Multi-omic Dissection of Oncogenically Active Epiproteomes Identifies Drivers of Proliferative and Invasive Breast Tumors

Abstract

Proliferative and invasive breast tumors evolve heterogeneously in individual patients, posing significant challenges in identifying new druggable targets for precision, effective therapy. Here we present a functional multi-omics method, interaction-Correlated Multi-omic Aberration Patterning (iC-MAP), which dissects intra-tumor heterogeneity and identifies in situ the oncogenic consequences of multi-omics aberrations that drive proliferative and invasive tumors. First, we perform chromatin activity-based chemoproteomics (ChaC) experiments on breast cancer (BC) patient tissues to identify genetic/transcriptomic alterations that manifest as oncogenically active proteins. ChaC employs a biotinylated small molecule probe that specifically binds to the oncogenically active histone methyltransferase G9a, enabling sorting/enrichment of a G9a-interacting protein complex that represents the predominant BC subtype in a tissue. Second, using patient transcriptomic/genomic data, we retrospectively identified some G9a interactor-encoding genes that showed individualized iC-MAP. Our iC-MAP findings represent both new diagnostic/prognostic markers to identify patient subsets with incurable metastatic disease and targets to create individualized therapeutic strategies.

Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics

“…For example, we identified the splicing factor SF3B1 and the 40S ribosomal protein Rps14 as ET-specific G9a interactors, and Bojkova et al found that emetine inhibition of Rps14 or pladienolide inhibition of SF3B1 significantly reduced SARS-CoV-2 replication 19 . Like our finding for G9a interactors in cancer patients with poor prognosis 16 , the ChaC-MS results for ET macrophages showed that certain genes upregulated by SARS-CoV-2 infection are fully translated to their encoded proteins as ET-specific G9a interactors. Thus, constitutively active G9a may coordinate these SARS-CoV-2 activated translation pathways.…”

“…Accordingly, we found by top-down mass spectrometry (MS), ChIP-PCR, and ChaC chemoprobe pull-down that the methylation activity of G9a was constitutively higher in chronically inflamed or endotoxin-tolerant (TL or ET) macrophages compared with acutely inflamed (NL) cells. 14 Thus, we performed label-free quantitation (LFQ), 25,26 UNC0965 ChaC experiments 16 on mouse Raw 264.7 macrophages under nonstimulated (N) and different inflammatory conditions (NL, TL) ( Fig. 1a ).…”

“…Similar to our recent report, 16 1 mg nuclear protein extracted from Raw 264.7 macrophage cells was incubated overnight at 4 o C with 2 nmole UNC0965 pre-coupled to 50 μ l neutravidin-agarose (Thermo Fisher), and washed three times with 1 ml lysis buffer to remove non-specific bound proteins. For on-beads sampling and processing, five additional washes with 50 mM Tris-HCl pH 8.0, 150 mM NaCl were used to remove residual detergents.…”

“…Further, we showed that inhibition of G9a enzyme activity mitigated or reversed ET, 14 which implicated active G9a in ET-related, SARS-CoV-2-induced pathogenesis. Thus, to dissect G9a-associated pathways and mechanisms in ET, we used our chromatin activity-based chemoproteomic (ChaC) approach, which consists of a biotinylated G9a inhibitor UNC0965 16 , to capture ET-phenotypic G9a protein complexes and identify their constituents by mass spectrometry (MS). Notably, ChaC-MS is superior to conventional immunoprecipitation-MS 17,18 that captures protein complexes based only on epitope abundance.…”

Novel brain-penetrant inhibitor of G9a methylase blocks Alzheimer’s disease proteopathology for precision medication

“…More broadly, combined ChaC-MS data predicted that, via AD-phenotypic interactions with key translation regulators such as HNRNPA2B1 and other regulators of ribosomal biogenesis, G9a has a noncanonical (nonepigenetic) function in translational and post-translational regulation of AD pathogenesis. Also, to determine the clinicopathological relevance of these ChaC findings, we used our multiomics approach (iC-MAP, interaction Correlated Multi-omic Aberration Patterning) 13 to retrospectively analyze the NIH Gene Expression Omnibus databases of AD patient blood (Accession: GSE63060) 33 . We identified 26 and 47 G9a interactor mRNAs that had interactioncorrelated overexpression patterns 34 in the blood of 80 MCI and 145 AD patients, respectively, compared with a healthy population (n=104) (fig.…”