We Study How Kinases Regulate Breast Cancer Growth And Drug Response

SHP2 is a phosphatase and a critical mediator of receptor tyrosine kinase (RTK)-driven RAS/mitogen-activated protein kinase (MAPK) signaling. Despite promising preclinical data, SHP2 inhibitors have shown minimal clinical efficacy, with no defined clinical mechanisms of primary resistance. Here, we elucidate phosphorylation of SHP2 at tyrosine 62 (pY62) as a hotspot phosphorylation site in the proteome and RTK-driven tumor types in patients. We demonstrate that SRC family kinases directly phosphorylate SHP2 at Y62, downstream of but not directly phosphorylated by RTKs. Using biochemical and biophysical analyses, we show that SHP2 Y62D enforces an open, active conformation, resulting in constitutive phosphatase activation that is sufficient to activate MAPK signaling and confer resistance to allosteric SHP2 inhibitors. These findings establish that SHP2 pY62 is a phosphorylation hotspot phenocopying mutational activation, a mechanism of primary resistance to SHP2 inhibitors, and a cancer drug target distinct from wildtype SHP2.

Based on the CAPItello-291 phase III trial results, capivasertib in combination with fulvestrant has been approved for patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer harboring one or more PIK3CA, AKT1, and/or PTEN alterations. Given the growing interest in circulating tumor DNA (ctDNA) next-generation sequencing (NGS) to detect PIK3CA/AKT1/PTEN alterations, we retrospectively compared blood-based FoundationOne®Liquid CDx versus tumor tissue-based FoundationOne®CDx real-world data from patients with various breast cancer subtypes. We utilized a database of patients profiled with FoundationOne®CDx and/or FoundationOne®Liquid CDx during routine clinical care. Analytical comparison of all pathogenic alterations in PIK3CA, AKT1, AKT2, AKT3, and PTEN, including alterations defined in the CAPItello-291 protocol (CAPItello-defined alterations), was performed in paired data from 289 patients with both tissue and liquid biopsies sampled within 90 days of each other. Overall positive percent agreement (PPA) for short variants across ctDNA tumor fraction (TF) subgroups in paired biopsy samples was: ctDNA TF ≥ 10%: PIK3CA, 93.9%; AKT1, 100%; PTEN, 100%; ctDNA TF 1%-10%: PIK3CA, 96.3%; AKT1, 100%; PTEN, 100%; ctDNA TF < 1%: PIK3CA, 34.7%; AKT1, 50.0%; PTEN, 37.5%. PPA for CAPItello-defined alterations was: ctDNA TF ≥ 10%: 92.5%; ctDNA TF 1%-10%: 97.1%; ctDNA TF < 1%: 33.9%. For PTEN homozygous deletions, PPA was 50.0% in cases with ctDNA TF ≥ 10%. Overall PPA for AKT2 and AKT3 copy number variations (CNVs) was 66.7% and 0%, respectively. Blood-based NGS could offer a minimally invasive option to identify clinically relevant PIK3CA/AKT1/PTEN short variants in cases with ctDNA TF ≥ 1%. Confirmatory tissue-based NGS should be performed when blood-based NGS results are negative, especially when ctDNA TF is < 1% and for enhanced detection of CNVs in general.

The purpose of this study is to comprehensively characterize the clinical and genomic landscapes of PIK3CA, AKT1, and PTEN alterations and examine their functional implications in AKT-driven breast cancer. Comprehensive genomic profiling of 51,767 breast tumors was performed with FoundationOne^® CDx or FoundationOne^®. We examined the genomic landscape of PIK3CA, PTEN, and AKT1 alterations and their distribution across clinical variables of interest. Prior deep mutational scanning (DMS) data was used to functionally characterize clinical PTEN variants. There were 29,157 total variants across PIK3CA, AKT1, and PTEN , including pathogenic variants and VUS. The most frequently altered gene was PIK3CA, followed by PTEN, then AKT1. The most common alterations in each gene were PIK3CA H1047R, E545K, and E542K; AKT1 E17K; and PTEN homozygous copy number deletion. PIK3CA alterations were less prevalent in patients of African genetic ancestry, while AKT1 and PTEN alterations were balanced across ancestries. PIK3CA, AKT1, and PTEN pathogenic alterations were all mutually exclusive to each other. Using available DMS data on missense PTEN mutations, we found that 32.5% showed discordant effects on protein stability and phosphatase activity, underscoring the need for functional validation beyond predicted loss-of-function. Here we present the landscape of PIK3CA, AKT1, and PTEN alterations in the largest clinical cohort examined to date. The functional implications of lesser-known variants in each gene warrant further investigation by tools such as deep mutational scanning.