Fiona Brown 1 , JiHyun Chung PhD - PowerPoint Presentation

1. Fiona Brown1, JiHyun Chung PhD1, Shelby Sloan1, Kris Vaddi PhD2, Rosa Lapalombella PhD1, Jihye Paik PhD3, and Robert A. Baiocchi MD PhD1 et al1 Division of Hematology, Department of Internal Medicine, Ohio State University Wexner Medical Center, Columbus, OH, USA 2 Prelude Therapeutics, Wilmington, DE, USA3 Weill Cornell Medicine, New York, NY, USAPRMT5 Inhibition Restarts a Pro-Apoptotic Program and Creates Vulnerability to Combination Treatment with BCL-2 Inhibitor Venetoclax in Mantle Cell LymphomaBackgroundMantle Cell LymphomaMantle Cell Lymphoma (MCL) is a CD19+/CD5+ B-cell malignancy.Arises in the mantle zone of the lymph nodes and can progress to extranodal disease.A major driver is the over expression of Cyclin D1. This is caused by the gene being translocated to the IgH promoter [t(11;14)(q13;q32)].There are approximately 5000 new cases in the US each year.The average patient is ~70 years of age and male.Frontline treatments include chemotherapy with secondary targeted therapy such as ibrutinib.Average survival is 5-8 years but for those who progress on targeted therapies, survival is 3-8 months.Currently considered incurable.PRMT5Protein Arginine Methyltransferase 5 (PRMT5) is an enzyme that mono- and symmetrically dimethylates proteins.Target proteins include many nuclear and cytosolic proteins such as p53, spliceosomal proteins, p65, and histones.Symmetric demethylation of arginine (SDMA) on histones is a repressive mark.Supports the survival and growth of many cancers including MCL. Prelude Therapeutics developed a novel PRMT5 inhibitor, PRT382, which is a tool compound for the PRMT5 inhibitor currently in phase 1 trials (PRT543).VenetoclaxTargeted therapy also known as ABT199.Venetoclax selectively inhibits BCL2 reducing the off target effects seen with other BH3 mimetics.Currently in trials for treatment of MCL and other Non-Hodgkin Lymphomas.SUMMARYConclusionsHere we show that PRMT5 inhibition with a novel inhibitor, PRT382, leads to enhanced vulnerability in mantle cell lymphoma to treatment with the BCL2 inhibitor venetoclax.PRMT5 inhibition lowers the activity of AKT including its ability to phosphorylate other proteins.PRMT5 inhibition causes AKT to lose its interaction with FOXO1 and lowers levels of inactive FOXO1.Active FOXO1 was recruited to pro-apoptotic BCL2 family genes, including BAX.With PRMT5 inhibition mRNA and protein levels of BAX rise.While only four MCL cell lines were sensitive to venetoclax, seven showed synergy between PRMT5 and BCL2 inhibitionThis synergy was also seen in two MCL PDX mouse models. SIGNIFICANCEPRMT5 inhibition provides vulnerabilities to secondary targeted therapies through reprograming of the cell at a genetic and protein level.Our observations show that PRMT5 and BCL2 inhibition could be combined to increase cell death and decrease dosages.This work will inform further clinical trials performed with the PRMT5 inhibitor PRT543.Figure 6 A) Experimental lay out of in vivo experiments. NSG mice were engrafted with passaged PDX cells and treated with vehicle, venetoclax, PRT382, or the combination (PO; 4 days on/3 days off). Venetoclax and PRT382 doses were subtherapeutic as determined by previous work. Treatment was ceased around day 100 in both experiments. B) Kaplan-Meier survival curves of two MCL PDX models. C) Disease progression as monitored by circulating huCD5+/huCD19+ in peripheral blood by flow cytometry.RESULTS Figure 1 A) Selectivity