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Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity. [Display omitted] • Chemical proteomic analysis of cysteine ligandability across 400+ cancer cell lines • Ligandability is impacted by cellular redox states, protein conformations, and mutations • Covalent ligands disrupt activity of oncogenic transcription factors NF-κB1 and SOX10 DrugMap serves as a roadmap to develop covalent ligands for oncogenic drivers. [ABSTRACT FROM AUTHOR]

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones—including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H 2 O 2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H 2 O 2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway—required to resolve nuclear H 2 O 2 accumulation and mediate resistance to platinum-based agents in ovarian cancers. [Display omitted] • Integrated chemical proteomics and CRISPRi screens identify ROS-target proteins • Nuclear H 2 O 2 oxidizes C408 in CHK1's autoinhibitory domain leading to its activation • CHK1 regulates mitochondrial translation by inhibiting mtDNA-binding protein SSBP1 • SSBP1 promotes resistance to platinum-based agents and nuclear H 2 O 2 in ovarian cancers Anticancer drug mechanism studies using an integrated proteogenomic framework reveal a nucleus-to-mitochondria ROS-sensing pathway that couples DNA damage response to the control of mitochondrial translation and may serve as a mechanism of resistance to platinum-based agents. [ABSTRACT FROM AUTHOR]

Summary: Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses. [ABSTRACT FROM AUTHOR]