Kevin P. Conlon

Activity and Cellular Functions of the Deubiquitinating Enzyme and Polyglutamine Disease Protein Ataxin-3 Are Regulated by Ubiquitination at Lysine 117

Deubiquitinating enzymes (DUbs) play important roles in many ubiquitin-dependent pathways, yet how DUbs themselves are regulated is not well understood. Here, we provide insight into the mechanism by which ubiquitination directly enhances the activity of ataxin-3, a DUb implicated in protein quality control and the disease protein in the polyglutamine neurodegenerative disorder, Spinocerebellar Ataxia Type 3. We identify Lys-117, which resides near the catalytic triad, as the primary site of ubiquitination in wild type and pathogenic ataxin-3. Further studies indicate that ubiquitin-dependent activation of ataxin-3 at Lys-117 is important for its ability to reduce high molecular weight ubiquitinated species in cells. Ubiquitination at Lys-117 also facilitates the ability of ataxin-3 to induce aggresome formation in cells. Finally, structure-function studies support a model of activation whereby ubiquitination at Lys-117 enhances ataxin-3 activity independent of the known ubiquitin-binding sites in ataxin-3, most likely through a direct conformational change in or near the catalytic domain.

Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK–mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma

The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

NPM-ALK signals through glycogen synthase kinase 3β to promote oncogenesis

Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70–80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3β. Expression of NPM-ALK in 293T cells led to an increase of pS9-GSK3β (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3β levels. Phosphorylation of pS9-GSK3β by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3β substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3β. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3β inhibition. Furthermore, stable knockdown of GSK3β conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS9-GSK3β and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS9-GSK3β and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S9-GSK3β by PI3K/AKT. The subsequent inhibition of GSK3β activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3β as a mediator of NPM-ALK oncogenesis.

Fusion Peptides from Oncogenic Chimeric Proteins as Putative Specific Biomarkers of Cancer

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

Global Phosphoproteomic Profiling Reveals Distinct Signatures in B-Cell Non-Hodgkin Lymphomas

Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of hematological malignancies; however, the extent to which deregulated phosphorylation may be involved in B-cell non-Hodgkin lymphoma (B-NHL) pathogenesis is largely unknown. To identify phosphorylation events important in B-NHLs, we performed mass spectrometry-based, label-free, semiquantitative phosphoproteomic profiling of 11 cell lines derived from three B-NHL categories: Burkitt lymphoma, follicular lymphoma, and mantle-cell lymphoma. In all, 6579 unique phosphopeptides, corresponding to 1701 unique phosphorylated proteins, were identified and quantified. The data are available via ProteomeXchange with identifier PXD000658. Hierarchical clustering highlighted distinct phosphoproteomic signatures associated with each lymphoma subtype. Interestingly, germinal center-derived B-NHL cell lines were characterized by phosphorylation of proteins involved in the B-cell receptor signaling. Of these proteins, phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) was identified with the most phosphorylated tyrosine peptides in Burkitt lymphoma and follicular lymphoma. PAG1 knockdown resulted in perturbation of the tyrosine phosphosignature of B-cell receptor signaling components. Significantly, PAG1 knockdown increased cell proliferation and response to antigen stimulation of these germinal center-derived B-NHLs. These data provide a detailed annotation of phosphorylated proteins in human lymphoid cancer. Overall, our study revealed the utility of unbiased phosphoproteome interrogation in characterizing signaling networks that may provide insights into pathogenesis mechanisms in B-cell lymphomas.

Functional proteogenomics reveals biomarkers and therapeutic targets in lymphomas

