Brian Raught

The SLIT–Roundabout signaling pathway regulates axon guidance and cell migration, and ROBO1 is a receptor for SLIT ligands. ROBO1 undergoes constitutive endocytosis, which is enhanced upon NSLIT2 binding, but the molecular mechanisms and functional consequences of this process are not well understood. Using pharmacologic inhibitors and molecular techniques, we found that clathrin-mediated endocytosis is necessary for NSLIT2-induced inhibition of cell spreading. To explore the underlying mechanisms, we performed proximity-dependent biotin identification to identify ROBO1-interacting proteins whose association with the cytoplasmic domain of ROBO1 is differentially regulated by NSLIT2. We discovered that adenomatous polyposis coli, a multifunctional tumor suppressor, is in close proximity to ROBO1 but dissociates upon binding of NSLIT2 and that this dissociation is necessary for clathrin-mediated endocytosis of ROBO1 and subsequent effects on cell morphology. These findings provide new insights into the functional mechanisms by which NSLIT2 binding to ROBO1 affects changes in actin cytoskeletal architecture.

Loss of expression of VIPAS39/VPS16B or the Sec1/Munc18 (SM) family protein VPS33B causes early lethal ARC syndrome in humans. These proteins have been shown to form a complex containing 2 opposed VPS33B molecules known as VPS33B-VPS16B. ARC patient-derived fibroblasts have dispersed Golgi elements and absence of ribbons, an abnormality also observed in VPS16B-knockout HEK293 cells. These observations indicate that VPS33B-VPS16B is involved in Golgi formation/stability. Immunofluorescence microscopy studies showed colocalization of VPS33B-VPS16B with several Golgi-associated proteins, and co-immunoprecipitation studies indicated interactions involving the Golgi tethering factor GRASP55/GORASP2. Interactions were also detected involving VPS33B-VPS16B and VPS51 and VPS53, components of the Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes. Abnormalities were observed in retrograde trafficking of mannose-6-phosphate receptors, and of cathepsin B between Golgi and endosomes in VPS33B-VPS16B deficient cells, along with abnormally large late endosomes/lysosomes. These observations suggest that VPS33B-VPS16B has previously unreported roles in key aspects of Golgi apparatus formation/stability, Golgi-endosome trafficking, and perhaps lysosome dynamics.

Supplementary Figure 1. Comprehensive RNA-Seq and CITE-Seq analysis of venetoclax sensitive versus resistant AML specimens. Supplementary Figure 2. Further Characterization of BCL-2/SERCA3 Relationship and Role of SERCA3 in Primary Human AML Cells. Supplementary Figure 3. BCL-2 Interactors and Characterization of additional MCU Inhibitors in Venetoclax Resistant ROS-low AML cells. Supplementary Figure 4. Effects of Chemotherapeutic agents on AML Function. Supplementary Figure 5. Effects of Mitoxantrone on In Vivo PDX Experiments. Supplementary Figure 6. Effects of Extracellular Calcium Addition on Mitochondrial Calcium Content in Primary Human AML cells.

Supplementary Table 1. List of Bonafide BCL-2 Interactors in HEK293T Cells. Supplementary Table 2. Characteristics of Patient Specimens. Supplementary Table 3. CiteSeq Antibodies Used for Clustering.

Genomic instability is a hallmark of cancer, driving oncogenic mutations that enhance tumor aggressiveness and drug resistance. MYC, a master transcription factor that is deregulated in nearly all human tumors, paradoxically induces replication stress and associated DNA damage while also increasing expression of DNA repair factors and mediating resistance to DNA-damaging therapies. Emerging evidence supports a nontranscriptional role for MYC in preserving genomic integrity at sites of active transcription and protecting stalled replication forks under stress. Understanding how MYC's genotoxic and genoprotective functions diverge may reveal new therapeutic strategies for MYC-driven cancers. Here, we identify a noncanonical role of MYC in DNA damage response (DDR) through its association with DNA breaks. We show that phosphorylation at serine 62 (pS62-MYC) is crucial for the efficient recruitment of MYC to damage sites, its interaction with repair factors BRCA1 and RAD51, and effective DNA repair to support cell survival under stress. Proteomic profiling of the MYC interactome confirms a conserved interaction with components of the DDR pathway. These findings establish pS62-MYC as a key regulator of genomic stability and a potential therapeutic target in cancers.

Patients with acute myeloid leukemia (AML) have a poor 5-year survival rate, highlighting the need for the identification of new approaches to target this disease. AML is highly dependent on glutathione (GSH) metabolism for survival. Although the metabolic role of GSH is well characterized in AML, the contribution of protein glutathionylation, a reversible modification that protects protein thiols from oxidative damage, remains largely unexplored. Therefore, we sought to elucidate the role of protein glutathionylation in AML pathogenesis. Here, we demonstrate that protein glutathionylation is essential for AML cell survival. Specifically, the loss of glutaredoxin 2 (GLRX2), an enzyme that removes GSH modifications, resulted in selective primary AML cell death while sparing normal human hematopoietic stem and progenitor cells. Unbiased proteomic analysis revealed increased mitochondrial protein glutathionylation upon GLRX2 depletion, accompanied by mitochondrial dysfunction, including impaired oxidative phosphorylation, reduced mitochondrial membrane potential, and increased opening of the mitochondrial permeability transition pore (mPTP). Further investigation identified adenosine triphosphate synthase subunit O (ATP5PO), a key regulator of mPTP opening and a component of the ATP synthase complex, as a critical GLRX2 target. Disruption of ATP5PO glutathionylation partially restored mPTP function and rescued AML cell viability after GLRX2 depletion. Moreover, both genetic and pharmacological inhibition of mPTP opening restored the leukemic potential of primary AML specimens in the absence of GLRX2. By disrupting glutathionylation-dependent mitochondrial homeostasis, this study reveals a novel vulnerability in AML that could inform future therapeutic strategies.

To probe drug mechanism of action (MOA) and interrogate the genetic architecture of human cells, we carried out isogenic genome-wide CRISPR/Cas9 knockout screens against 310 diverse drugs, bioactive compounds, and stress conditions. Stringent statistical correction for gene knockout fitness defects yielded a large-scale matrix of >12,000 high confidence chemical-genetic interactions (CGIs). This dataset revealed many previously unappreciated off-target effects for well-characterized compounds and novel MOAs for uncharacterized compounds. The CGI matrix uncovered dense genetic modules that yielded new biological insights into phospholipidosis, mitotic regulation, metabolism, the DNA damage response, and mTOR signaling. The dataset allowed identification of multi-drug sensitization and resistance mechanisms, inference of gene function, elaboration of cross-process connectivity, evaluation of the cell type specificity of CGIs, prediction of chemical synergism, and extensive annotation of understudied genes. This resource provides a map of the genetic landscape in human cells and a framework to help guide drug discovery.

Here we describe ProtacID, a flexible BioID (proximity-dependent biotinylation)-based approach to identify PROTAC-proximal proteins in living cells. ProtacID analysis of VHL- and CRBN-recruiting PROTACs targeting a number of different proteins (localized to chromatin or cellular membranes, and tested across six different human cell lines) demonstrates how this technique can be used to validate PROTAC degradation targets and identify non-productive (i.e. non-degraded) PROTAC-interacting proteins, addressing a critical need in the field of PROTAC development. We also demonstrate that ProtacID can be used to characterize native, endogenous multiprotein complexes without the use of antibodies, or modification of the protein of interest with epitope tags or biotin ligase tagging.

Characterization of the pathogenic variants of SMARCA4 retaining protein expression

Top 31 compounds identified from the FDA drug screen in BIN-67 cells.