial (NCT06026410). Jovylyn G. Gasendo, Stacia Chan, Hetika Vora Patel, Alison Smith, Linda Kessler, Francis Burrows, Shivani Malik. Farnesyl transferase inhibitor KO-2806 enhances the antitumor activity of cabozantinib in ccRCC tumors that progress on anti-VEGFR agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6370.

Members of the KMT2C/D–KDM6A complex are recurrently mutated in urothelial carcinoma and in histologically normal urothelium. Here, using genetically engineered mouse models, we demonstrate that Kmt2c/d knockout in the urothelium led to impaired differentiation, augmented responses to growth and inflammatory stimuli and sensitization to oncogenic transformation by carcinogen and oncogenes. Mechanistically, KMT2D localized to active enhancers and CpG-poor promoters that preferentially regulate the urothelial lineage program and Kmt2c/d knockout led to diminished H3K4me1, H3K27ac and nascent RNA transcription at these sites, which leads to impaired differentiation. Kmt2c/d knockout further led to KMT2A–menin redistribution from KMT2D localized enhancers to CpG-high and bivalent promoters, resulting in derepression of signal-induced immediate early genes. Therapeutically, Kmt2c/d knockout upregulated epidermal growth factor receptor signaling and conferred vulnerability to epidermal growth factor receptor inhibitors. Together, our data posit that functional loss of Kmt2c/d licenses a molecular ‘field effect’ priming histologically normal urothelium for oncogenic transformation and presents therapeutic vulnerabilities. Loss of Kmt2c or Kmt2d in mice drives the redeployment of KMT2A–menin to bivalent promoters, leading to changes in gene expression. This primes cells for transformation and elicits sensitivity to EGFR inhibitors.

Abstract Urothelial carcinoma arises from a “field” of precancerous but histologically normal urothelium. The KMT2C/D-KDM6A complexes known to bind and activate enhancers are frequently mutated in urothelial carcinoma and in histologically normal urothelium. Here, using a genetically engineered mouse model, we demonstrate that knockout of Kmt2c and/or Kmt2d induced a histologically normal, pre-tumorigenic state characterized by impaired differentiation from basal state and by augmented responses to growth and inflammatory stimuli. This sensitized the urothelium to transformation by known oncogenic drivers in urothelial carcinoma. Mechanistically, Kmt2d localized to both active enhancers and a small group of CpG-poor promoters and Kmt2c/d knockout led to diminished H3K4me1, H3K27ac and nascent RNA transcription at these sites, which together led to downregulation of the urothelial differentiation program. We further observed that Menin distributed extensively to Kmt2d localized enhancers and Kmt2c/d knockout led to redistribution of Menin to CpG-high and particularly bivalent promoters, resulting in transcriptional de-repression of bivalent genes. Blockade of Kmt2a/b-Menin interaction partially rescued the transcriptional changes that induced by Kmt2c/d deletion. Therapeutically, Kmt2c/d knockout maintained a basal state that upregulated EGFR signaling and conferred vulnerability to EGFR inhibitors. Together, our data posits that functional loss of Kmt2c/d licenses a molecular “field effect” priming histologically normal urothelium for oncogenic transformation and leading to therapeutic vulnerabilities to EGFR signaling pathway inhibition. Citation Format: Naitao Wang, Mohini Pachai, Dan Li, Cindy Lee, Sarah Warda, Makhzuna Khudoynazarova, Woo Hyun Cho, Guojia Xie, Fengshen Kuo, Sagar Shah, Li Yao, Cheng Qian, Elissa Wong, Juan Yan, Fanny Tomas, Wenhuo Hu, Sizhi Gao, Alison Smith, Ming Han, Dong Gao, Kai Ge, Haiyuan Yu, Sarat Chandarlapaty, Gopakumar Iyer, Jonathan Rosenberg, David Solit, Hikmat AI-Ahmadie, Ping Chi, Yu Chen. Kmt2c/d loss primes urothelium for tumorigenesis and redistributes Menin to bivalent promoters [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A012.

