Biology subjects

Flower, C. T.

Publications and source records attributed to Flower, C. T..

5 recordsLinked to original sources

bioRxiv2025

Tyrosine phosphoproteome profiling identifies cell-intrinsic signals limiting the efficacy of tyrosine kinase inhibitor therapies

Tyrosine kinases (TKs) are frequently mutated or overexpressed in cancer, and TK inhibitors (TKIs) are an important therapeutic modality against TK-driven cancers, but many patients show an underwhelming response to TKIs prescribed on the basis of tumor genotype. To find cell-intrinsic TK signaling patterns which might be predictive of poor response to TKI exposure, we used high-sensitivity multiplexed mass spectrometry to quantify endogenous levels of 1,222 phosphotyrosine (pY) sites across the proteomes of TK-driven human cancer cell lines with variable response to genotype-matched TKIs. In direct comparisons between TKI-tolerant and TKI-sensitive lines with a common driver TK, we found that TKI treatment was equally effective at blocking driver TK signaling, and higher basal activity of the driver TK did not always predict higher sensitivity to TKI. All tolerant lines showed a dampened proteome-wide pY response to TKI exposure compared to sensitive lines, suggesting tumor cells with more robust TK signaling are less vulnerable to driver TK blockade. We found that each tolerant line depends on a unique set of compensatory TKs and signaling axes but are unified by hyperactivity of at least one of the SRC family kinases (SFKs) or the related ABL1/2 kinases, both at rest and under TKI treatment, despite absence of SFK/ABL genetic mutations. In time- and dose-resolved drug combination experiments, SFK/ABL inhibitors were potently synergistic with all TKIs tested, demonstrating that elevated SFK/ABL signaling is a conserved bottleneck for maximal TKI efficacy which could be exploited therapeutically. SignificanceThe last twenty-five years have seen a remarkable number of new anti-cancer therapies specifically targeting tyrosine kinases, which frequently drive tumorigenesis when hyperactive. While these therapies have brought undeniable improvements in survival time and quality of life for a large fraction of patients, many still do not respond to inhibitors aimed at their presumed driver kinase. By profiling tumor cell-intrinsic signaling networks using phosphotyrosine immunoaffinity pulldown and high-sensitivity mass spectrometry, we directly compare the signaling networks of drug-tolerant and drug-sensitive cancer cell lines across several oncogenic driver contexts and find both unique and shared signals promoting therapy tolerance.

cancer biology↗

bioRxiv2024

Uncovering the signaling networks of disseminated glioblastoma cells in vivo with INSIGHT

Dysregulation of intracellular signaling networks underpins cancer. However, a systems-level elucidation of how signaling networks within distinct cell subpopulations drive cancer progression in vivo has been unattainable due to technical limitations. We developed INSIGHT (INvestigating SIGnaling network of specific cell subpopulation in Heterogeneous Tissue), a new platform technology combining fluorescence-activated cell sorting with ultra-sensitive mass spectrometry to enable phosphoproteomic characterization of rare and discrete cell subpopulations from fixed tissues. We demonstrated the broad utility of INSIGHT by analyzing the oligodendroglial cell-specific signaling network in the mouse brain. We then applied INSIGHT to investigate the rare, disseminated tumor cell subpopulation in glioblastoma patient-derived xenograft models. INSIGHT uncovered a global rewiring of signaling networks with tumor cell dissemination, marked by a transition from proliferation-associated signaling in the primary tumor cells to signaling associated with postsynapse, neuronal migration, and ion homeostasis in disseminated tumor cells. We reveal interconnections between signaling circuitries within the networks, with numerous proteins, including GluA2, exhibiting altered phosphorylation without protein expression changes, emphasizing the role of post-translational modifications in glioblastoma dissemination. We validated key phosphorylation changes and inferred differentially active kinases with tumor spread to offer new systems-level insights into glioblastoma dissemination mechanisms in vivo. INSIGHT is generally applicable to a wide range of biological systems without genetic engineering and provides quantitative phosphorylation and protein expression data for selected cell subpopulations from heterogeneous tissues.

systems biology↗

bioRxiv2024

Structural and systems characterization of phosphorylation on metabolic enzymes identifies sex-specific metabolic reprogramming in obesity

