Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

RLY-4008, the First Highly Selective FGFR2 Inhibitor with Activity across FGFR2 Alterations and Resistance Mutations.

Oncogenic activation of fibroblast growth factor receptor 2 (FGFR2) drives multiple cancers and represents a broad therapeutic opportunity, yet selective targeting of FGFR2 has not been achieved. Although the clinical efficacy of pan-FGFR inhibitors (pan-FGFRi) validates FGFR2 driver status in FGFR2 fusion-positive intrahepatic cholangiocarcinoma, their benefit is limited by incomplete target coverage due to FGFR1- and FGFR4-mediated toxicities (hyperphosphatemia and diarrhea, respectively) and the emergence of FGFR2 resistance mutations. RLY-4008 is a highly selective, irreversible FGFR2 inhibitor designed to overcome these limitations. In vitro, RLY-4008 demonstrates >250- and >5,000-fold selectivity over FGFR1 and FGFR4, respectively, and targets primary alterations and resistance mutations. In vivo, RLY-4008 induces regression in multiple xenograft models-including models with FGFR2 resistance mutations that drive clinical progression on current pan-FGFRi-while sparing FGFR1 and FGFR4. In early clinical testing, RLY-4008 induced responses without clinically significant off-isoform FGFR toxicities, confirming the broad therapeutic potential of selective FGFR2 targeting. SIGNIFICANCE: Patients with FGFR2-driven cancers derive limited benefit from pan-FGFRi due to multiple FGFR1-4-mediated toxicities and acquired FGFR2 resistance mutations. RLY-4008 is a highly selective FGFR2 inhibitor that targets primary alterations and resistance mutations and induces tumor regression while sparing other FGFRs, suggesting it may have broad therapeutic potential. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.

Avapritinib in advanced PDGFRA D842V-mutant gastrointestinal stromal tumour (NAVIGATOR): a multicentre, open-label, phase 1 trial.

BACKGROUND: Targeting of KIT and PDGFRA with imatinib revolutionised treatment in gastrointestinal stromal tumour; however, PDGFRA Asp842Val (D842V)-mutated gastrointestinal stromal tumour is highly resistant to tyrosine kinase inhibitors. We aimed to assess the safety, tolerability, and antitumour activity of avapritinib, a novel KIT and PDGFRA inhibitor that potently inhibits PDGFRA D842V, in patients with advanced gastrointestinal stromal tumours, including patients with KIT and PDGFRA D842V-mutant gastrointestinal stromal tumours (NAVIGATOR). METHODS: NAVIGATOR is a two-part, open-label, dose-escalation and dose-expansion, phase 1 study done at 17 sites across nine countries (Belgium, France, Germany, Poland, Netherlands, South Korea, Spain, the UK, and the USA). Patients aged 18 years or older, with an Eastern Cooperative Oncology Group performance status of 2 or less, and with adequate end-organ function were eligible to participate. The dose-escalation part of the study included patients with unresectable gastrointestinal stromal tumours. The dose-expansion part of the study included patients with an unresectable PDGFRA D842V-mutant gastrointestinal stromal tumour regardless of previous therapy or gastrointestinal stromal tumour with other mutations that either progressed on imatinib and one or more tyrosine kinase inhibitor, or only received imatinib previously. On the basis of enrolment trends, ongoing review of study data, and evolving knowledge regarding the gastrointestinal stromal tumour treatment paradigm, it was decided by the sponsor's medical director together with the investigators that patients with PDGFRA D842V mutations would be analysed separately; the results from this group of patients is reported in this Article. Oral avapritinib was administered once daily in the dose-escalation part (starting dose of 30 mg, with increasing dose levels once daily in continuous 28-day cycles until the maximum tolerated dose or recommended phase 2 dose was determined; in the dose-expansion part, the starting dose was the maximum tolerated dose from the dose-escalation part). Primary endpoints were maximum tolerated dose, recommended phase 2 dose, and safety in the dose-escalation part, and overall response and safety in the dose-expansion part. Safety was assessed in all patients from the dose-escalation part and all patients with PDGFRA D842V-mutant gastrointestinal stromal tumour in the dose-expansion part, and activity was assessed in all patients with PDGFRA D842V-mutant gastrointestinal stromal tumour who received avapritinib and who had at least one target lesion and at least one post-baseline disease assessment by central radiology. This study is registered with ClinicalTrials.gov, NCT02508532. FINDINGS: Between Oct 26, 2015, and Nov 16, 2018 (data cutoff), 46 patients were enrolled in the dose-escalation part, including 20 patients with a PDGFRA D842V-mutant gastrointestinal stromal tumour, and 36 patients with a PDGFRA D842V-mutant gastrointestinal stromal tumour were enrolled in the dose-expansion part. At data cutoff (Nov 16, 2018), 38 (46%) of 82 patients in the safety population (median follow-up of 19·1 months [IQR 9·2-25·5]) and 37 (66%) of the 56 patients in the PDGFRA D842V population (median follow-up of 15·9 months [IQR 9·2-24·9]) remained on treatment. The maximum tolerated dose was 400 mg, and the recommended phase 2 dose was 300 mg. In the safety population (patients with PDGFRA D842V-mutant gastrointestinal stromal tumour from the dose-escalation and dose-expansion parts, all doses), treatment-related grade 3-4 events occurred in 47 (57%) of 82 patients, the most common being anaemia (14 [17%]); there were no treatment-related deaths. In the PDGFRA D842V-mutant population, 49 (88%; 95% CI 76-95) of 56 patients had an overall response, with five (9%) complete responses and 44 (79%) partial responses. No dose-limiting toxicities were observed at doses of 30-400 mg per day. At 600 mg, two patients had dose-limiting toxicities (grade 2 hypertension, dermatitis acneiform, and memory impairment in patient 1, and grade 2 hyperbilirubinaemia in patient 2). INTERPRETATION: Avapritinib has a manageable safety profile and has preliminary antitumour activity in patients with advanced PDGFRA D842V-mutant gastrointestinal stromal tumours. FUNDING: Blueprint Medicines.

