Ian D. Ferguson's research while affiliated with Stanford University and other places

One-Sentence Summary Raf1 is present within the mitochondrial matrix, where it binds GLS to regulate glutamine catabolism and tumorigenesis. In cancer, Raf1 activation occurs via mechanisms that include mutation of upstream regulators, such as receptor tyrosine kinases and Ras GTPases, as well as by mutations that affect RAF1 itself, including via gene amplification ( 1 – 4 ). Once recruited to the plasma membrane ( PM ) Raf1 can engage downstream mitogen-activated protein kinase ( MAPK ) pathway signaling through phosphorylation of the MEK kinases ( 5 ). In addition to Raf1, A-Raf and B-Raf can also activate MEK and these other two Raf isoforms can compensate for MAPK activation in the event of Raf1 loss ( 6 , 7 ). Despite this, Raf1 remains essential for the development and maintenance of some tumors through mechanisms independent of MAPK activity ( 7 , 8 ). In this regard, Raf1 has well-described interactions outside the canonical MAPK pathway, including several with outer mitochondrial membrane ( OMM ) proteins ( 9 , 10 ), although Raf1 has not been previously identified inside mitochondria. Mitochondria comprise a hub for various metabolic processes modulated in cancer cells to accommodate rapid proliferation. One such process is glutaminolysis, which involves the catabolism of glutamine to generate both ATP as well as precursors for the synthesis of fatty acids, nucleotides, and nonessential amino acids ( 11 – 13 ). Glutaminase ( GLS ) proteins, which catalyze the first and rate-limiting step of this process by converting glutamine to glutamate, are often upregulated in cancer ( 14 – 16 ). GLS activation has been previously associated with tumors driven by Ras, upstream regulators of Raf kinases ( 13 , 17 ). Here we identify Raf1 protein inside mitochondria where Raf1 associates with GLS in the mitochondrial matrix to enable glutamine catabolism and tumorigenic growth. Raf kinases play vital roles in normal mitogenic signaling and cancer, however, the identities of functionally important Raf-proximal proteins throughout the cell are not fully known. Raf1 proximity proteomics/BioID in Raf1-dependent cancer cells unexpectedly identified Raf1-adjacent proteins known to reside in the mitochondrial matrix. Inner-mitochondrial localization of Raf1 was confirmed by mitochondrial purification and super-resolution microscopy. Inside mitochondria, Raf1 associated with glutaminase (GLS) in diverse human cancers and enabled glutaminolysis, an important source of biosynthetic precursors in cancer. These impacts required Raf1 kinase activity and were independent of canonical MAP kinase pathway signaling. Kinase-dead mitochondrial matrix-localized Raf1 impaired glutaminolysis and tumorigenesis in vivo. These data indicate that Raf1 localizes inside mitochondria where it interacts with GLS to engage glutamine catabolism and support tumorigenesis.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation. Graphical a

The cell surface proteome (“surfaceome”) serves as the interface between diseased cells and their local microenvironment. In cancer, this compartment is critical not only for defining tumor biology but also serves as a rich source of potential therapeutic targets and diagnostic markers. Recently, we profiled the surfaceome of the blood cancer multiple myeloma, an incurable plasma cell malignancy. While available small molecule agents can drive initial remissions in myeloma, resistance inevitably occurs. Several new classes of immunotherapies targeting myeloma surface antigens, including antibody therapeutics and chimeric antigen receptor (CAR) T-cells, can further prolong survival. However, new approaches are still needed for those who relapse. We thus applied the glycoprotein cell surface capture (CSC) methodology to panel of multiple myeloma cell lines, identifying key surface protein features of malignant plasma cells. We characterized the most abundant surface proteins on plasma cells, nominating CD48 as a high-density antigen favorable for a possible avidity-based strategy to enhance CAR-T efficacy. After chronic resistance to proteasome inhibitors, a first-line therapy, we found significant alterations in the surface profile of myeloma cells, including down-regulation of CD50, CD361/EVI2B, and CD53, while resistance to another first-line therapy, lenalidomide, drove increases in CD33 and CD45/PTPRC. In contrast, short-term treatment with lenalidomide led to upregulation of the surface antigen MUC-1, thereby enhancing efficacy of MUC-1 targeting CAR-T cells. Integrating our proteomics data with available transcriptome datasets, we developed a scoring system to rank potential standalone immunotherapy targets. Novel targets of interest included CCR10, TXNDC11, and LILRB4. We developed proof-of-principle CAR-T cells versus CCR10 using its natural ligand, CCL27, as an antigen recognition domain. Finally, we developed a “miniaturized” version of the CSC methodology and applied it to primary myeloma patient specimens. Overall, our work creates a unique resource for the myeloma community. This study also supports unbiased surface proteomic profiling as a fruitful strategy for identifying new therapeutic targets and markers of drug resistance, that could have utility in improving myeloma patient outcomes. Similar approaches could be readily applied to additional tumor types or even models/tissues derived from other diseases.

