Faculty Bibliography

The 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 co-transcriptional 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 (TF) activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced (AS) isoforms linked to proliferation, metabolism, and splicing in PIK3CA mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from PIK3CA-mutated breast cancer patients 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.

PURPOSE/OBJECTIVE:To update recommendations on appropriate use of breast cancer biomarker assay results to guide adjuvant endocrine and chemotherapy decisions in early-stage breast cancer. METHODS:An updated literature search identified randomized clinical trials and prospective-retrospective studies published from January 2016 to October 2021. Outcomes of interest included overall survival and disease-free or recurrence-free survival. Expert Panel members used informal consensus to develop evidence-based recommendations. RESULTS:The search identified 24 studies informing the evidence base. RECOMMENDATIONS/CONCLUSIONS:in patients with node-negative ER+ and HER2- breast cancer. Current data suggest that premenopausal patients with 1-3 positive nodes benefit from chemotherapy regardless of genomic assay result. There are no data on use of genomic tests to guide adjuvant chemotherapy in patients with ≥ 4 positive nodes. Ki67 combined with other parameters or immunohistochemistry 4 score may be used in postmenopausal patients without access to genomic tests to guide adjuvant therapy decisions. BCI may be offered to patients with 0-3 positive nodes who received 5 years of endocrine therapy without evidence of recurrence to guide decisions about extended endocrine therapy. None of the assays are recommended for treatment guidance in individuals with HER2-positive or triple-negative breast cancer. Treatment decisions should also consider disease stage, comorbidities, and patient preferences.Additional information is available at www.asco.org/breast-cancer-guidelines.

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER+) breast cancer patients. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2 and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacological inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA-mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance.

PURPOSE/OBJECTIVE:biodistribution, pharmacokinetics, metabolism, and safety; and the feasibility of epichaperome-targeted tumor imaging. EXPERIMENTAL DESIGN/METHODS:I-PU-H71 tracer (201±12 MBq, <25 μg) IV bolus followed by PET/CT scans and blood radioassays. RESULTS:I-PU-H71 was retained by tumors for several days while it cleared rapidly from bones, healthy soft tissues, and blood. Radiation dosimetry is favorable and patients suffered no adverse effects. CONCLUSIONS:I-PU-H71 PET, supporting clinical development of PU-H71 and other epichaperome-targeted therapeutics.

Alpelisib is a selective inhibitor of PI3Kα, shown to improve outcomes for PIK3CA mutant, hormone receptor positive (HR+) metastatic breast cancers (MBC) when combined with antiestrogen therapy. To uncover mechanisms of resistance, we conducted a detailed, longitudinal analysis of tumor and plasma circulating tumor DNA among such patients from a phase I/II trial combining alpelisib with an aromatase inhibitor (AI) (NCT01870505). The trial's primary objective was to establish safety with maculopapular rash emerging as the most common grade 3 adverse event (33%). Among 44 evaluable patients, the observed clinical benefit rate was 52%. Correlating genetic alterations with outcome, we identified loss-of-function PTEN mutations in 25% of patients with resistance. ESR1 activating mutations also expanded in number and allele fraction during treatment and were associated with resistance. These data indicate that genomic alterations that mediate resistance to alpelisib or antiestrogen may promote disease progression and highlight PTEN loss as a recurrent mechanism of resistance to PI3Kα inhibition.

Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.

Activating mutations in PIK3CA are frequent in human breast cancer, and phosphoinositide 3-kinase alpha (PI3Kα) inhibitors have been approved for therapy. To characterize determinants of sensitivity to these agents, we analyzed PIK3CA-mutant cancer genomes and observed the presence of multiple PIK3CA mutations in 12 to 15% of breast cancers and other tumor types, most of which (95%) are double mutations. Double PIK3CA mutations are in cis on the same allele and result in increased PI3K activity, enhanced downstream signaling, increased cell proliferation, and tumor growth. The biochemical mechanisms of dual mutations include increased disruption of p110α binding to the inhibitory subunit p85α, which relieves its catalytic inhibition, and increased p110α membrane lipid binding. Double PIK3CA mutations predict increased sensitivity to PI3Kα inhibitors compared with single-hotspot mutations.

The 'Competing interests' statement of this Article has been updated; please see the accompanying Amendment. The original Article has not been corrected online.

We integrated the genomic sequencing of 1,918 breast cancers, including 1,501 hormone receptor-positive tumors, with detailed clinical information and treatment outcomes. In 692 tumors previously exposed to hormonal therapy, we identified an increased number of alterations in genes involved in the mitogen-activated protein kinase (MAPK) pathway and in the estrogen receptor transcriptional machinery. Activating ERBB2 mutations and NF1 loss-of-function mutations were more than twice as common in endocrine resistant tumors. Alterations in other MAPK pathway genes (EGFR, KRAS, among others) and estrogen receptor transcriptional regulators (MYC, CTCF, FOXA1, and TBX3) were also enriched. Altogether, these alterations were present in 22% of tumors, mutually exclusive with ESR1 mutations, and associated with a shorter duration of response to subsequent hormonal therapies.

Somatic mutations of ERBB2 and ERBB3 (which encode HER2 and HER3, respectively) are found in a wide range of cancers. Preclinical modelling suggests that a subset of these mutations lead to constitutive HER2 activation, but most remain biologically uncharacterized. Here we define the biological and therapeutic importance of known oncogenic HER2 and HER3 mutations and variants of unknown biological importance by conducting a multi-histology, genomically selected, 'basket' trial using the pan-HER kinase inhibitor neratinib (SUMMIT; clinicaltrials.gov identifier NCT01953926). Efficacy in HER2-mutant cancers varied as a function of both tumour type and mutant allele to a degree not predicted by preclinical models, with the greatest activity seen in breast, cervical and biliary cancers and with tumours that contain kinase domain missense mutations. This study demonstrates how a molecularly driven clinical trial can be used to refine our biological understanding of both characterized and new genomic alterations with potential broad applicability for advancing the paradigm of genome-driven oncology.