Anagh A. Sahasrabuddhe

Integrated genomic sequencing reveals mutational landscape of T-cell prolymphocytic leukemia.

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK–STAT pathway in Sézary syndrome

Sézary syndrome (SS) is an aggressive leukaemia of mature T cells with poor prognosis and limited options for targeted therapies. The comprehensive genetic alterations underlying the pathogenesis of SS are unknown. Here we integrate whole-genome sequencing (n=6), whole-exome sequencing (n=66) and array comparative genomic hybridization-based copy-number analysis (n=80) of primary SS samples. We identify previously unknown recurrent loss-of-function aberrations targeting members of the chromatin remodelling/histone modification and trithorax families, including ARID1A in which functional loss from nonsense and frameshift mutations and/or targeted deletions is observed in 40.3% of SS genomes. We also identify recurrent gain-of-function mutations targeting PLCG1 (9%) and JAK1, JAK3, STAT3 and STAT5B (JAK/STAT total ∼11%). Functional studies reveal sensitivity of JAK1-mutated primary SS cells to JAK inhibitor treatment. These results highlight the complex genomic landscape of SS and a role for inhibition of JAK/STAT pathways for the treatment of SS.

A novel recurrent NPM1-TYK2 gene fusion in cutaneous CD30-positive lymphoproliferative disorders

The spectrum of cutaneous CD30-positive lymphoproliferative disorders (LPDs) includes lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Chromosomal translocations targeting tyrosine kinases in CD30-positive LPDs have not been described. Using whole-transcriptome sequencing, we identified a chimeric fusion involving NPM1 (5q35) and TYK2 (19p13) that encodes an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. Fluorescence in situ hybridization revealed NPM1-TYK2 fusions in 2 of 47 (4%) primary cases of CD30-positive LPDs and was absent in other mature T-cell neoplasms (n = 151). Functionally, NPM1-TYK2 induced constitutive TYK2, signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5 activation. Conversely, a kinase-defective NPM1-TYK2 mutant abrogated STAT1/3/5 signaling. Finally, short hairpin RNA-mediated silencing of TYK2 abrogated lymphoma cell growth. This is the first report of recurrent translocations involving TYK2, and it highlights the novel therapeutic opportunities in the treatment of CD30-positive LPDs with TYK2 translocations.

Oncogenic Y641 mutations in EZH2 prevent Jak2/β-TrCP-mediated degradation

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase β-TrCP (FBXW1). β-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs β-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of β-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2Y641 mutants recurrently implicated in lymphoma pathogenesis are unable to bind β-TrCP. Further, endogenous EZH2Y641 mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that β-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

Recurrent reciprocal RNA chimera involving YPEL5 and PPP1CB in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in the Western hemisphere. Tumor-specific chromosomal translocations, characteristic findings in several human malignancies that directly lead to malignant transformation, have not been identified in CLL. Using paired-end transcriptome sequencing, we identified recurrent and reciprocal RNA chimeras involving yippee like 5 (YPEL5) and serine/threonine-protein phosphatase PP1-beta-catalytic subunit (PPP1CB) in CLL. Two of seven index cases (28%) harbored the reciprocal RNA chimeras in our initial screening. Using quantitative real-time PCR (q real-time PCR), YPEL5/PPP1CB and PPP1CB/YPEL5 fusion transcripts were detected in 97 of 103 CLL samples (95%) but not in paired normal samples, benign lymphocytes, or various unrelated cancers. Whole-genome sequencing and Southern blotting demonstrated no evidence for a genomic fusion between YPEL5 and PPP1CB. YPEL5/PPP1CB chimera, when introduced into mammalian cells, expressed a truncated PPP1CB protein that demonstrated diminished phosphatase activity. PPP1CB silencing resulted in enhanced proliferation and colony formation of MEC1 and JVM3 cells, implying a role in the pathogenesis of mature B-cell leukemia. These studies uncover a potential role for recurrent RNA chimeras involving phosphatases in the pathogenesis of a common form of leukemia.

