Abstract Glioblastoma Multiforme (GBM) is associated with a dismal prognosis despite intensive multimodal therapy, highlighting the need for novel therapeutic approaches. Chromatin remodeling complexes and associated co-repressors such as histone deacetylases (HDAC), DNA methyltransferases (DNMT) and protein arginine methyltransferase 5 (PRMT5) are involved in silencing tumor suppressor and regulatory gene expression and may contribute to glial cell transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8) and works more efficiently when associated with other co-repressor enzymes. We have previously identified PRMT5 over-expression in GBM cell lines, primary GBM tumors, and GBM that spontaneously develop in a pre-clinical mouse model. The degree of PRMT5 over-expression inversely correlated with survival of GBM patients (r=-0.57, p=0.0001) and correlated with proliferation of GBM cell lines (r=0.81, p<0.0001), PRMT5 works concertedly with HDAC2, methyl-CpG binding domain protein 2 (MBD2) and DNMT3a to silence genes with anti-cancer and immune modulatory activities. PRMT5 silending in GBM cell lines leads to growth arrest, apoptosis and loss of HDAC2, DNMT3a and PRMT5 co-repressor complex recruitment at chemokine gene promoters. These findings provide evidence that PRMT5 is a central repressive element required for maintenance of target gene silencing. Thus, we explored methods to inhibit PRMT5 activity as a novel experimental therapeutic strategy for GBM. A rational design of small molecule compounds to inhibit PRMT5 activity led us to construct an in silico model of the human PRMT5 catalytic domain based on available homologous crystal structures. We screened a library of 10,000 compounds and eight small molecules were identified based on binding energy in the PRMT5 catalytic site. Enzyme inhibition assays showed that a lead compound (BLL1) was capable of selectively inhibiting PRMT5 and not PRMT1 or PRMT7 activity (p<0.0001). BLL1 interfered with maintenance of S2Me-H4R3 and S2Me-H3R8 in GBM cell lines by western blot and confocal microscopy. Dose titration experiments with BLL1 showed a dose-dependent response of inhibition of cellular proliferation in GBM cell lines. Combination treatment of GBM cells with subtoxic doses of BLL1, hypomethylating agent 5-azacitidine, and HDAC inhibitor TSA showed synergistic induction of cell death, loss of the epigenetic mark S2Me-H4R3, and de-repression of the immune modulating chemokine CXCL10. Preclinical in vivo studies have shown favorable toxicity and pharmacokinetic profiles for BLL1. We have successfully developed a first in class drug to selectively target dysregulated PRMT5 enzymatic activity in GBM. We are currently developing drugs with improved selectivity and potency. Citation Format: Fengting Yan, Kate Gordon, Kiran Mahasenan, Mark Lustberg, Lapo Alinari, Christian T. Earl, Balveen Kaur, Chenglong Li, Robert A. Baiocchi. Developing a first in class of drug to inhibit protein arginine methyltransferase 5 (PRMT5) enzyme dysregulation in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4637. doi:10.1158/1538-7445.AM2013-4637
Only a minority of patients with high risk lymphoma will be cured with autologous transplant, so maintenance with vorinostat, an oral agent with activity in relapsed lymphoma, was studied starting day + 60 for 21 consecutive days followed by a week off for up to 11 cycles. Twenty-three patients with lymphoma were treated. Ten patients completed the full 11-cycle treatment plan per protocol, four patients were removed due to progressive disease and seven withdrew or were removed from the study due to toxicities. Despite Prevnar vaccine administration every 2 months for three injections, the mean antibody concentration never reached protective levels (> 0.35 μg/mL). Fatigue and functional well-being measured by Brief Fatigue Inventory and Functional Assessment of Cancer Therapy-General improved significantly from cycle 1 to cycle 7, but depression scores from the Center for Epidemiologic Studies Depression scale did not change. Given the toxicities observed, this broad-spectrum deacetylase inhibitor at this schedule is not optimal for prolonged maintenance therapy.
Abstract PURPOSE: TNBC is an aggressive disease with limited effective targeted therapies. Protein arginine methyltransferase 5 (PRMT5) is an enzyme that symmetrically methylates arginine residues of both histone and non-histone proteins that regulate cellular growth and drive malignant transformation. PRMT5 is an oncogenic marker for aggressive cancers with limited treatment options like glioblastoma multiforme (GBM) and mantle cell lymphoma (MCL) and PRMT5 knockdown has been shown to improve survival in a preclinical model of GBM. The expression profile of PRMT5 in TNBC has not been previously evaluated. METHODS: We obtained a clinical database of TNBC patients that correlates to tissue samples collected after resection of the original breast tumor (n = 106). Tissue microarrays (TMA) were stained for PRMT5 and graded based on level of cytoplasmic and nuclear staining. The association of PRMT5 expression with patient overall survival (OS) and recurrent free survival (RFS) was analyzed using a log-rank test. Multivariate COX regression model was used to evaluate PRMT expression level as independent predictor of OS and RFS with histopathologic factors such as lymph node involvement and tumor size. RESULTS: High cytoplasmic levels of PRMT5 in TNBC correlated with improvement in RFS (p = 0.043, CI 0.218-0.976), while showing no correlation with OS. High levels of nuclear PRTM5 did not show a significant correlation with OS or RFS. Patients with tumors showing high nuclear and low cytoplasmic levels of PRMT5 had the worst OS and RFS (p<0.05). When accounting for stage, lymph node status, and nuclear or cytoplasmic PRMT5 levels, higher stages predicted worse RFS with stage 3 being the worst (p<0.01). Having high PRMT5 in the cytoplasm alone was associated with an improved RFS trend (p = 0.08), but TNBC with low cytoplasmic PRMT5 and high nuclear PRMT5 levels was significant for predicting worse RFS (p = 0.005). Most TNBC with high cytoplasmic levels of PRMT5 were Stage 2 (65%) and most TNBC with low nuclear levels of PRMT5 were also Stage 2 (64%). In stage 3 cancers, 55% had low cytoplasmic levels of PRMT5 while 44% had high nuclear levels of PRMT5. The levels of PRMT5 in the cytoplasm or nucleus were no different depending on the presence or absence of positive lymph nodes at the time of diagnosis. CONCLUSIONS: PRMT5 is variably expressed in TNBC and the cellular location of PRMT5 correlates with RFS and OS. Having association between tumors with low cytoplasmic and high nuclear levels with significantly worse RFS implies that high cytoplasmic PRMT5 may have a protective effect. These results prompt further investigation into the biologic role of PRMT5 in TNBC and whether it can serve as a potential therapeutic target. Citation Format: Elaine P. Alexander, Christian T. Earl, Xiaokui Mo, Konstantin Shilo, Robert A. Baiocchi, Maryam B. Lustberg. Cellular localization of PRMT5 correlates with poor recurrent free survival in triple-negative breast cancer (TNBC). [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 4786. doi:10.1158/1538-7445.AM2015-4786
