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Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer., Competing Interests: Declaration of interests M. Sallaku is an employee of Loxo Oncology. M. Scaltriti has ownership interest (including patents) in Medendi.org, reports other support from AstraZeneca, and is an employee of AstraZeneca. H.M.C. and T.C. are employees of AstraZeneca. P.C. reports personal fees from Venthera outside the submitted work. P.R. has received institutional grants/funding from Grail, Novartis, AstraZeneca, EpicSciences, Invitae/ArcherDx, Biothernostics, Tempus, Neogenomics, Biovica, Guardant, Personalis, and Myriad and has shares/ownership interest in Odyssey Biosciences as well as consultation/ad-board/honoraria from Novartis, AstraZeneca, Pfizer, Lilly/Loxo, Prelude Therapeutics, Epic Sciences, Daiichi-Sankyo, Foundation Medicine, Inivata, Tempus, SAGA Diagnostics, Paige.ai, Guardant, and Myriad. E.T. reports grants and personal fees from AstraZeneca and consulting from Menarini., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The phosphoinositide 3-kinase (PI3K) pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and cotranscriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced isoforms linked to proliferation, metabolism, and splicing in PIK3CA-mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from patients with PIK3CA-mutated breast cancer undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer., Significance: Transcriptomic analysis reveals a key role for the PI3K pathway in regulating RNA splicing, uncovering new mechanisms by which PI3K regulates proliferation and metabolism in breast cancer. See related commentary by Claridge and Hopkins, p. 2216., (©2022 The Authors; Published by the American Association for Cancer Research.)

The efficacy of the highly selective RET inhibitor selpercatinib is now established in RET-driven cancers, and we sought to characterize the molecular determinants of response and resistance. We find that the pre-treatment genomic landscape does not shape the variability of treatment response except for rare instances of RAS-mediated primary resistance. By contrast, acquired selpercatinib resistance is driven by MAPK pathway reactivation by one of two distinct routes. In some patients, on- and off-target pathway reactivation via secondary RET solvent front mutations or MET amplifications are evident. In other patients, rare RET-wildtype tumor cell populations driven by an alternative mitogenic driver are selected for by treatment. Multiple distinct mechanisms are often observed in the same patient, suggesting polyclonal resistance may be common. Consequently, sequential RET-directed therapy may require combination treatment with inhibitors targeting alternative MAPK effectors, emphasizing the need for prospective characterization of selpercatinib-treated tumors at the time of monotherapy progression., (© 2022. The Author(s).)

On-target resistance to next-generation TRK inhibitors in TRK fusion-positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKA G667 and TRKC G696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations. SIGNIFICANCE: In TRK fusion-positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation. This article is highlighted in the In This Issue feature, p. 1 ., (©2020 American Association for Cancer Research.)

Mutations in the pioneer transcription factor FOXA1 are a hallmark of estrogen receptor-positive (ER + ) breast cancers. Examining FOXA1 in ∼5,000 breast cancer patients identifies several hotspot mutations in the Wing2 region and a breast cancer-specific mutation SY242CS, located in the third β strand. Using a clinico-genomically curated cohort, together with breast cancer models, we find that FOXA1 mutations associate with a lower response to aromatase inhibitors. Mechanistically, Wing2 mutations display increased chromatin binding at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility. In contrast, SY242CS shows neomorphic properties that include the ability to open distinct chromatin regions and activate an alternative cistrome and transcriptome. Structural modeling predicts that SY242CS confers a conformational change that mediates stable binding to a non-canonical DNA motif. Taken together, our results provide insights into how FOXA1 mutations perturb its function to dictate cancer progression and therapeutic response., Competing Interests: Declaration of Interests J.L.V.M. is an employee at Inzen Therapeutics. M.Sallaku has received funds from Puma Biotechnology, AstraZeneca, Daiichi-Sankio, Immunomedics, Targimmune, and Menarini Ricerche, is a cofounder of Medendi.org and is on the advisory board of Menarini Ricerche. J.B. is an employee and shareholder of AstraZeneca, Board of Directors member of Foghorn Therapeutics, and is a past board member of Varian Medical Systems, Bristol-Myers Squibb, Grail, Aura Biosciences, and Infinity Pharmaceuticals. He has performed consulting and/or advisory work for Grail, PMV Pharma, ApoGen, Juno, Lilly, Seragon, Novartis, and Northern Biologics. He has stock or other ownership interests in PMV Pharma, Grail, Juno, Varian, Foghorn, Aura, Infinity, ApoGen, as well as Tango and Venthera, of which he is a co-founder. He has previously received Honoraria or Travel Expenses from Roche, Novartis, and Lilly. C.L.S. serves on the board of directors of Novartis, is a co-founder of ORIC Pharm and co-inventor of enzalutamide and apalutamide. He is a science advisor to Agios, Beigene, Blueprint, Column Group, Foghorn, Housey Pharma, Nextech, KSQ, Petra, and PMV. He was a co-founder of Seragon (purchased by Genentech/Roche, 2014). J.S.R.-F. is a consultant of Goldman Sachs and REPARE Therapeutics, a member of the Scientific Advisory Board of VolitionRx and Paige.AI, and an ad hoc member of the Scientific Advisory Board of Ventana Medical Systems, Roche, Genentech, Novartis, and InviCRO. C.S.V. has been the recipient of research grants from Ipsen Pharmaceuticals, MSD International, and Proctor & Gamble. E.T. has received Honoraria from AstraZeneca for invited lectures. P.R. has received consultation fees from Novartis, AstraZeneca, Foundation Medicine and institutional research funds from Grail, Novartis and Illumina. W.R.K. is a patent holder with KNAW, Organoid Technology shared with Hans Clevers.S.Kannan and C.S.V. are founder directors of SiNOPSEE Therapeutics and Aplomex. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)