John T. Patton

Genetic Validation of the Protein Arginine Methyltransferase PRMT5 as a Candidate Therapeutic Target in Glioblastoma

Glioblastoma is the most common and aggressive histologic subtype of brain cancer with poor outcomes and limited treatment options. Here, we report the selective overexpression of the protein arginine methyltransferase PRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatory genes by catalyzing symmetric dimethylation of arginine residues on histone tails. PRMT5 overexpression in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival. Genetic attenuation of PRMT5 led to cell-cycle arrest, apoptosis, and loss of cell migratory activity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemotherapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitation identified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associated with reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led to restored expression of ST7 and cell growth inhibition. Finally, PRMT5 attenuation enhanced glioblastoma cell survival in a mouse xenograft model of aggressive glioblastoma. Together, our findings defined PRMT5 as a candidate prognostic factor and therapeutic target in glioblastoma, offering a preclinical justification for targeting PRMT5-driven oncogenic pathways in this deadly disease.

Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV(+) lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas.

The Epstein-Barr virus lytic protein BZLF1 as a candidate target antigen for vaccine development

Hartlage, Liu, and colleagues show that vaccination of hu-PBL-SCID mice with EBV protein BZLF1-pulsed dendritic cells induced specific cellular immunity and prolonged survival from fatal EBV-driven lymphoproliferative disease, identifying BZLF1 as a prophylactic vaccine candidate for EBV-associated diseases. The Epstein–Barr virus (EBV) is an oncogenic, γ-herpesvirus associated with a broad spectrum of disease. Although most immune-competent individuals can effectivley develop efficient adaptive immune responses to EBV, immunocompromised individuals are at serious risk for developing life-threatening diseases, such as Hodgkin lymphoma and posttransplant lymphoproliferative disorder (PTLD). Given the significant morbidity associated with EBV infection in high-risk populations, there is a need to develop vaccine strategies that restore or enhance EBV-specific immune responses. Here, we identify the EBV immediate-early protein BZLF1 as a potential target antigen for vaccine development. Primary tumors from patients with PTLD and a chimeric human-murine model of EBV-driven lymphoproliferative disorder (EBV-LPD) express BZLF1 protein. Pulsing human dendritic cells (DC) with recombinant BZLF1 followed by incubation with autologous mononuclear cells led to expansion of BZLF1-specific CD8+ T cells in vitro and primed BZLF1-specific T-cell responses in vivo. In addition, vaccination of hu-PBL-SCID mice with BZLF1-transduced DCs induced specific cellular immunity and significantly prolonged survival from fatal EBV-LPD. These findings identify BZLF1 as a candidate target protein in the immunosurveillance of EBV and provide a rationale for considering BZLF1 in vaccine strategies to enhance primary and recall immune responses and potentially prevent EBV-associated diseases. Cancer Immunol Res; 3(7); 787–94. ©2015 AACR.

Complete and Durable Responses in Primary Central Nervous System Posttransplant Lymphoproliferative Disorder with Zidovudine, Ganciclovir, Rituximab, and Dexamethasone

Purpose: Primary central nervous system posttransplant lymphoproliferative disorder (PCNS-PTLD) is a complication of solid organ transplantation with a poor prognosis and typically associated with Epstein–Barr virus (EBV). We hypothesized EBV lytic-phase protein expression would allow successful treatment with antiviral therapy. Patients and Methods: Thirteen patients were treated with zidovudine (AZT), ganciclovir (GCV), dexamethasone, and rituximab in EBV+ PCNS-PTLD. Twice-daily, intravenous AZT 1,500 mg, GCV 5 mg/kg, and dexamethasone 10 mg were given for 14 days. Weekly rituximab 375 mg/m2 was delivered for the first 4 weeks. Twice-daily valganciclovir 450 mg and AZT 300 mg started day 15. Lytic and latent protein expression was assessed using in situ hybridization and immunohistochemistry. Immunoblot assay assessed lytic gene activation. Cells transfected with lytic kinase vectors were assessed for sensitivity to our therapy using MTS tetrazolium and flow cytometry. Results: The median time to response was 2 months. Median therapy duration was 26.5 months. Median follow-up was 52 months. The estimated 2-year overall survival (OS) was 76.9% (95% CI, 44.2%–91.9%). Overall response rate (ORR) was 92% (95% CI, 64%–100%). BXLF1/vTK and BGLF4 expression was found in the seven tumor biopsies evaluated. Lytic gene expression was induced in vitro using the four-drug regimen. Transfection with viral kinase cDNA increased cellular sensitivity to antiviral therapy. Conclusions: EBV+ PCNS-PTLD expressed lytic kinases and therapy with AZT, GCV, rituximab and dexamethasone provided durable responses. Induction of the lytic protein expression and increased cellular sensitivity to antiviral therapy after transfection with viral kinase cDNA provides a mechanistic rationale for our approach. Clin Cancer Res; 24(14); 3273–81. ©2018 AACR.

