Fengting Yan

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.

Combination anti-CD74 (milatuzumab) and anti-CD20 (rituximab) monoclonal antibody therapy has in vitro and in vivo activity in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmunotherapy. Rituximab, a chimeric anti-CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatuzumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malignancies. Because rituximab and milatuzumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochondrial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab-mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-κB pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL.

FTY720 Shows Promising In vitro and In vivo Preclinical Activity by Downmodulating Cyclin D1 and Phospho-Akt in Mantle Cell Lymphoma

Purpose: Despite the progress that has been made in the treatment of mantle cell lymphoma (MCL), all patients invariably relapse with the currently available therapies. Because of the absence of curative therapy for MCL, we explored FTY720 as a novel agent against MCL. Experimental Design: The cytotoxic effect of FTY720 in primary MCL tumor cells and cell lines were evaluated in vitro. The effects of FTY720 on caspase activation, generation of reactive oxygen species, and modulation of Cyclin D1 and Akt, which are implied in the pathogenesis of MCL, were investigated. The in vivo efficacy of FTY720 was evaluated in a Jeko-severe combined immunodeficient xenograft model of human MCL. Results: FTY720 mediated time- and dose-dependent cytotoxicity in primary MCL tumor cells and MCL cell lines in vitro. FTY720-induced cytotoxicity occured independent of caspase activation but dependent on the generation of ROS in MCL. In addition, FTY720 treatment resulted in the time-dependent downmodulation of Cyclin D1 and accumulation of cells in G0-G1 and G2-M phases of the cell cycle with concomitant decrease in S-phase entry. Furthermore, concentrations of FTY720 that induced cytotoxicity led to decreased phospho-Akt in primary MCL cells and cell lines. Most importantly, the in vivo therapeutic activity of FTY720 was shown in severe combined immunodeficient mice engrafted with the Jeko MCL cell line. Conclusions: These results provide the first evidence for a potential use of FTY720 in targeting key pathways that are operable in the pathogenesis of MCL and warrant further investigation of FTY720 in clinical trials to treat patients with MCL. Clin Cancer Res; 16(12); 3182–92. ©2010 AACR.

Strong inverse correlation between microRNA-125b and human papillomavirus DNA in productive infection

Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, −99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis.

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.

PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27Kip1

Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27Kip1 expression in human melanoma cells.

Protein arginine methyltransferase 5 (PRMT5) inhibition induces lymphoma cell death through reactivation of the retinoblastoma tumor suppressor pathway and polycomb repressor complex 2 (PRC2) silencing.

Background: PRMT5, PRC2, and cyclin D1 are overexpressed in non-Hodgkin lymphoma (NHL). Results: PRMT5 expression inversely correlates with levels of hypophospho-RB1 and RBL2. Conclusion: PRMT5 inhibition reactivates RB1 and RBL2 and silences PRC2 and cyclin D1. Significance: PRMT5 inhibition results in NHL growth arrest and cell death. Epigenetic regulation mediated by lysine- and arginine-specific enzymes plays an essential role in tumorigenesis, and enhanced expression of the type II protein arginine methyltransferase PRMT5 as well as the polycomb repressor complex PRC2 has been associated with increased cell proliferation and survival. Here, we show that PRMT5 is overexpressed in three different types of non-Hodgkin lymphoma cell lines and clinical samples as well as in mouse primary lymphoma cells and that it up-regulates PRC2 expression through inactivation of the retinoblastoma proteins RB1 and RBL2. Although PRMT5 epigenetically controls RBL2 expression, it indirectly promotes RB1 phosphorylation through enhanced cyclin D1 expression. Furthermore, we demonstrate that PRMT5 knockdown in non-Hodgkin lymphoma cell lines and mouse primary lymphoma cells leads to RBL2 derepression and RB1 reactivation, which in turn inhibit PRC2 expression and trigger derepression of its CASP10, DAP1, HOXA5, and HRK pro-apoptotic target genes. We also show that reduced PRMT5 expression leads to cyclin D1 transcriptional repression via loss of TP53K372 methylation, which results in decreased BCL3 expression and enhanced recruitment of NF-κB p52-HDAC1 repressor complexes to the cyclin D1 promoter. These findings indicate that PRMT5 is a master epigenetic regulator that governs expression of its own target genes and those regulated by PRC2 and that its inhibition could offer a promising therapeutic strategy for lymphoma patients.

