LINCS MCF 10A Common Project: Fixed-time-point sensitivity measures of the MCF 10A breast cell line to 8 small molecule perturbagens. Repeat performed by Scientist B in 2019 to assess reproducibility. Dataset 2 of 2: Calculated dose response metrics.
Project description:Metastasis poses a major challenge in cancer management, including EML4-ALK-rearranged non-small cell lung cancer (NSCLC). As cell migration is a critical step during metastasis, we assessed the anti-migratory activities of several clinical ALK inhibitors in NSCLC cells and observed differential anti-migratory capabilities despite similar ALK inhibition, with brigatinib displaying superior anti-migratory effects over other ALK inhibitors. Applying an unbiased in-situ mass spectrometry-based chemoproteomics approach, we determined the proteome-wide target profile of brigatinib in EML4-ALK+ NSCLC cells. Dose-dependent and cross-competitive chemoproteomics suggested MARK2 and MARK3 as relevant brigatinib kinase targets. Functional validation showed that combined pharmacological inhibition or genetic modulation of MARK2/3 inhibited cell migration. Consistently, brigatinib treatment induced inhibitory YAP1 phosphorylation downstream of MARK2/3. Collectively, our data suggest that brigatinib exhibits unusual cross-phenotype polypharmacology as despite similar efficacy for inhibiting EML4-ALK-dependent cell proliferation as other ALK inhibitors, it more effectively prevented migration of NSCLC cells due to co-targeting of MARK2/3.
Project description:We implemented an integrative systems-level analysis of multi-region postmortem human brain proteomics derived from the Religious Order and Rush Memory and Aging Project (ROSMAP) to identify proteins and pathways significantly altered in resilient cases. ROSMAP is an information-rich longitudinal cohort-based study in which participants enroll without dementia, undergo annual cognitive and clinical assessments and donate their brains at death (12). Multiplex tandem mass tag mass spectrometry (TMT-MS)-based proteomic data was implemented for a correlation network analysis. Data from an independent brain proteome wide association study (PWAS) of cognitive trajectory was integrated with the brain network to robustly prioritize protein communities associated with cognitive resilience. This revealed proteins linked to synaptic biology and cellular energetics. Neuritin (NRN1) was prioritized as a hub that co-expressed with a community of proteins with high correlation to cognitive stability in life and is known for important roles in synaptic maturation and stability.
Project description:Comparing the performance of methylamine and hydroxylamine on phospho-peptide analysis, particularly in relation to the identification of more complex dose-dependent patterns.
Project description:Osimertinib is the first-line therapy for EGFR-mutated non-small cell lung cancer, but acquired resistance commonly develops, linked to a drug-tolerant persister (DTP) cell state that promotes survival and eventual resistance. Using data-independent acquisition mass spectrometry (DIA-MS), we mapped dynamic proteomic and phosphoproteomic changes in DTPs. While osimertinib initially blocks EGFR signaling, DTPs show activation in ribosome synthesis and protein translation pathways, with resistance emerging via EGFR reactivation and anti-apoptotic mechanisms, including YAP1 and mTOR-BAD hyperphosphorylation. Elevated phosphorylation of CDK1 substrates in DTPs was observed, and inhibiting CDK1-mediated SAMHD1 activation impaired DTP growth. This study highlights key DTP mechanisms, suggesting biomarkers and therapeutic targets to delay resistance.
Project description:Lung cancer is associated with high prevalence and mortality, and despite significant successes with targeted drugs in genomically defined subsets of lung cancer and immunotherapy, the majority of patients currently does not benefit from these therapies. Through a targeted drug screen, we found the recently approved multi-kinase inhibitor midostaurin to have potent activity in several lung cancer cells independent of its intended target, PKC, or a specific genomic marker. To determine the underlying mechanism of action we applied a layered functional proteomics approach and a new data integration method. Using chemical proteomics, we identified multiple midostaurin kinase targets in these cells. Network-based integration of these targets with quantitative tyrosine and global phosphoproteomics data using protein-protein interactions from the STRING database suggested multiple targets are relevant for the mode of action of midostaurin. Subsequent functional validation using RNA interference and selective small molecule probes showed that simultaneous inhibition of TBK1, PDK1 and AURKA was required to elicit midostaurin’s cellular effects. Immunoblot analysis of downstream signaling nodes showed that combined inhibition of these targets altered PI3K/AKT and cell cycle signaling pathways that in part converged on PLK1. Furthermore, rational combination of midostaurin with the more potent PLK1 inhibitor BI2536, which is in advanced clinical trials, elicited strong synergy. Our results demonstrate that combination of complementary functional proteomics approaches and subsequent network-based data integration can reveal novel insight into the complex mode of action of multi-kinase inhibitors, actionable targets for drug discovery and cancer vulnerabilities. Finally, we illustrate how this knowledge can be utilized for the rational design of synergistic drug combinations with high potential for clinical translation.