Biology subjects

Farhi, J.

Publications and source records attributed to Farhi, J..

3 recordsLinked to original sources

bioRxiv2023

Histone H3 E50K mutation confers oncogenic activity and supports an EMT phenotype

Sequencing of human patient tumors has identified recurrent missense mutations in genes encoding core histones. We report that mutations that convert histone H3 amino acid 50 from a glutamate to a lysine (H3E50K) support an oncogenic phenotype in human cells. Expression of H3E50K is sufficient to transform human cells as evidenced by a dramatic increase in cell migration and invasion, and a statistically significant increase in proliferation and clonogenicity. H3E50K also increases the invasive phenotype in the context of co-occurring BRAF mutations, which are present in patient tumors characterized by H3E50K. H3E50 lies on the globular domain surface in a region that contacts H4 within the nucleosome. We find that H3E50K perturbs proximal H3 post-translational modifications globally and dysregulates gene expression, activating the epithelial to mesenchymal transition. Functional studies using S. cerevisiae reveal that, while yeast cells that express H3E50K as the sole copy of histone H3 show sensitivity to cellular stressors, including caffeine, H3E50K cells display some genetic interactions that are distinct from the characterized H3K36M oncohistone yeast model. Taken together, these data suggest that additional histone H3 mutations have the potential to be oncogenic drivers and function through distinct mechanisms that dysregulate gene expression. SummaryO_LIRecurrent missense mutation that alter histone H3E50 were identified in patient tumors C_LIO_LIH3E50K expression dysregulates global H3 post-translational modification, gene expression and may govern a transcriptional program associated with metastatic phenotypes C_LIO_LIhht2-E50K yeast cells exhibit growth defects in the presence of DNA damaging agents C_LI

cancer biology↗

bioRxiv2022

Dynamic in vivo mapping of the methylproteome using a chemoenzymatic approach

Dynamic protein post-translation methylation is essential for cellular function, highlighted by the essential role of methylation in transcriptional regulation and its aberrant dysregulation in diseases including cancer. This underscores the importance of cataloging the cellular methylproteome. However, comprehensive analysis of the methylproteome remains elusive due to limitations in current enrichment and analysis pipelines. Here, we employ an L-Methionine analogue, ProSeMet, that is chemoenzymatically converted to the SAM analogue ProSeAM in cells and in vivo to tag proteins with a biorthogonal alkyne that can be directly detected via LC-MS/MS, or functionalized for subsequent selective enrichment and LC-MS/MS identification. Without enrichment, we identify lysine mono-, di-, and trimethylation, histidine methylation, and arginine methylation with site specific resolution on proteins including heat shock protein HSPA8, for which methylation is implicated in human disease. With enrichment, we identify 486 proteins known to be methylated and 221 proteins with novel methylation sites encompassing diverse cellular functions. Systemic ProSeMet delivery in mice pseudomethylates proteins across organ systems with blood-brain barrier penetrance and identifies site-specific pseudomethylation in vivo with LC-MS/MS. Leveraging these pipelines to define the cellular methylproteome may have broad applications for understanding the methylproteome in the context of disease.

biochemistry↗

bioRxiv2021

Combined PI3K and MLL inhibition synergizes in models of HR+ breast cancer

The high frequency of aberrant PI3K pathway activation in hormone receptor-positive (HR+) breast cancer has led to the development, clinical testing, and approval of the p110-selective PI3K inhibitor alpelisib. The limited clinical efficacy of alpelisib and other PI3K inhibitors is partially attributed to the functional antagonism between PI3K and estrogen receptor (ER) signaling, which is mitigated via combined PI3K inhibition and endocrine therapy. We and others have previously demonstrated a chromatin-associated mechanism by which PI3K supports cancer development and antagonizes ER signaling through the modulation of H3K4 methylation. Here we show that inhibition of the H3K4 histone methyltransferase MLL1 in combination with PI3K inhibition impairs HR+ breast cancer clonogenicity and cell proliferation. While combined PI3K/MLL1 inhibition reduces AKT effector signaling and H3K4 methylation, MLL1 inhibition increases PI3K effector signaling and upregulates the expression of receptor tyrosine kinase signaling cascades upstream of and including AKT. These data reveal a feedback loop between MLL1 and AKT in which MLL1 inhibition reactivates AKT. We additionally show that combined PI3K and MLL1 inhibition synergizes to cause cell death in in vitro and in vivo models of HR+ breast cancer, which is enhanced by the additional genetic ablation of the H3K4 methyltransferase and AKT target MLL4. Together, our data provide evidence of a feedback mechanism connecting histone methylation with AKT and may support the preclinical development and testing of pan-MLL inhibitors. Significance StatementPharmacological inhibition of PI3K provides limited efficacy in PIK3CA-mutated, HR+ breast cancers. Here the authors leverage PI3K/AKT-driven chromatin modification to identify MLL histone methyltransferases as a therapeutic target. Dual PI3K and MLL inhibition synergize to reduce clonogenicity and cell proliferation while enhancing apoptosis in in vitro models, and induces tumor regression in xenograft models of PI3K-activated, HR+ breast cancer. Furthermore, MLL1 inhibition reveals a feedback loop leading to AKT hyperactivation, which is relieved with combined PI3K/MLL inhibition. These findings demonstrate the utility of MLL inhibitors for the treatment of some solid cancers, as patients with HR+ breast cancer characterized by PIK3CA mutation may derive clinical benefit from combined PI3K/MLL inhibition.

cancer biology↗