Selective KRAS G12C inhibitors, including AMG510 (sotorasib) and MRTX849 (adagrasib), have exhibited clinical activity in patients with non-small cell lung cancer (NSCLC); however, drug resistance and relapse inevitably occur through various mechanisms, which limits the ability of these therapeutic agents to achieve durable responses. Resistance mechanisms include feedback reactivation of compensatory, oncogenic signaling pathways and the attainment of a drug-tolerant state, each of which allows cancer cells to survive the anti-tumor effects of these highly active drugs. Compensatory mechanisms ameliorating KRAS blockade include HRAS/NRAS and the PI3K-AKT-mTOR pathway. Tipifarnib is a clinical stage farnesyltransferase inhibitor known to inhibit multiple farnesylation-dependent proteins in tumor cells. We have recently shown that tipifarnib can prevent feedback-mediated adaptive resistance to the PI3Kα inhibitor, alpelisib, through inhibition of both HRAS and RHEB, allowing simultaneous blockade of two key nodes of the oncogenic MAPK- and PI3K-signaling pathways. In another example, tipifarnib has been demonstrated by others to inhibit the ability of tumor cells to enter a drug-tolerant state induced by the EGFR inhibitor, osimertinib, in EGFR-mutant NSCLC models. In these models, inhibition of farnesylation of certain RHO proteins appear to contribute to the death of drug-tolerant cells prior to the emergence of acquired resistance mutations. Building on these two mechanisms of how tipifarnib may prevent adaptive resistance, we propose that tipifarnib can be used as an effective partner drug to delay or prevent the onset of adaptive resistance to KRAS G12C inhibitors. We have conducted in vitro 2D and 3D viability and regrowth experiments using combinations of tipifarnib with KRAS G12C inhibitors and have observed synergistic, anti-proliferative effects in KRAS G12C NSCLC cell lines as well as enhanced activity of combination in a KRAS G12C NSCLC PDX model. We are currently expanding the scope of in vitro and in vivo combination studies to further evaluate the molecular mechanism(s) of resistance that tipifarnib targets when combined with KRAS G12C inhibitors. Citation Format: Hetika Vora Patel, Alison Smith, Stacia Chan, Linda Kessler, Francis Burrows, Shivani Malik. Combination of tipifarnib with KRAS G12C inhibitors to prevent adaptive resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1079.

Abstract Emerging clinical and preclinical data have shown that responses to KRASG12C inhibition are limited by the activation of compensatory signaling proteins, including receptor tyrosine kinases (RTKs) and mTOR. Combination therapeutic strategies co-targeting these nodes are needed to fully realize the potential of breakthrough KRASG12C-selective inhibitors. We propose that, given its multipronged mechanism of action, KO-2806, a next-generation farnesyltransferase inhibitor (FTI) with increased potency and improved pharmacokinetic properties relative to earlier FTI candidates that is poised to enter a first-in-human clinical trial, is a uniquely suited combination partner for KRASG12C inhibitors. Here, we evaluate the therapeutic potential of combined KO-2806 and adagrasib in preclinical models of KRASG12C non-small cell lung cancer (NSCLC). Our previous work indicated that FTIs amplify the antitumor effects of PI3Kα inhibition by blocking compensatory mTOR signaling. As activation of compensatory signaling is a well-described bypass mechanism to escape KRAS inhibition, we asked whether KO-2806 could improve the in vivo efficacy of adagrasib. Combination of KO-2806 with adagrasib led to markedly enhanced antitumor effects in KRASG12C-mutant NSCLC cell line- and patient-derived xenograft models compared to adagrasib alone. Tumor regression in the combination group corresponded to increased apoptosis, as evidenced