of PRT382 among methyltransferases. B) IC50 of MCL cell lines treated with PRT382. Data was collected at day 9 by annexin v/PI staining and flow cytometry. Error bars show standard deviation. C) Survival curve of an ibrutinib resistant MCL PDX mouse model treated with ibrutinib, the standard of care for relapsed or refractory MCL, or PRT382. Activity of a Novel PRMT5 Inhibitor (PRT382) in MCL ModelsFigure 2 A) Nuclear and cytosolic fractions of CCMCL MCL cells with and without PRMT5 inhibited. The phosphorylated active form of AKT decreases with treatment while the non-phosphorylated active form of FOXO1 increases. B) AKT was immunoprecipitated and a loss of interaction with FOXO1 was seen with PRMT5 inhibition.PRMT5 Inhibition Reduces AKT Activity and pFOXO1 LevelsFigure 4 A) MCL cell line response to venetoclax at 24hrs as measured by annexin V/PI staining and flow cytometry. Starred cell lines were defined as sensitive lines with an IC50 <1uM. B) Synergy score for MCL cell lines pretreated with PRT382 for 6 days and co-treated with PRT382 and venetoclax. Viability was measured for multiple combinations via MTS assay and data analyzed through Combenefit software. -10 to 0 Mild antagonism, 0-10 mild synergy, 10-20 synergistic, >20 highly synergisticFigure 3 A) FOXO1 was immunoprecipitated and the resulting chromosomal material was sequenced. Shown are traces of FOXO1 recruitment with and without PRMT5 inhibition to the promoter region of several pro-apoptotic BCL-2 family proteins. B) mRNA levels by q-rtPCR of three pro-apoptotic BCL-2 family proteins. C) Protein expression of BAX by western blot. Measured and quantified by image studio using GAPDH as a control.PRMT5 Inhibition Allows FOXO1 to Promote BCL-2 Family ProteinsSynergistic Cell Death is Intrinsic and Dependent on BAX Figure 5 A) Western blots of Z-138 cells dosed with DMSO, venetoclax, PRT382, or the combination for two, four, or six days. The level of cleaved caspase 9 was highest in the combination treated cells, indicative of intrinsic apoptosis. B) shRNA was used to knock down BAX or BAK1 in Jeko or Z-138 cells. C) Viability of scramble, BAX, or BAK1 shRNA cells treated with DMSO, venetoclax, PRT382, or a combination. Viability was measured at day four by annexin V/PI staining and flow cytometry. BAX knock down rescued both cell lines from venetoclax and combination treatment while BAK1 was only protective in Jeko.PRMT5 Inhibition is Synergistic with Venetoclax In VitroAAAACACFigure 7 Model for synergy observed with PRMT5 and BCL2 inhibition. PRMT5 inhibition allows for the upregulation of BAX while BCL2 inhibition prevents BAX from being blocked.PRMT5 Inhibition Synergizes with Venetoclax through BAX Up-regulation and BCL-2 InhibitionBPRMT5 Inhibition Synergizes with Venetoclax In VivoFunding : NIH/NCI P01 CA214274-01A1 & Leukemia Lymphoma Society MCL Research Initiative OSU COM SIB Training FellowshipBABRG1α-TubulinFOXO1p-FOXO1 (Thr24)/FOXO3A (Thr32)p-FOXO1 (Ser256)N C N CDMSO PRMT5iDMSO PRMT5iBRG1p-AKT (S450)p-AKT (S473)N C N CDMSO AKTPRMT5iα-TubulinDMSO PRMT5iBCPDX.AA.MCL IR PRMT5iP<0.01BAXBAK1[0-10]PRTCONBIM (BCL2L11)PRTCON[0-10]BIKBBC3PRTCONNOXA1BMF[0-10]Z EVZ BAX v1Z BAK1 V1Z BAK1 V2J EVJ BAX V1J BAK1 V2GapdhGapdhBAXBAK11.0.91.46.641.01.1.911.0.001.0.811.0.01.661.0.88.681.21.0.81.401.0.84.09.071.01.0.17GAPDHProcaspase 9Cleaved Caspase 9Cleaved Caspase 9DMSO Day 2DMSO Day 4DMSO Day 6Ven Day 2Ven Day 4Ven Day 6PRT382 Day 2PRT382 Day 4PRT382 Day 6Combo Day 2Combo Day 4Combo Day 6BBBC