Biology subjects

Stauffer, P. E.

Publications and source records attributed to Stauffer, P. E..

3 recordsLinked to original sources

bioRxiv2025

Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma using Data-Independent Acquisition Mass Spectrometry

Cells rely on the Unfolded Protein Response (UPR) to maintain ER protein homeostasis (proteostasis) when faced with elevated levels of misfolded and aggregated proteins. The UPR is comprised of three main branches--ATF6, IRE1, and PERK--that coordinate the synthesis of proteins involved in folding, trafficking, and degradation of nascent proteins to restore ER function. Dysregulation of the UPR is linked to numerous diseases, including neurodegenerative disorders, cancer, and diabetes. Despite its importance, identifying UPR targets has been challenging due to their heterogeneous induction, which varies by cell type and tissue. Additionally, defining the magnitude and range of UPR-regulated genes is difficult because of intricate temporal regulation, feedback between UPR branches, and extensive cross-talk with other stress-signaling pathways. To comprehensively identify UPR-regulated proteins and determine their branch specificity, we developed a data-independent acquisition (DIA) liquid-chromatography mass spectrometry (LC-MS) pipeline. Our optimized workflow improved identifications of low-abundant UPR proteins and leveraged an automated SP3-based protocol on the Biomek i5 liquid handler for label-free peptide preparation. Using engineered stable cell lines that enable selective pharmacological activation of each UPR branch without triggering global UPR activation, we identified branch-specific UPR proteomic targets. These targets were subsequently applied to investigate proteomic changes in multiple patient-derived BRAF-mutant melanoma cell lines treated with a BRAF inhibitor (PLX4720, i.e., vemurafenib). Our findings revealed differential regulation of the XBP1s branch of the UPR in the BRAF-mutant melanoma cell lines after PLX4720 treatment, likely due to calcium activation, suggesting that the UPR plays a role as a non-genetic mechanism of drug tolerance in melanoma. In conclusion, the validated branch-specific UPR proteomic targets identified in this study provide a robust framework for investigating this pathway across different cell types, drug treatments, and disease conditions in a high-throughput manner.

systems biology↗

bioRxiv2023

Purinergic calcium signaling drives drug tolerance through ERK reactivation in BRAF-mutant melanoma

Drug tolerance is a major cause of relapse after cancer treatment. In spite of intensive efforts1-9, its molecular basis remains poorly understood, hampering actionable intervention. We report a previously unrecognized signaling mechanism supporting drug tolerance in BRAF-mutant melanoma treated with BRAF inhibitors that could be of general relevance to other cancers. Its key features are cell-intrinsic intracellular Ca2+ signaling initiated by P2X7 receptors (purinergic ligand-gated cation channels), and an enhanced ability for these Ca2+ signals to reactivate ERK1/2 in the drug-tolerant state. Extracellular ATP, virtually ubiquitous in living systems, is the ligand that can initiate Ca2+ spikes via P2X7 channels. ATP is abundant in the tumor microenvironment and is released by dying cells, ironically implicating treatment-initiated cancer cell death as a source of trophic stimuli that leads to ERK reactivation and drug tolerance. Such a mechanism immediately offers an explanation of the inevitable relapse after BRAFi treatment in BRAF-mutant melanoma, and points to actionable strategies to overcome it.

cancer biology↗

bioRxiv2022

A heterogeneous drug tolerant persister state in BRAF-mutant melanoma is characterized by ion channel dysregulation and susceptibility to ferroptosis

Drug-tolerant persisters (DTPs) represent a major obstacle to durable responses in targeted cancer therapy. DTPs are commonly described as distinct single-cell states that survive drug treatment via reversible, non-genetic mechanisms and drive tumor recurrence. Recent work demonstrates that multiple DTPs can coexist, reflecting diversity in lineage, signaling programs, or stress responses. However, each DTP is still generally viewed as a uniform cellular phenotype. Building on our prior work describing a population-level DTP termed "idling" [Paudel et al., Biophys. J. (2018) 114, 1499-1511], here we present evidence supporting a fundamentally different view: that DTPs are not single-cell states, but rather heterogeneous populations composed of multiple sub-states with distinct division and death rates that balance to produce near-zero net population growth. Using single-cell transcriptomics and lineage barcoding, we identify multiple phenotypic states within idling DTP populations, with reduced heterogeneity compared to untreated populations, and find that idling DTP cells emerge from nearly all lineages. Transcriptomic and functional analyses further reveal altered ion-channel activity in idling DTPs, which we confirm experimentally. Moreover, drug-response assays reveal increased susceptibility of idling DTPs to ferroptosis, a non-apoptotic form of regulated cell death, indicating the emergence of vulnerabilities associated with drug tolerance. Altogether, our results support a population-level view of tumor drug tolerance in which DTPs comprise stable collections of phenotypic states, shaped by treatment-defined phenotypic landscapes, which are potentially vulnerable to subsequent interventions. This perspective implies that eradicating DTPs will require a fundamental shift away from cell-type-centric strategies toward sequential treatments that progressively reduce phenotypic heterogeneity by modulating the molecular and cellular processes that establish the DTP landscape, an approach previously termed "targeted landscaping."

systems biology↗