Phase I/II Study of Combined BCL-xL and MEK Inhibition with Navitoclax and Trametinib in KRAS or NRAS Mutant Advanced Solid Tumors.

PURPOSE: MEK inhibitors (MEKi) lack monotherapy efficacy in most RAS-mutant cancers. BCL-xL is an anti-apoptotic protein identified by a synthetic lethal shRNA screen as a key suppressor of apoptotic response to MEKi. PATIENTS AND METHODS: We conducted a dose escalation study (NCT02079740) of the BCL-xL inhibitor navitoclax and MEKi trametinib in patients with RAS-mutant tumors with expansion cohorts for: pancreatic, gynecologic (GYN), non-small cell lung cancer (NSCLC), and other cancers harboring KRAS/NRAS mutations. Paired pretreatment and day 15 tumor biopsies and serial cell-free (cf)DNA were analyzed. RESULTS: A total of 91 patients initiated treatment, with 38 in dose escalation. Fifty-eight percent had ≥3 prior therapies. A total of 15 patients (17%) had colorectal cancer, 19 (11%) pancreatic, 15 (17%) NSCLC, and 32 (35%) GYN cancers. The recommended phase II dose (RP2D) was established as trametinib 2 mg daily days 1 to 14 and navitoclax 250 mg daily days 1 to 28 of each cycle. Most common adverse events included diarrhea, thrombocytopenia, increased AST/ALT, and acneiform rash. At RP2D, 8 of 49 (16%) evaluable patients achieved partial response (PR). Disease-specific differences in efficacy were noted. In patients with GYN at the RP2D, 7 of 21 (33%) achieved a PR and median duration of response 8.2 months. No PRs occurred in patients with colorectal cancer, NSCLC, or pancreatic cancer. MAPK pathway inhibition was observed in on-treatment tumor biopsies. Reductions in KRAS/NRAS mutation levels in cfDNA correlated with clinical benefit. CONCLUSIONS: Navitoclax in combination with trametinib was tolerable. Durable clinical responses were observed in patients with RAS-mutant GYN cancers, warranting further evaluation in this population.

The Irreversible FGFR Inhibitor KIN-3248 Overcomes FGFR2 Kinase Domain Mutations.

PURPOSE: FGFR2 and FGFR3 show oncogenic activation in many cancer types, often through chromosomal fusion or extracellular domain mutation. FGFR2 and FGFR3 alterations are most prevalent in intrahepatic cholangiocarcinoma (ICC) and bladder cancers, respectively, and multiple selective reversible and covalent pan-FGFR tyrosine kinase inhibitors (TKI) have been approved in these contexts. However, resistance, often due to acquired secondary mutations in the FGFR2/3 kinase domain, limits efficacy. Resistance is typically polyclonal, involving a spectrum of different mutations that most frequently affect the molecular brake and gatekeeper residues (N550 and V565 in FGFR2). EXPERIMENTAL DESIGN: Here, we characterize the activity of the next-generation covalent FGFR inhibitor, KIN-3248, in preclinical models of FGFR2 fusion+ ICC harboring a series of secondary kinase domain mutations, in vitro and in vivo. We also test select FGFR3 alleles in bladder cancer models. RESULTS: KIN-3248 exhibits potent selectivity for FGFR1-3 and retains activity against various FGFR2 kinase domain mutations, in addition to being effective against FGFR3 V555M and N540K mutations. Notably, KIN-3248 activity extends to the FGFR2 V565F gatekeeper mutation, which causes profound resistance to currently approved FGFR inhibitors. Combination treatment with EGFR or MEK inhibitors potentiates KIN-3248 efficacy in vivo, including in models harboring FGFR2 kinase domain mutations. CONCLUSIONS: Thus, KIN-3248 is a novel FGFR1-4 inhibitor whose distinct activity profile against FGFR kinase domain mutations highlights its potential for the treatment of ICC and other FGFR-driven cancers.

Sotorasib Is a Pan-RASG12C Inhibitor Capable of Driving Clinical Response in NRASG12C Cancers.