DNA–protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide, including CD53, CD10, EVI2B, and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally, we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological, therapeutic, and diagnostic understanding of myeloma. The myeloma cell surface proteome regulates plasma cell biology and delineates therapy targets. Here, the authors profile the myeloma surfaceome at baseline and in drug resistance, finding the potential target CCR10, and include a streamlined approach to primary sample analysis.

Background Targeting surface antigens upregulated on malignant plasma cells is one of the most promising approaches to improve outcomes for multiple myeloma (MM) patients. Surface markers with approved therapeutics include CD38, BCMA, and SLAMF7, with many more under development. However, none of these therapies are known to be curative. Furthermore, the “surfaceome” of cancer cells regulates tumor proliferation, migration, and endogenous immune cell interactions. There remains a need to identify new surface antigens that can serve as new immunotherapeutic targets and/or reveal surface protein biology in MM. Methods We used glycoprotein “Cell Surface Capture” proteomics to define the MM “surfaceome”. Across four cell line models, we quantified 1245 proteins annotated as membrane-spanning in Uniprot; 530 are high-confidence plasma membrane proteins. We integrated our proteomic data with publicly-available mRNA datasets and bioinformatic prediction algorithms to create a ranking system for possible single-antigen immunotherapy targets. Primary patient samples were obtained under an IRB-approved tissue banking protocol. Results Four of the top six targets by our ranking are already being clinically investigated in MM: BCMA, TACI, ITGB7, and SLAMF7. We thus probed other high-scoring proteins found in our proteomic data that, to our knowledge, have not yet been explored as therapeutic targets. To this end, we found the chemokine receptor CCR10 to be robustly expressed on MM cells per the CCLE but with minimal expression on other tumor cell lines. Data from GTEx and the Human Blood Atlas also suggest low mRNA expression on non-hematopoietic tissues and markedly higher mRNA expression on plasmablasts than other hematopoietic cells. By flow cytometry we verified markedly increased CCR10 expression on MM models compared to B-cell cancers. In patient bone marrow aspirates we also confirmed CCR10 expression on patient tumor cells as well as T-regulatory cells. We developed proof-of-concept Chimeric Antigen Receptor (CAR) constructs using CCL27, the native chemokine ligand of CCR10. We found these CAR’s could robustly activate Jurkat T-cells when co-cultured with CCR10+ MM cell lines, suggesting CAR functionality. However, we found that while peripheral blood CD8+ T-cells do not express detectable CCR10 at baseline, T-cell activation during CAR-T production leads to CCR10 upregulation and thus fratricide with functional cyotoxic CAR-T’s. Current efforts involve T-cell engineering to avoid this fratricide and thus develop preclinical therapeutic candidates targeting CCR10. Conclusion Our surface proteomic profiling provides a powerful resource to discover new biology and immunotherapeutic strategies in MM. CCR10 serves as potential immunotherapeutic target in this disease. CCR10 upregulation on plasma cells warrants further investigation into the role of the CCL27-CCR10 axis in MM pathology.

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.