Significance An important goal in precision oncology is the identification of biomarkers and therapeutic targets. We identified and annotated a compendium of N-glycoproteins from diverse human lymphoid neoplasia, an attractive class of proteins with potential to serve as cancer biomarkers and therapeutic targets. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an underappreciated and targetable ALK-regulated cytokine/receptor signaling network highlighting the utility of functional proteogenomics for discovery of cancer biomarkers and therapeutic targets. Identification of biomarkers and therapeutic targets is a critical goal of precision medicine. N-glycoproteins are a particularly attractive class of proteins that constitute potential cancer biomarkers and therapeutic targets for small molecules, antibodies, and cellular therapies. Using mass spectrometry (MS), we generated a compendium of 1,091 N-glycoproteins (from 40 human primary lymphomas and cell lines). Hierarchical clustering revealed distinct subtype signatures that included several subtype-specific biomarkers. Orthogonal immunological studies in 671 primary lymphoma tissue biopsies and 32 lymphoma-derived cell lines corroborated MS data. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an ALK-regulated cytokine/receptor signaling network, including vulnerabilities corroborated by a genome-wide clustered regularly interspaced short palindromic screen. Functional targeting of IL-31 receptor β, an ALCL-enriched and ALK-regulated N-glycoprotein in this network, abrogated ALK+ALCL growth in vitro and in vivo. Our results highlight the utility of functional proteogenomic approaches for discovery of cancer biomarkers and therapeutic targets.

RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1

Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ. In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.

Differential proteomic analysis of endemic and sporadic Epstein-Barr virus-positive and negative Burkitt lymphoma

BACKGROUND Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children worldwide and the most common paediatric malignancy in sub-Saharan Africa. The endemic (eBL) and sporadic (sBL) variants have distinct epidemiologic and virologic characteristics. Although gene expression studies have defined the transcriptional profiles of both, their proteomic signatures have not been studied. METHODS We compared the proteomic expression profiles using differential mass spectrometry-based isotope tag for relative and absolute quantitation (iTRAQ) analysis of a cell line representing Epstein-Barr virus (EBV)+ eBL, EBV+ and EBV- sBL, and EBV+/- normal B cells from healthy donors. RESULTS In total, there were 144 differentially expressed proteins with a statistically significant false discovery rate (FDR) of ⩽0.2. Results revealed over-expression of specific proteins with well-established links to lymphomagenesis such as TUBB2C (FDR 0.05), UCHL1 (FDR 0.05) and HSP90AB1 (FDR 0.1). Distinct characteristics based upon the epidemiologic and virologic subtypes of BL were also identified. In sBL, PCNA (FDR 0.05) and SLC3A2 (FDR 0.1) were significantly over-expressed. In eBL, C1QBP (FDR 0.1) and ENO1 (FDR 0.25) were significantly over-expressed. Comparison of EBV+ to EBV- BL cell lines and B cells revealed significant over-expression of DDX3X (FDR 0.1). Proteins were validated using Western blot analysis. CONCLUSION Our results suggest unique signal transduction pathways associated with EBV infection and epidemiological subtype of BL that may contribute to lymphomagenesis. These proteomic findings provide potential diagnostic, prognostic and therapeutic links to BL.

SUMOylation of ROR-γt inhibits IL-17 expression and inflammation via HDAC2

Dysregulated ROR-γt-mediated IL-17 transcription is central to the pathogenesis of several inflammatory disorders, yet the molecular mechanisms that govern the transcription factor activity of ROR-γt in the regulation of IL-17 are not fully defined. Here we show that SUMO-conjugating enzyme Ubc9 interacts with a conserved GKAE motif in ROR-γt to induce SUMOylation of ROR-γt and suppress IL-17 expression. Th17 cells expressing SUMOylation-defective ROR-γt are highly colitogenic upon transfer to Rag1–/– mice. Mechanistically, SUMOylation of ROR-γt facilitates the binding of HDAC2 to the IL-17 promoter and represses IL-17 transcription. Mice with conditional deletion of HDAC2 in CD4+ T cells have elevated IL-17 expression and severe colitis. The identification of the Ubc9/ROR-γt/HDAC2 axis that governs IL-17 expression may open new venues for the development of therapeutic measures for inflammatory disorders.Interleukin-17 (IL-17)-secreting CD4 T cells (Th17) are induced by the master transcription factor RORγt, and are important for anti-fungal immunity and inflammatory responses. Here the authors show that Ubc9-mediated SUMOylation of RORγt induces HDAC2 binding to IL-17 promoter for suppressing IL-17 production in Th17 cells.

A comparative global phosphoproteomics analysis of obinutuzumab (GA101) versus rituximab (RTX) against RTX sensitive and resistant Burkitt lymphoma (BL) demonstrates differential phosphorylation of signaling pathway proteins after treatment

We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.