Publications

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.

Immune checkpoint inhibitors have transformed treatment paradigms in multiple tumor types including triple-negative breast cancer. However, clinical benefit in metastatic disease is restricted to patients with PD-L1-positive tumors, and primary and acquired resistance remains common among treated patients. These challenges have prompted interest in novel immune checkpoints such as V-domain immunoglobulin suppressor of T-cell activation (VISTA), a B7 family protein with complex immunoregulatory functions. Although VISTA is expressed in multiple tumor types and increasingly explored as a therapeutic target, its exact signaling mechanisms remain unclear. In this issue of Cancer Research, Zhao and colleagues demonstrated that VISTA suppresses tumor growth in a subset of VISTA-expressing triple-negative breast cancers via a cell-intrinsic, immune-independent mechanism. They discovered a conserved four-amino acid motif (NPGF) in VISTA’s intracellular domain that mediates its antiproliferative effects by recruiting and sequestering the adapter protein NUMB at endosomes, thereby impairing trafficking of multiple growth factor receptors. VISTA-expressing tumors exhibit marked sensitivity to VISTA-blocking antibodies, a response that depends on the presence of the NPGF motif. Together, these findings redefine VISTA as a cell-intrinsic regulator of tumor growth and introduce new potential therapeutic avenues that extend beyond receptor-ligand disruption to interfere with checkpoint-mediated intracellular trafficking and signaling.

Diabetes (DM) and hyperglycemia during chemotherapy increase the risk of toxicity, yet the prevalence and patterns of hyperglycemia in early-stage breast cancer (ESBC) patients undergoing chemotherapy with concurrent dexamethasone remain poorly understood. We conducted a prospective single-arm study using FreeStyle Libre Pro continuous glucose monitoring in patients with ESBC receiving chemotherapy from 12/2020-2/2022. Sensors measured interstitial glucose every 15 min and were reapplied every 2-3 weeks. Primary endpoints were (1) prevalence of hyperglycemia, and (2) for those with hyperglycemia, the proportion of time spent hyperglycemic. Secondary endpoints included baseline glucose tolerance by A1c, changes in glucose-related biomarkers, and changes in patient-reported neuropathy, quality of life, and fatigue. Analysis was stratified by baseline A1c. Among 20 evaluable patients, common chemotherapy regimens included docetaxel/cyclophosphamide, paclitaxel/trastuzumab, paclitaxel then doxorubicin/cyclophosphamide, docetaxel/carboplatin/trastuzumab/pertuzumab, and cyclophosphamide/methotrexate/fluorouracil. All patients received Dexamethasone. At baseline, 10 patients were euglycemic, 7 had pre-DM, and 3 had DM. All experienced hyperglycemia. All patients receiving chemotherapy for ESBC experienced hyperglycemia, with time spent hyperglycemic varying significantly by baseline A1c. Future research should explore approaches to and benefits of improving glycemic control during treatment in patients with baseline dysglycemia.

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.

Colorectal cancer (CRC) remains a leading cause of cancer death because of metastatic spread. LIN28B is overexpressed in 30% of CRCs and promotes metastasis, yet its mechanisms remain unclear. In this study, we genetically modified CRC cell lines to overexpress LIN28B, resulting in enhanced PI3K/AKT pathway activation and liver metastasis in mice. We developed genetically modified mouse models with constitutively active Pik3ca that form intestinal tumors progressing to liver metastases with an intact immune system, addressing the limitations of previous Pik3ca-mutant models, including long tumor latency, mixed histology, and lack of distant metastases. The PI3Kα-specific inhibitor alpelisib reduced migration and invasion in vitro and metastasis in vivo. We present a comprehensive analysis of vertical inhibition of the PI3K/AKT pathway in CRC using the FDA-approved drugs alpelisib and capivasertib (an AKT inhibitor) in combination with LY2584702 (a ribosomal protein S6 kinase inhibitor) in CRC cell lines and mouse- and patient-derived organoids. Tissue microarrays from patients with CRC verified that LIN28B and PI3K/AKT pathway activation correlate with CRC progression. These findings highlight the critical role of the LIN28B-mediated PI3K/AKT pathway in CRC metastasis, the therapeutic potential of targeted inhibition, and the promise of patient-derived organoids in precision medicine in metastatic CRC.

Approximately 20% of patients living with colorectal cancer (CRC) have activating mutations in their tumors in the PIK3CA oncogene. Two or more activating mutations (multi-hit) for the PIK3CA allele increase PI3K⍺ signaling compared to single-point mutations, resulting in exceptional response to PI3K⍺ inhibition. We aimed to identify the prevalence of PIK3CA multi-hit mutations in metastatic CRC to identify patients who may benefit from PI3K inhibitors. The Foundation Medicine database (Boston, MA, USA) was analyzed for patients with CRC who underwent genomic profiling on tumor DNA isolated during routine clinical care from 2013 to 2021. Molecular and clinical variables were abstracted for patients with PIK3CA mutations. We identified 49 051 patients with CRC who underwent Foundation Medicine testing. 710/41154 (1.7%) patients had multi-hit PIK3CA mutations, of which 53% were male with a median age of 60. Microsatellite status was available for 697 patients with multi-hit PIK3CA and 17.6% were microsatellite instability-high. Clinically relevant mutations in KRAS and BRAF^V600E were seen in 459/710 and 65/710, respectively. The 4 most common PIK3CA variants were H1047R, E545K, E542K, and R88Q. The most common variant pair was E542K-E545K.

Tumors harboring two or more PIK3CA short variant (SV) (“multi-hit”) mutations have been linked to improved outcomes with anti-PIK3CA-targeted therapies in breast cancer. The landscape and clinical implications of multi-hit PIK3CA alterations in clinically advanced prostate cancer (CAPC) remain elusive. The Foundation Medicine FoundationCore database was used to identify 19,978 CAPC tumors that underwent hybrid capture-based comprehensive genomic profiling to evaluate all classes of genomic alterations (GA) and determine tumor mutational burden (TMB), microsatellite instability (MSI), genomic ancestry, single-base substitution mutational signatures, and homologous recombination deficiency signature (HRDsig). Tumor cell PD-L1 expression was determined by IHC. 18,741 tumors were PIK3CA wild type (WT), 1155 featured single PIK3CA SV, and 82 featured multi-hit PIK3CA SVs. Single-hit and multi-hit featured more driver GA per tumor than PIK3CA WT CAPC, as well as higher prevalence of MMR mutational signature, MSI high status, and TMB levels versus PIK3CA WT. Other differences in GA included higher frequencies of GA in BRCA2 in multi-hit versus WT, ATM in multi-hit versus WT and PTEN in single-hit versus WT. Homologous recombination deficiency signatures were higher in PIK3CA WT versus single-hit. There were no significant differences in PD-L1 expression among the three groups. Identification of multi-hit PIK3CA GA in CAPC highlights a potentially unique phenotype that may be associated with response to anti-PIK3CA targeted therapy and checkpoint inhibition, supporting relevant clinical trial designs.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.