Role of the ubiquitin proteasome system in hematologic malignancies

Ubiquitination is a post‐translational modification process that regulates several critical cellular processes. Ubiquitination is orchestrated by the ubiquitin proteasome system (UPS), which constitutes a cascade of enzymes that transfer ubiquitin onto protein substrates. The UPS catalyzes the destruction of many critical protein substrates involved in cancer pathogenesis. This review article focuses on components of the UPS that have been demonstrated to be deregulated by a variety of mechanisms in hematologic malignancies. These include E3 ubiquitin ligases and deubiquitinating enzymes. The prospects of specific targeting of key enzymes in this pathway that are critical to the pathogenesis of particular hematologic neoplasia are also discussed.

Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival

Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.

NPM-ALK phosphorylates WASp Y102 and contributes to oncogenesis of anaplastic large cell lymphoma

Mechanisms by which NPM-ALK signaling regulates cell migration, invasion and contributes to the oncogenesis of anaplastic large cell lymphoma (ALCL) are not completely understood. In an attempt to identify novel actin signaling pathways regulated by NPM-ALK, a comprehensive phosphoproteome analysis of ALCL cell lines was performed in the presence or absence of NPM-ALK activity. Numerous phosphoproteins involved in actin dynamics including Wiskott–Aldrich syndrome protein (WASp) were regulated by NPM-ALK. Network analysis revealed that WASp is a central component of the NPM-ALK-dependent actin signaling pathway. Here we show that NPM-ALK phosphorylates WASp at its known activation site (Y290) as well as at a novel residue (Y102). Phosphorylation of WASp at Y102 negatively regulates its interaction with Wiskott–Aldrich interacting protein and decreases its protein stability. Phosphorylation of WASp at Y102 enhances anchorage-independent growth and tumor growth in an in vivo xenograft model and enhances invasive properties of ALCL. We show that knock-down of WASp or expression of Y102F mutant of WASp decreases colony formation and in vivo tumor growth. Our results show that WASp is a novel substrate of ALK and has a critical role in regulating invasiveness and oncogenesis of ALCL.

Fbxo45 inhibits calcium-sensitive proteolysis of N-cadherin and promotes neuronal differentiation.

Background: Fbxo45 is an atypical E3 ligase that plays an important role in neuronal development. Results: Fbxo45 binds to N-cadherin intracellularly and prevents its degradation. Conclusion: By protecting N-cadherin from proteolysis, Fbxo45 plays a key role in promoting N-cadherin-mediated neuronal differentiation. Significance: This study reveals a novel mechanism of Fbxo45-mediated neuronal differentiation. Fbxo45 is an atypical E3 ubiquitin ligase, which specifically targets proteins for ubiquitin-mediated degradation. Fbxo45 ablation results in defective neuronal differentiation and abnormal formation of neural connections; however, the mechanisms underlying these defects are poorly understood. Using an unbiased mass spectrometry-based proteomic screen, we show here that N-cadherin is a novel interactor of Fbxo45. N-cadherin specifically interacts with Fbxo45 through two consensus motifs overlapping the site of calcium-binding and dimerization of the cadherin molecule. N-cadherin interaction with Fbxo45 is significantly abrogated by calcium treatment. Surprisingly, Fbxo45 depletion by RNAi-mediated silencing results in enhanced proteolysis of N-cadherin. Conversely, ectopic expression of Fbxo45 results in decreased proteolysis of N-cadherin. Fbxo45 depletion results in dramatic reduction in N-cadherin expression, impaired neuronal differentiation, and diminished formation of neuronal processes. Our studies reveal an unanticipated role for an F-box protein that inhibits proteolysis in the regulation of a critical biological process.

The role of aberrant proteolysis in lymphomagenesis.

Purpose of reviewDeregulated proteolysis is increasingly being implicated in pathogenesis of lymphoma. In this review, we highlight the major cellular processes that are affected by deregulated proteolysis of critical substrates that promote lymphoproliferative disorders. Recent findingsEmerging evidence supports the role of aberrant proteolysis by the ubiquitin proteasome system (UPS) in lymphoproliferative disorders. Several UPS mediators are identified to be altered in lymphomagenesis. However, the precise role of their alteration and comprehensive knowledge of their target substrate critical for lymphomagenesis is far from complete. SummaryMany E3 ligase and deubiquitinases that contribute to regulated proteolysis of substrates critical for major cellular processes are altered in various lineages of lymphoma. Understanding of the proteolytic regulatory mechanisms of these major cellular pathways may offer novel biomarkers and targets for lymphoma therapy.