Functional glycoproteomics by integrated network assembly and partitioning.

The post-translational modification (PTM) of proteins by O-linked ß-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 Networking of Interactors and SubstratEs (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.

The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance.

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.

CRISPR-based screens uncover determinants of immunotherapy response in multiple myeloma.

Cancer cells commonly develop resistance to immunotherapy 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 B-cell maturation antigen (BCMA). We discovered that pharmacologic inhibition of HDAC7 and the Sec61 complex increased cell surface BCMA, including in primary patient cells. Pharmacologic Sec61 inhibition enhanced the antimyeloma efficacy of a BCMA-targeted antibody-drug conjugate. A CRISPR interference chimeric antigen receptor T cells (CAR-T cells) coculture screen enabled us to identify both antigen-dependent and antigen-independent mechanisms controlling response of myeloma cells to BCMA-targeted CAR-T cells. Thus, our study shows the potential of CRISPR screens to uncover mechanisms controlling response of cancer cells to immunotherapy and to suggest potential combination therapies.

Proteasome inhibitor-induced modulation reveals the spliceosome as a specific therapeutic vulnerability in multiple myeloma.

Enhancing the efficacy of proteasome inhibitors (PI) is a central goal in myeloma therapy. We proposed that signaling-level responses after PI may reveal new mechanisms of action that can be therapeutically exploited. Unbiased phosphoproteomics after treatment with the PI carfilzomib surprisingly demonstrates the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation is invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrates broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings lead us to evaluate direct spliceosome inhibition in myeloma, which synergizes with carfilzomib and shows potent anti-tumor activity. Functional genomics and exome sequencing further support the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.

Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies.

T-cell-recruiting bispecific antibodies (T-BsAbs) have shown potent tumor killing activity in humans, but cytokine release-related toxicities have affected their clinical utility. The use of novel anti-CD3 binding domains with more favorable properties could aid in the creation of T-BsAbs with improved therapeutic windows. Using a sequence-based discovery platform, we identified new anti-CD3 antibodies from humanized rats that bind to multiple epitopes and elicit varying levels of T-cell activation. In T-BsAb format, 12 different anti-CD3 arms induce equivalent levels of tumor cell lysis by primary T-cells, but potency varies by a thousand-fold. Our lead CD3-targeting arm stimulates very low levels of cytokine release, but drives robust tumor antigen-specific killing in vitro and in a mouse xenograft model. This new CD3-targeting antibody underpins a next-generation T-BsAb platform in which potent cytotoxicity is uncoupled from high levels of cytokine release, which may lead to a wider therapeutic window in the clinic.

Seeking Convergence and Cure with New Myeloma Therapies.

For over a decade, the mainstay of multiple myeloma therapy has been small molecules that directly attack malignant plasma cell biology. However, potent immunotherapies have recently emerged, transforming the myeloma therapeutic landscape. Here we first review new promising strategies to target plasma cells through protein homeostasis and epigenetic modulators. We then discuss emerging immunotherapy strategies that are leading to dramatic results in patients. Finally, we focus on recent preclinical data suggesting that enforcing cell-surface antigen expression through small molecules may enhance immunotherapy efficacy and avoid resistance. We argue that these emerging observations point the way toward potential convergence between drug classes. With recent rapid progress we may finally be on the verge of the 'C' word: a cure for myeloma.

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection.

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays.

DiGeorge syndrome gene tbx1 functions through wnt11r to regulate heart looping and differentiation.

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.

Zebrafish screen identifies novel compound with selective toxicity against leukemia.

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells.

Alcama mediates Edn1 signaling during zebrafish cartilage morphogenesis.

The zebrafish pharyngeal cartilage is derived from the pharyngeal apparatus, a vertebrate-specific structure derived from all three germ layers. Developmental aberrations of the pharyngeal apparatus lead to birth defects such as Treacher-Collins and DiGeorge syndromes. While interactions between endoderm and neural crest (NC) are known to be important for cartilage formation, the full complement of molecular players involved and their roles remain to be elucidated. Activated leukocyte cell adhesion molecule a (alcama), a member of the immunoglobulin (Ig) superfamily, is among the prominent markers of pharyngeal pouch endoderm, but to date no role has been assigned to this adhesion molecule in the development of the pharyngeal apparatus. Here we show that alcama plays a crucial, non-autonomous role in pharyngeal endoderm during zebrafish cartilage morphogenesis. alcama knockdown leads to defects in NC differentiation, without affecting NC specification or migration. These defects are reminiscent of the phenotypes observed when Endothelin 1 (Edn1) signaling, a key regulator of cartilage development is disrupted. Using gene expression analysis and rescue experiments we show that Alcama functions downstream of Edn1 signaling to regulate NC differentiation and cartilage morphogenesis. In addition, we also identify a role for neural adhesion molecule 1.1 (nadl1.1), a known interacting partner of Alcama expressed in neural crest, in NC differentiation. Our data shows that nadl1.1 is required for alcama rescue of NC differentiation in edn1(-/-) mutants and that Alcama interacts with Nadl1.1 during chondrogenesis. Collectively our results support a model by which Alcama on the endoderm interacts with Nadl1.1 on NC to mediate Edn1 signaling and NC differentiation during chondrogenesis.

Recurrent abortions in Asian Indians: no role of factor V Leiden Hong Kong/Cambridge mutation and MTHFR polymorphism.

Recurrent fetal loss is a frequent health problem. Data accumulated over the past few years have suggested a possible correlation between thrombophilia and fetal loss. Although a clear association has been established between fetal loss and certain thrombophilic states, such as antiphospholipid antibody syndromes, antithrombin deficiency, and combined defects, reports on the prevalence of inherited prothrombotic defects such as factor V Leiden mutation and methylene tetrahydrofolate reductase C677T polymorphism in fetal loss are contradictory. The prevalence of these 2 mutations in Asian Indians with recurrent fetal loss has not yet been studied. In light of this, the present study looked at the prevalence of these mutations in 85 patients with spontaneous recurrent abortion and 31 controls. The authors did not find any significant role of these mutations in the development of recurrent abortion.

Mapping the interaction site of Rpb4 and Rpb7 subunits of RNA polymerase II in Saccharomyces cerevisiae.

Rpb4 and Rpb7, the fourth and the seventh largest subunits of RNA polymerase II, form a heterodimer in Saccharomyces cerevisiae. To identify the site of interaction between these subunits, we constructed truncation mutants of both these proteins and carried out yeast two hybrid analysis. Deletions in the amino and carboxyl terminal domains of Rpb7 abolished its interaction with Rpb4. In comparison, deletion of up to 49 N-terminal amino acids of Rpb4 reduced its interaction with Rpb7. Complete abolishment of interaction between Rpb4 and Rpb7 occurred by truncation of 1-106, 1-142, 108-221, 172-221 or 198-221 amino acids of Rpb4. Use of the yeast two-hybrid analysis in conjunction with computational analysis of the recently reported crystal structure of Rpb4/Rpb7 sub-complex allowed us to identify regions previously not suspected to be involved in the functional interaction of these proteins. Taken together, our results have identified the regions that are involved in interaction between the Rpb4 and Rpb7 subunits of S. cerevisiae RNA polymerase II in vivo.