by higher cleaved caspase 3 staining in combination-treated tumors compared to single agent groups. Intriguingly, adagrasib induced expression of the RTK HER3, a potential driver of bypass signaling, while HER3 expression in combination-treated tumors was lower than in vehicle-treated tumors. To further interrogate the mechanistic basis of the observed in vivo synergy, we cultured KRASG12C-mutant cell lines as 3D tumor spheroids and exposed them to KO-2806 and/or adagrasib. Consistent with the xenograft tumor data, spheroids exposed to the combination expressed lower levels of the upstream receptor HER3 (total and phosphorylated forms) and higher levels of apoptotic markers cleaved PARP and cleaved caspase 3. Concurrently, activity of the downstream effectors ERK and mTOR was reduced. Co-exposure to KO-2806 and adagrasib potently inhibited phosphorylation of ERK and its substrate p90 RSK as well as phosphorylation of mTOR substrates p70 S6K, 4EBP1, and S6. The mTOR signaling reduction paralleled defarnesylation of the farnesylation-dependent mTOR activator RHEB, so we depleted RHEB expression in spheroids and assayed their growth. Genetic depletion of RHEB phenocopied the antiproliferative effects of KO-2806 in combination with adagrasib, implicating RHEB as an important farnesylated target in KRASG12C NSCLC. These preclinical results suggest that KO-2806 is an effective combination partner for KRASG12C inhibitors by simultaneously targeting an upstream activator (HER3) and a downstream effector (RHEB/mTOR) of KRAS, leading to improved antitumor activity in KRASG12C-mutant NSCLC models. Citation Format: Hetika V Patel, Alison Smith, Stacia Chan, Linda Kessler, Francis Burrows, Shivani Malik. The next generation farnesyltransferase inhibitor, KO-2806, blocks oncogenic signaling at multiple nodes to enhance the antitumor efficacy of KRASG12C inhibitor adagrasib in KRASG12C non-small cell lung carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B025.

Abstract Outcomes for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are poor, with median overall survival (OS) ranging from 6 to 18 months. For those who progress on standard-of-care (chemo)immunotherapy, treatment options are limited, necessitating the development of rational therapeutic strategies. Toward this end, we targeted the key HNSCC drivers PI3K–mTOR and HRAS via the combination of tipifarnib, a farnesyltransferase (FTase) inhibitor, and alpelisib, a PI3Kα inhibitor, in multiple molecularly defined subsets of HNSCC. Tipifarnib synergized with alpelisib at the level of mTOR in PI3Kα- or HRAS-dependent HNSCCs, leading to marked cytotoxicity in vitro and tumor regression in vivo. On the basis of these findings, the KURRENT-HN trial was launched to evaluate the effectiveness of this combination in PIK3CA-mutant/amplified and/or HRAS-overexpressing R/M HNSCC. Preliminary evidence supports the clinical activity of this molecular biomarker-driven combination therapy. Combined alpelisib and tipifarnib has potential to benefit >45% of patients with R/M HNSCC. By blocking feedback reactivation of mTORC1, tipifarnib may prevent adaptive resistance to additional targeted therapies, enhancing their clinical utility. Significance: The mechanistically designed, biomarker-matched strategy of combining alpelisib and tipifarnib is efficacious in PIK3CA- and HRAS-dysregulated head and neck squamous carcinoma and could improve outcomes for many patients with recurrent, metastatic disease. See related commentary by Lee et al., p. 3162