KRASG12C inhibitors, like sotorasib and adagrasib, potently and selectively inhibit KRASG12C through a covalent interaction with the mutant cysteine, driving clinical efficacy in KRASG12C tumors. Because amino acid sequences of the three main RAS isoforms-KRAS, NRAS, and HRAS-are highly similar, we hypothesized that some KRASG12C inhibitors might also target NRASG12C and/or HRASG12C, which are less common but critical oncogenic driver mutations in some tumors. Although some inhibitors, like adagrasib, were highly selective for KRASG12C, others also potently inhibited NRASG12C and/or HRASG12C. Notably, sotorasib was five-fold more potent against NRASG12C compared with KRASG12C or HRASG12C. Structural and reciprocal mutagenesis studies suggested that differences in isoform-specific binding are mediated by a single amino acid: Histidine-95 in KRAS (Leucine-95 in NRAS). A patient with NRASG12C colorectal cancer treated with sotorasib and the anti-EGFR antibody panitumumab achieved a marked tumor response, demonstrating that sotorasib can be clinically effective in NRASG12C-mutated tumors. SIGNIFICANCE: These studies demonstrate that certain KRASG12C inhibitors effectively target all RASG12C mutations and that sotorasib specifically is a potent NRASG12C inhibitor capable of driving clinical responses. These findings have important implications for the treatment of patients with NRASG12C or HRASG12C cancers and could guide design of NRAS or HRAS inhibitors. See related commentary by Seale and Misale, p. 698. This article is featured in Selected Articles from This Issue, p. 695.

Landscape of Clinical Resistance Mechanisms to FGFR Inhibitors in FGFR2-Altered Cholangiocarcinoma.

PURPOSE: FGFR inhibitors are effective in FGFR2-altered cholangiocarcinoma, leading to approval of reversible FGFR inhibitors, pemigatinib and infigratinib, and an irreversible inhibitor, futibatinib. However, acquired resistance develops, limiting clinical benefit. Some mechanisms of resistance have been reported, including secondary FGFR2 kinase domain mutations. Here, we sought to establish the landscape of acquired resistance to FGFR inhibition and to validate findings in model systems. EXPERIMENTAL DESIGN: We examined the spectrum of acquired resistance mechanisms detected in circulating tumor DNA or tumor tissue upon disease progression following FGFR inhibitor therapy in 82 FGFR2-altered cholangiocarcinoma patients from 12 published reports. Functional studies of candidate resistance alterations were performed. RESULTS: Overall, 49 of 82 patients (60%) had one or more detectable secondary FGFR2 kinase domain mutations upon acquired resistance. N550 molecular brake and V565 gatekeeper mutations were most common, representing 63% and 47% of all FGFR2 kinase domain mutations, respectively. Functional studies showed different inhibitors displayed unique activity profiles against FGFR2 mutations. Interestingly, disruption of the cysteine residue covalently bound by futibatinib (FGFR2 C492) was rare, observed in 1 of 42 patients treated with this drug. FGFR2 C492 mutations were insensitive to inhibition by futibatinib but showed reduced signaling activity, potentially explaining their low frequency. CONCLUSIONS: These data support secondary FGFR2 kinase domain mutations as the primary mode of acquired resistance to FGFR inhibitors, most commonly N550 and V565 mutations. Thus, development of combination strategies and next-generation FGFR inhibitors targeting the full spectrum of FGFR2 resistance mutations will be critical.

Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary PIK3CA Mutations.

PIK3CA mutations occur in ∼8% of cancers, including ∼40% of HR-positive breast cancers, where the PI3K-alpha (PI3Kα)-selective inhibitor alpelisib is FDA approved in combination with fulvestrant. Although prior studies have identified resistance mechanisms, such as PTEN loss, clinically acquired resistance to PI3Kα inhibitors remains poorly understood. Through serial liquid biopsies and rapid autopsies in 39 patients with advanced breast cancer developing acquired resistance to PI3Kα inhibitors, we observe that 50% of patients acquire genomic alterations within the PI3K pathway, including PTEN loss and activating AKT1 mutations. Notably, although secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Kα inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket. Some mutations had differential effects on PI3Kα-selective versus pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Kα-inhibitor RLY-2608. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. SIGNIFICANCE: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Kα inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Kα inhibitor. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 240 . This article is featured in Selected Articles from This Issue, p. 201.

Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

Ultrasensitive detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.