Abstract LB-218: Protein arginine methyltransferase inhibition of malignant gliomas leads to restored chemokine expression and enhanced immune effector function

Patients with Glioblastoma Multiforme (GBM) face a poor prognosis despite multimodal therapy, thus, there is an unmet need for discovery of novel therapeutic targets and approaches. The immune-privileged nature of the central nervous system and down-modulation of cytokines and chemokines in GBM tumors led us to explore epigenetic approaches to restore expression of immune-relevant genes. PRMT5 is a type II arginine methyltransferase that catalyzes symmetric dimethylation of arginine residues on histone proteins leading to transcriptional repression. Our previous work showed PRMT5 overexpression correlates with poor clinical outcome of GBM patients and that PRMT5 silencing inhibited tumor growth in vitro and in vivo. Microarray transcriptional studies following PRMT5 depletion identified potential PRMT5 target genes interferon-inducible protein 10 (CXCL10) and interferon-inducible T cell α chemoattractant (CXCL11). Increased secretion of CXCL10 and CXCL11 has been shown to facilitate homing of innate and adaptive immune-effector populations that promote anti-GBM activity in vivo. Thus we further investigated the significance of restored CXCL10 and CXLC11 with PRMT5 inhibition in GBM. CXCL10 protein expression was found to be significantly lower in GBM tumors (N = 15) by immunochemistry staining, compared with grade I gliomas (N = 7) (p = 0.0001). PRMT5 knockdown led to increased mRNA and protein expression of CXCL10 and CXCL11. Chromatin immunoprecipitation (ChIP) experiments identified CXCL10 and CXCL11 promoters to be directly targeted by PRMT5 repressive complexes. Previous work has shown that PRMT5 associates with other co-repressor molecules including HDAC2, DNA methyltransferase 3a (DNMT3a) and methyl binding domain protein 2 (MBD2). ChIP studies confirmed that PRMT5, HDAC2, DNMT3a and MBD2 proteins were all recruited to the promoters of CXCL10 and CXCL11. Interestingly, when two patient-derived GBM cell lines were transfected with siRNA targeting PRMT5, recruitment of all co-repressor proteins, PRMT5, HDAC2, DNMT3a, and MBD2 was lost on CXCL10 and CXCL11 promoters. These findings suggest that PRMT5 is a master transcriptional repressor that plays a central role in coordinating and assembling co-repressor chromatin remodeling complexes on PRMT5 target gene promoters. PRMT5 silencing resulted in secretion of biologically relevant levels of CXCL10 and CXCL11 in culture medium that promoted immune cell recruitment in transwell migration assays toward target glioma cells. Our data suggests that in addition to restoring regulatory and tumor suppressor gene expression that promotes direct GBM cell death, PRMT5 silencing may enhance immune-mediated anti-tumor activity. These findings further justify the development experimental therapeutic strategies targeting PRMT5 to modulate activity of immune-relevant genes. Citation Format: Fengting Yan, Yeshavanth Banasavadi-Siddegowda, John T. Patton, Mark Lustberg, Xin Wu, Balveen Kaur, Rober A. Baiocchi. Protein arginine methyltransferase inhibition of malignant gliomas leads to restored chemokine expression and enhanced immune effector function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-218. doi:10.1158/1538-7445.AM2015-LB-218