Background:The introduction of anti-HER2 therapies, trastuzumab and pertuzumab (HP) to the treatment of metastatic HER2-positive breast cancer has had a transformational impact on the outcomes for this disease. Nevertheless, disease progression is ultimately observed in most patients within 3 years carrying the potential for morbidity and potentially more toxic therapies. To identify selective strategies to prevent disease progression, we sought to elucidate mechanisms of resistance to anti-HER2 therapies by analyzing a large cohort of genomically and clinically annotated HER2+ breast cancers. Methods: Patients with advanced HER2+ breast cancer who underwent prospective clinical tumor sequencing utilizing MSK-IMPACT assay between April 2014 and February 2021 were included in the analysis. Clinical HER2 positivity was defined as per ASCO/CAP guidelines. Cox proportional hazard models were used to determine the association between genomic alterations and progression-free survival (PFS) on 1st line HP and taxane-based therapy (THP). Only patients with a sequenced pre-treatment tumor sample were included in the survival analysis. Recurrent mutations identified were modeled in HER2+ breast cancer cell lines using short hairpin RNAs and CRISPR/Cas9, and the sensitivity of these isogenic pairs to HER2-targeted therapies was evaluated via metabolic and colony formation assays of cell proliferation. Results: We identified 733 ERBB2-amplified primary (n=385) and metastatic (n=348) that underwent sequencing. Concurrent PIK3CA mutations were identified in 30% of the tumors. Pathogenic activating alterations involving the MAPK pathway were observed in 12.8% of tumors with the most frequent alterations being NF1 loss, ERBB2 and RAS activating mutations. MAPK alterations were significantly enriched in the metastatic tumors (16.6%) compared to the treatment-naïve primaries (9.8%, p=0.020). The outcome analysis included 145 patients with advanced clinically HER2+ breast cancer whose tumors were sequenced prior to starting 1st line THP. Twenty percent (29/145) of tumors did not show genomic ERBB2 amplification as detected by NGS and had a significantly worse outcome (median PFS of 9.4 months [95% CI 5.5-19] and 23 months [95% CI 17-30], respectively; p=0.015). PIK3CA mutations were also associated with a shorter PFS (mutant: 13 months [95%CI: 7.7-18] vs wild type: 23 months [95%CI 17-16], p=0.0013). We further found reduced PFS in MAPK altered tumors (median PFS 9.9 months; 95% CI: 5.5-17) compared to the rest of the population (median PFS 21 months; 95% CI: 17-30; p=0.01). On multivariable analysis adjusted for estrogen receptor status, and presence of PIK3CA/AKT1/PTEN mutations and genomic ERBB2 amplification, MAPK pathway alterations were independently associated with worse outcome (HR: 2.25; 95% CI: 1.29, 3.93; multivariate p = 0.0043). To establish a causal role for MAPK alterations in reducing efficacy of anti-HER2 therapy, we depleted NF1 expression or expressed mutant KRAS or BRAF in a panel of HER2+ breast cancer cell lines. Consistently, MAPK-altered cell lines exhibited resistance to FDA approved HER2 inhibitors in vitro and in vivo. Conclusions: This clinicogenomic analysis of mechanisms of resistance to anti-HER2 therapy demonstrated that PIK3CA activating mutations and lack of genomic ERBB2 amplification as detected by tumor sequencing are associated with shortened PFS on HP-based therapy. Our analysis uniquely identified MAPK pathway alterations as additional potential drivers of resistance to anti-HER2 therapy. Inhibition of the PI3K or MAPK pathway in such tumors may represent a new therapeutic strategy to extend H/P benefit. Citation Format: Emanuela Ferraro, Alison Smith, Anton Safonov, Paulino Tallon De Lara, Cristina Bernado', Enrique J. Arenas Lahuerta, Joaquín Arribas, David Solit, Jorge S. Reis-Filho, Neal Rosen, Larry Norton, Shanu Modi, Mark E. Robson, Chau T. Dang, Giuseppe Curigliano, Sarat Chandarlapaty, Pedram Razavi. Genomic analysis of 733 HER2+ breast cancers identifies recurrent pathways alterations associated with anti-HER2 resistance and new therapeutic vulnerabilities [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS3-03.

