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The post-translational modification (PTM) of proteins by O-linked β- N -acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe N etworking of I nteractors and S ubstrat E s (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.

Cancer cells commonly develop resistance to immunotherapy, commonly by loss of antigen expression. Combinatorial treatments that increase levels of the target antigen on the surface of cancer cells have the potential to restore efficacy to immunotherapy. Here, we use our CRISPR interference and CRISPR activation-based functional genomics platform to systematically identify pathways controlling cell-surface expression of the multiple myeloma immunotherapy antigen BCMA. We discovered that inhibition of HDAC7 and the Sec61 complex increased cell-surface BCMA, including in primary patient cells. Importantly, pharmacological Sec61 inhibition enhanced the anti-myeloma efficacy of a BCMA-targeted antibody-drug conjugate. A CRISPR interference CAR-T coculture screen enabled us to identify both antigen-dependent and -independent mechanisms controlling response of myeloma cells to BCMA-targeted CAR-T cells. Thus, our study demonstrates the potential of CRISPR screens to uncover mechanisms controlling response of cancer cells to immunotherapy and to suggest potential combination therapies. Statement of significance Cancer patients can acquire resistance to immunotherapy, often by reducing expression of the targeted antigen. Using CRISPR screens, we systematically identified mechanisms increasing expression of the immunotherapy target BCMA and efficacy of a BCMA-directed antibody drug conjugate. We also identified antigen-independent mechanisms regulating response of cancer cells to CAR-T cells.

DiGeorge syndrome, characterized by congenital heart disease, craniofacial and immune system abnormalities, is the most common microdeletion syndrome with an incidence of 1 in 4000 live births. Congenital cardiac defects including conotruncal and ventricular septal defects are the leading cause of mortality. Experiments in mouse have identified Tbx1 as the gene responsible for DiGeorge syndrome, and these findings were subsequently corroborated in human patients. However, the underlying pathways of Tbx1 regulating heart, craniofacial and thymus development remain to be fully characterized. My dissertation focuses on using the zebrafish tbx1-/- mutant with craniofacial and immune defects similar to DiGeorge syndrome patients, to study the role and pathways of tbx1 regulating heart and cartilage development. I found that tbx1-/- mutants have defects in heart morphology, looping, function and differentiation. I showed that tbx1 likely affects heart looping and differentiation by regulating cardiomyocyte proliferation and shape. I further demonstrated that tbx1 regulates heart looping and differentiation via wnt11r and its downstream gene alcama. In addition to heart defects alcama morphants present cartilage abnormalities similar to those obtained by knockdown of edn1, a gene previously identified to act downstream of tbx1 in cartilage development. Further investigation revealed that alcama functions downstream of Edn1 signaling in regulating neural crest differentiation and cartilage formation. In addition, I showed that Alcama on endoderm interacts with Nadl1.1 on iv neural crest to mediate Edn1 signaling and regulate cartilage morphogenesis. Hence, I have demonstrated that Tbx1 regulates Alcama via Wnt11r in the heart and via Edn1 in pharyngeal endoderm.

DiGeorge syndrome (DGS) is the most common microdeletion syndrome, and is characterized by congenital cardiac, craniofacial and immune system abnormalities. The cardiac defects in DGS patients include conotruncal and ventricular septal defects. Although the etiology of DGS is critically regulated by TBX1 gene, the molecular pathways underpinning TBX1's role in heart development are not fully understood. In this study, we characterized heart defects and downstream signaling in the zebrafish tbx1^(−/−) mutant, which has craniofacial and immune defects similar to DGS patients. We show that tbx1^(−/−) mutants have defective heart looping, morphology and function. Defective heart looping is accompanied by failure of cardiomyocytes to differentiate normally and failure to change shape from isotropic to anisotropic morphology in the outer curvatures of the heart. This is the first demonstration of tbx1's role in regulating heart looping, cardiomyocyte shape and differentiation, and may explain how Tbx1 regulates conotruncal development in humans. Next we elucidated tbx1's molecular signaling pathway guided by the cardiac phenotype of tbx1^(−/−) mutants. We show for the first time that wnt11r (wnt11 related), a member of the non-canonical Wnt pathway, and its downstream effector gene alcama (activated leukocyte cell adhesion molecule a) regulate heart looping and differentiation similarly to tbx1. Expression of both wnt11r and alcama are downregulated in tbx1^(−/−) mutants. In addition, both wnt11r^(−/−) mutants and alcama morphants have heart looping and differentiation defects similar to tbx1^(−/−) mutants. Strikingly, heart looping and differentiation in tbx1^(−/−) mutants can be partially rescued by ectopic expression of wnt11r or alcama, supporting a model whereby heart looping and differentiation are regulated by tbx1 in a linear pathway through wnt11r and alcama. This is the first study linking tbx1 and non-canonical Wnt signaling and extends our understanding of DGS and heart development.

The post-translational modification (PTM) of proteins by O-linked β- N -acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe N etworking of I nteractors and S ubstrat E s (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states., Competing Interests: DECLARATION OF INTERESTS S.D.G. is founder and CEO/CTO of Proteas Bioanalytics, Inc.