Acquired On-Target Clinical Resistance Validates FGFR4 as a Driver of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide with no clinically confirmed oncogenic driver. Although preclinical studies implicate the FGF19 receptor FGFR4 in hepatocarcinogenesis, the dependence of human cancer on FGFR4 has not been demonstrated. Fisogatinib (BLU-554) is a potent and selective inhibitor of FGFR4 and demonstrates clinical benefit and tumor regression in patients with HCC with aberrant FGF19 expression. Mutations were identified in the gatekeeper and hinge-1 residues in the kinase domain of FGFR4 upon disease progression in 2 patients treated with fisogatinib, which were confirmed to mediate resistance in vitro and in vivo. A gatekeeper-agnostic, pan-FGFR inhibitor decreased HCC xenograft growth in the presence of these mutations, demonstrating continued FGF19-FGFR4 pathway dependence. These results validate FGFR4 as an oncogenic driver and warrant further therapeutic targeting of this kinase in the clinic. SIGNIFICANCE: Our study is the first to demonstrate on-target FGFR4 kinase domain mutations as a mechanism of acquired clinical resistance to targeted therapy. This further establishes FGF19-FGFR4 pathway activation as an oncogenic driver. These findings support further investigation of fisogatinib in HCC and inform the profile of potential next-generation inhibitors.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.

First-in-Human Phase I Study of Fisogatinib (BLU-554) Validates Aberrant FGF19 Signaling as a Driver Event in Hepatocellular Carcinoma.

Outcomes for patients with advanced hepatocellular carcinoma (HCC) remain poor despite recent progress in drug development. Emerging data implicate FGF19 as a potential HCC driver, suggesting its receptor, FGFR4, as a novel therapeutic target. We evaluated fisogatinib (BLU-554), a highly potent and selective oral FGFR4 inhibitor, in a phase I dose-escalation/dose-expansion study in advanced HCC using FGF19 expression measured by IHC as a biomarker for pathway activation. For dose escalation, 25 patients received 140 to 900 mg fisogatinib once daily; the maximum tolerated dose (600 mg once daily) was expanded in 81 patients. Fisogatinib was well tolerated; most adverse events were manageable, grade 1/2 gastrointestinal events, primarily diarrhea, nausea, and vomiting. Across doses, the overall response rate was 17% in FGF19-positive patients [median duration of response: 5.3 months (95% CI, 3.7-not reached)] and 0% in FGF19-negative patients. These results validate FGFR4 as a targetable driver in FGF19-positive advanced HCC. SIGNIFICANCE: Fisogatinib elicited clinical responses in patients with tumor FGF19 overexpression in advanced HCC. These results validate the oncogenic driver role of the FGFR4 pathway in HCC and the use of FGF19 as a biomarker for patient selection.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.

MM-151 overcomes acquired resistance to cetuximab and panitumumab in colorectal cancers harboring EGFR extracellular domain mutations.