The PI3K-AKT-mTOR signaling cascade is the most frequently activated pathway in the head and neck squamous cell carcinomas (HNSCC). PIK3CA (encoding PI3K’s α catalytic subunit), is activated by gain-of-function mutation or amplification in approximately 30% of HNSCCs, making PI3Kα an attractive therapeutic candidate. While the PI3Kα inhibitor alpelisib has shown some promise in HNSCC in a phase I setting, its single agent efficacy will likely be limited by feedback reactivation of PI3K or compensatory parallel pathways, necessitating the development of rational combination strategies. Here, we utilize cell line and patient-derived xenograft (PDX) models to evaluate the therapeutic potential of the farnesyltransferase inhibitor (FTI) tipifarnib, in combination with alpelisib in the PIK3CA-dysregulated subset of HNSCC. Because FTIs potentially block hyperactivated growth factor signaling at multiple nodes, including HRAS and RHEB, we examined tipifarnib’s impact on growth of PIK3CA-altered HNSCC models in vitro and in vivo. In cell lines harboring PIK3CA mutation or amplification, tipifarnib reduced proliferation of both monolayer and spheroid cultures and when combined with alpelisib, induced cytotoxicity. Consistently, in PIK3CA mutant/amplified PDX models, the tipifarnib-alpelisib doublet led to deeper antitumor responses compared to alpelisib monotherapy. Simultaneous administration was superior to split intermittent dosing, hinting at cooperativity between the mechanistic targets of tipifarnib and alpelisib. To interrogate the mechanistic underpinnings of this synergy, we exposed HNSCC cell lines to tipifarnib, alpelisib, or the combination, and assessed their effect on RAS/PI3K pathway activity. In PIK3CA dysregulated lines, single agent tipifarnib or alpelisib reduced phosphorylation of p90 RSK, and mTOR substrates, particularly S6 kinase and ribosomal protein S6. Combination treatment effects were more robust and induced rapid apoptosis. In cells exposed to alpelisib alone, marked rebound of RSK and mTOR substrate phosphorylation occurred after 24 hours, correlating with restored AKT activity. In contrast, although AKT activity rebounded in cells treated with the combination, RSK phosphorylation and markers of mTOR activity (including 4EBP1) remained suppressed. Thus, tipifarnib appears to blunt both MAPK and mTOR reactivation following PI3K inhibition. This dual effect implies that the efficacy of tipifarnib in this context may stem from simultaneous defarnesylation of multiple targets, likely HRAS and RHEB, which converge upon mTOR and synergize with alpelisib to durably block tumor growth. We contend that combination of alpelisib and tipifarnib holds therapeutic potential for treatment of recurrent/metastatic HNSCCs harboring dysregulated PIK3CA, which will be evaluated in the recently initiated KURRENT clinical trial (NCT04997902). Citation Format: Shivani Malik, Alison Smith, Stacia Chan, Asako McCloskey, Hetika Vora, Quinn Reilly, Francis Burrows. Tipifarnib potentiates the antitumor effects of PI3Ka blockade in HNSCC via convergent inhibition of mTOR activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1120.

Urothelial carcinoma (UC) is widely recognized to arise from a “field” of precancerous but histologically normal urothelium (HNU). The molecular mechanism that licenses the “field effect” remains elusive. Recent studies revealed prevalent KMT2C and KMT2D loss-of-function (LOF) mutations in HNU and cancer adjacent urothelium, suggesting their potential involvement in UC initiation. Here, we demonstrated that knockout (KO) of Kmt2c and/or Kmt2d in murine urothelial cells induced drastic alterations of cellular states by single cell RNA analysis, but was insufficient to induce robust histological changes. Kmt2c/d loss enhanced organoid formation efficiency, induced epithelial-mesenchymal transition (EMT), and impaired urothelial differentiation, indicating the augmented lineage plasticity after Kmt2c/d KO. Additionally, we identified that Kmt2c/d KO induced a pre-tumorigenic transcriptome with increased enrichments of gene sets associated with inflammation and decreased enrichments of gene sets associated with differentiation. Consequently, loss of Kmt2c/d sensitized urothelial cells to common oncogenic mutations to initiate UC in mouse models. We further observed that KO of Kmt2c/d increased tumorigenic susceptibility in carcinogen-induced UC model. Mechanistically, we observed decreased H3K4me1, H3K27Ac histone marks and decreased enhancer RNA production at the majority of enhancers that correlate with downregulation of urothelial specific lineage gene expression after Kmt2c/d KO. Furthermore, we observed increased Menin deposition on promoters of up-regulated genes. Blockade of Menin-KMT2A complex by small molecule inhibitor partially rescued the EMT and basal differentiation induced by Kmt2c/d KO. Together, our data posit that Kmt2c/d represents a key molecular determinant and their functional loss licenses a molecular “field effect” which primes the urothelium for oncogenic transformation. Citation Format: Naitao Wang, Mohini R. Pachai, Dan Li, Cindy Lee, Sarah Warda, Guojia Xie, Cheng Qian, Wai Pung E. Wong, Juan Yan, Wenhuo Hu, Alison Smith, Kai Ge, Sarat Chandarlapaty, Gopakumar V. Iyer, Jonathan E. Rosenberg, David B. Solit, Hikmat A. AI-Ahmadie, Ping Chi, Yu Chen. Inactivation mutations of Kmt2c/d license a molecular “field effect” and prime the urothelium for tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B003.