Coordination of adaptive metabolism through cellular signaling networks and metabolic response is essential for balanced flow of energy and homeostasis. Post-translational modifications such as phosphorylation offer a rapid, efficient, and dynamic mechanism to regulate metabolic networks. Although numerous phosphorylation sites have been identified on metabolic enzymes, much remains unknown about their contribution to enzyme function and systemic metabolism. In this study, we stratify phosphorylation sites on metabolic enzymes based on their location with respect to functional and dimerization domains. Our analysis reveals that the majority of published phosphosites are on oxidoreductases, with particular enrichment of phosphotyrosine (pY) sites in proximity to binding domains for substrates, cofactors, active sites, or dimer interfaces. We identify phosphosites altered in obesity using a high fat diet (HFD) induced obesity model coupled to multiomics, and interrogate the functional impact of pY on hepatic metabolism. HFD induced dysregulation of redox homeostasis and reductive metabolism at the phosphoproteome and metabolome level in a sex-specific manner, which was reversed by supplementing with the antioxidant butylated hydroxyanisole (BHA). Partial least squares regression (PLSR) analysis identified pY sites that predict HFD or BHA induced changes of redox metabolites. We characterize predictive pY sites on glutathione S-transferase pi 1 (GSTP1), isocitrate dehydrogenase 1 (IDH1), and uridine monophosphate synthase (UMPS) using CRISPRi-rescue and stable isotope tracing. Our analysis revealed that sites on GSTP1 and UMPS inhibit enzyme activity while the pY site on IDH1 induces activity to promote reductive carboxylation. Overall, our approach provides insight into the convergence points where cellular signaling fine-tunes metabolism. Summary StatementBy employing a multi-disciplinary approach we stratify structural features of phosphorylation sites on metabolic enzymes, map the systems level changes induced by obesity, identify key pathways with sex specific phosphoproteomic responses, and validate the functional role of phosphorylation sites for select enzymes.

biochemistry↗

bioRxiv2024

Signaling and transcriptional dynamics underlying early adaptation to oncogenic BRAF inhibition

A major contributor to poor sensitivity to anti-cancer kinase inhibitor therapy is drug-induced cellular adaptation, whereby remodeling of signaling and gene regulatory networks permits a drug-tolerant phenotype. Here, we resolve the scale and kinetics of critical subcellular events following oncogenic kinase inhibition and preceding cell cycle re-entry, using mass spectrometry-based phosphoproteomics and RNA sequencing to capture molecular snapshots within the first minutes, hours, and days of BRAF kinase inhibitor exposure in a human BRAF-mutant melanoma model of adaptive therapy resistance. By enriching specific phospho-motifs associated with mitogenic kinase activity, we monitored the dynamics of thousands of growth- and survival-related protein phosphorylation events under oncogenic BRAF inhibition and drug removal. We observed early and sustained inhibition of the BRAF-ERK axis, gradual downregulation of canonical cell cycle-dependent signals, and three distinct and reversible phase transitions toward quiescence. Statistical inference of kinetically-defined signaling and transcriptional modules revealed a concerted response to oncogenic BRAF inhibition and a dominant compensatory induction of SRC family kinase (SFK) signaling, which we found to be at least partially driven by accumulation of reactive oxygen species via impaired redox homeostasis. This induction sensitized cells to co-treatment with an SFK inhibitor across a panel of patient-derived melanoma cell lines and in an orthotopic mouse xenograft model, underscoring the translational potential for measuring the early temporal dynamics of signaling and transcriptional networks under therapeutic challenge.

systems biology↗

bioRxiv2020

High-density, targeted monitoring of tyrosine phosphorylation reveals activated signaling networks in human tumors

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed "SureQuant pTyr," coupling low input pTyr enrichment with a panel of isotopically labeled, tyrosine phosphorylated internal standard (IS) peptides. Using internal standard guided acquisition, the real-time detection of IS peptides during the analysis initiates the sensitive and selective quantitation of endogenous pTyr targets. This framework allows for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, enhances target detection success rates, and improves the robustness and usability of targeted acquisition. We establish the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient specific oncogenic driving mechanisms. While in some cases pTyr profiles align with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlight instances of new insights gained using pTyr characterization and emphasize the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enable pTyr-based measurements for applications in precision medicine. SummaryA targeted, mass spectrometry-based method, termed "SureQuant pTyr," enables highly sensitive and reproducible profiling of tyrosine phosphorylation levels in human colorectal tumors and reveals dysregulated signaling networks for enhanced tumor characterization and biomarker identification.

bioengineering↗