The anti-epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab are used to treat RAS wild-type colorectal cancers (CRCs), but their efficacy is limited by the emergence of acquired drug resistance. After EGFR blockade, about 20% of CRCs develop mutations in the EGFR extracellular domain (ECD) that impair antibody binding and are associated with clinical relapse. We hypothesized that EGFR ECD-resistant variants could be targeted by the recently developed oligoclonal antibody MM-151 that binds multiple regions of the EGFR ECD. MM-151 inhibits EGFR signaling and cell growth in preclinical models, including patient-derived cells carrying mutant EGFR. Upon MM-151 treatment, EGFR ECD mutations decline in circulating cell-free tumor DNA (ctDNA) of CRC patients who previously developed resistance to EGFR blockade. These data provide molecular rationale for the clinical use of MM-151 in patients who become resistant to cetuximab or panitumumab as a result of EGFR ECD mutations.

Enhanced Targeting of the EGFR Network with MM-151, an Oligoclonal Anti-EGFR Antibody Therapeutic.

Although EGFR is a validated therapeutic target across multiple cancer indications, the often modest clinical responses to current anti-EGFR agents suggest the need for improved therapeutics. Here, we demonstrate that signal amplification driven by high-affinity EGFR ligands limits the capacity of monoclonal anti-EGFR antibodies to block pathway signaling and cell proliferation and that these ligands are commonly coexpressed with low-affinity EGFR ligands in epithelial tumors. To develop an improved antibody therapeutic capable of overcoming high-affinity ligand-mediated signal amplification, we used a network biology approach comprised of signaling studies and computational modeling of receptor-antagonist interactions. Model simulations suggested that an oligoclonal antibody combination may overcome signal amplification within the EGFR:ERK pathway driven by all EGFR ligands. Based on this, we designed MM-151, a combination of three fully human IgG1 monoclonal antibodies that can simultaneously engage distinct, nonoverlapping epitopes on EGFR with subnanomolar affinities. In signaling studies, MM-151 antagonized high-affinity EGFR ligands more effectively than cetuximab, leading to an approximately 65-fold greater decrease in signal amplification to ERK. In cell viability studies, MM-151 demonstrated antiproliferative activity against high-affinity EGFR ligands, either singly or in combination, while cetuximab activity was largely abrogated under these conditions. We confirmed this finding both in vitro and in vivo in a cell line model of autocrine high-affinity ligand expression. Together, these preclinical studies provide rationale for the clinical study of MM-151 and suggest that high-affinity EGFR ligand expression may be a predictive response marker that distinguishes MM-151 from other anti-EGFR therapeutics.

On the edge of validation--cancer protease fibroblast activation protein.

Numerous studies implicate the prolyl peptidase, fibroblast activation protein (FAP) in tumorigenesis; however, FAP-selective inhibitors have not yet been developed to fully validate FAP as a therapeutic target. Herein, we review recent efforts aimed at validating and inhibiting FAP for cancer therapy and highlight future directions for successful targeting of this prolyl peptidase.

Ala657 and conserved active site residues promote fibroblast activation protein endopeptidase activity via distinct mechanisms of transition state stabilization.

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.

Synthesis and structure-activity relationship of N-acyl-Gly-, N-acyl-Sar- and N-blocked-boroPro inhibitors of FAP, DPP4, and POP.

The structure-activity relationship of various N-acyl-Gly-, N-acyl-Sar-, and N-blocked-boroPro derivatives against three prolyl peptidases was explored. Several N-acyl-Gly- and N-blocked-boroPro compounds showed low nanomolar inhibitory activity against fibroblast activation protein (FAP) and prolyl oligopeptidase (POP) and selectivity against dipeptidyl peptidase-4 (DPP4). N-Acyl-Sar-boroPro analogs retained selectivity against DPP4 and potent POP inhibitory activity but displayed decreased FAP inhibitory activity.

Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving specificity.

Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's P(4)-P(2)(') specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in alpha(2)-antiplasmin (TSGP-NQ). FAP required substrates with Pro at P(1) and Gly or d-amino acids at P(2) and preferred small, uncharged amino acids at P(3), but tolerated most amino acids at P(4), P(1)(') and P(2)('). These substrate preferences allowed design of peptidyl-chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase-4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.

Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity.

Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P(2)-Pro(1) and acetyl (Ac)-P(2)-Pro(1) dipeptide substrate libraries, where P(2) was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P(2)-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P(2) residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P(2)-Pro(1) library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K(i) of 23 +/- 3 nm. This was approximately 9- to approximately 5400-fold lower than the K(i) values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.

Apoptosis: letting slip the dogs of war.

Recent studies have shown that, during cell death, the protein Omi is released from the mitochondrial intermembrane space into the cytosol, where it augments caspase-dependent apoptosis by blocking inhibitors and may induce caspase-independent cell death via its serine protease activity.