Background: Understanding the interplay between novel molecular drivers is critical to optimize biomarker-driven clinical studies for patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). This study used real world data, in vitro signaling, phenotypic assays and xenograft models to investigate HRAS overexpression, PIK3CA mutation and amplification as targets for combination therapy with tipifarnib, a farnesyl transferase inhibitor, and alpelisib, a PI3Ka inhibitor. Methods: Real world clinical and genomic data (RWD) from >1000 patients with HNSCC with NGS and whole transcriptome RNAseq at Tempus Labs was used to determine the prevalence of PIK3CA and HRAS biomarkers. A CLIA-validated IHC assay was developed to assess HRAS expression in FFPE tissue. Results: In a RWD dataset of >1000 HNSCC tumors, ∼45% harbored an actionable PIK3CA and/or HRAS biomarker: PIK3CA mutation and amplification: 20% and 5.6%, respectively; HRAS mutations, 4.7%; and HRAS overexpression (defined as HRAS expression 1 standard deviation above the mean), ∼15%. Since HRAS-MAPK and PI3K-AKT-mTOR signaling cascades are two interdependent, frequently dysregulated pathways in HNSCC, we explored if dual inhibition of these pathways with tipifarnib and alpelisib would be an effective anti-tumor strategy in these defined genetic subtypes. As reported previously in PIK3CA-dysregulated models, the combination synergistically induced cytotoxicity in HNSCC cell lines with HRAS overexpression relative to HRAS-low cell lines. siRNA-mediated depletion of HRAS and RHEB demonstrated that these farnesylated targets are essential to tipifarnib-mediated inhibition of mTOR and the compensatory reactivation of MAPK and mTOR pathways induced by alpelisib monotherapy. In vivo, combination therapy induced deeper antitumor responses in HNSCC PDXs with HRAS overexpression ± co-occurring mutations in PIK3CA. We validated an HRAS IHC assay to CAP/ CLIA standards to investigate HRAS overexpression as a potential biomarker of response. In 46 HNSCC tumors selected to match patients in ongoing trials, 16/46 (35%) displayed intense and widespread HRAS staining, equivalent to a tumor proportion score ≥50. The preclinical and mechanistic data showing antitumor activity of the targeted combination regimen and an ability to prescreen patients for HRAS overexpression by IHC prompted the opening of KO-TIP-013, a P1/2 trial of tipifarnib plus alpelisib in HNSCC patients who failed prior therapy (NCT04997902). Conclusions: Nearly half of HSNCC tumors harbor genomic alterations that suggest dependence on dysregulation of HRAS-MAPK and PI3K-AKT-mTOR pathways. Preclinical data point to a mechanistic synergy between tipifarnib and alpelisib in PIK3CA- and HRAS-dysregulated HNSCC that may overcome the lack of efficacy observed in monotherapy trials with these agents. Conflict of interest: Ownership: All authors are employed by Kura Oncology

Background: Breast cancer (BC) is the second most common cancer in the world with over 1.67 million new cases yearly. 30% of women with early breast cancer develop metastatic breast cancer (MBC). Despite advances in treatment strategies, MBC remains largely incurable and is the primary cause of over 600 000 deaths annually worldwide.