Abstract 933: The expression of PRMT5 methyltransferase mediates cell survival and metastatic phenotype in malignant melanoma

Post-translational modification of proteins is involved at all levels of cellular regulation. The PRMT5 enzyme is a type II arginine methyltransferase that catalyzes the transfer of a methyl group to two of three guanidino nitrogen atoms within the arginine molecule. This enzyme has been shown to methylate histone H3 at arginine 8 (H3R8) and H4R3 to trigger silencing of tumor suppressor genes. In addition, PRMT5 has been shown to interact with p53, TRAIL receptor, and the CDK4 complex to regulate the cell cycle or apoptosis. Although prior studies have provided insight into these mechanistic features of the PRMT enzymes, most data are derived from a limited panel of cell lines. PRMT5 over-expression has recently been shown to influence progression of leukemia and lymphoma. However, few, if any publications exist which document the role of PRMT enzymes in melanoma. We hypothesized that PRMT5 expression promotes metastasis and contributes to reduced immunologic recognition of melanoma cells. Immunoblot analysis and confocal microscopy revealed that PRMT5 is expressed in a panel of melanoma cell lines regardless of the mutational status of B-Raf, NRas, or p53. siRNA-mediated inhibition of PRMT5 led to apoptosis and restored CXCL10 chemokine expression. PRMT5 expression was next determined by immunohistochemical (IHC) analysis of formalin-fixed samples from patients with melanoma (n=56 primary; n=20 metastases) or benign nevi (n=24). The specimens were obtained as a commercially-available tissue microarray, or procured as de-identified primary patient samples (IRB #20100071; P.I. Lesinski; OSU IRB # 2002H0089; co-PI: Peters). PRMT5 expression was significantly increased in the nucleus of melanoma cells as compared to normal epidermis or benign nevi (p=0.001). The level of cytoplasmic PRMT5 expression was also elevated in melanoma lesions, however its distribution in benign tissues was bimodal, and expressed in a subset of benign samples. Notably, nuclear PRMT5 expression increased as melanoma cells invaded the dermis or in melanoma lesions exhibiting pagetoid spread. Finally, we have identified a specific inhibitor of PRMT5 arginine methyltransferase activity from a small molecule library. This lead compound (BLL-1) blocked the dimethylation of arginine 3 on histone H4 (H4R3me2s). A series of titration experiments revealed reduction of both H4R3me2s and proliferation rate. Treatment of multiple melanoma cell lines with BLL-1 (24 hr) led to significantly increased apoptosis. These data represent the first report of PRMT5 and its inhibition in melanoma. Together, this suggests PRMT5 inhibition as a potential therapeutic strategy by virtue of its ability to promote apoptosis and restore immunomodulatory gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 933. doi:10.1158/1538-7445.AM2011-933

Abstract LB-254: PRMT5 is upregulated in malignant and metastatic melanoma, and regulates expression of the MITF transcription factor

Protein arginine methyltransferase 5 (PRMT5) is an enzyme which catalyzes the covalent attachment of methyl groups to arginine residues of various proteins. PRMT5 binds and/or methylates both nuclear (e.g. histones) and cytoplasmic (e.g. p53, CRAF) proteins to regulate cellular functions including gene expression, cell cycle, and apoptosis among others. PRMT5 has been characterized in leukemia, lymphoma, glioma, and breast cancer; however little is known regarding its role in malignant melanoma. We hypothesized that PRMT5 plays a unique role in regulating melanoma cell biology. PRMT5 expression was measured by IHC analysis of formalin-fixed samples from patients with melanoma (n=133 primary; n=66 metastases), benign nevi (n=24), and normal adjacent epidermis (n=21). PRMT5 expression was significantly elevated in melanoma samples compared to normal adjacent epidermis (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-254. doi:1538-7445.AM2012-LB-254

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.