A 10-year-old intact male Golden Retriever was presented to The Ohio State University Veterinary Medical Center for acute, non-painful facial swelling of the right mandibular region. On physical examination, the right mandibular swelling was found to represent marked lymphadenopathy of the submandibular lymph node. At this time, marked lymphadenopathy of the prescapular and popliteal lymph nodes was also appreciated. Abdominal lymphadenopathy with suspected splenic and hepatic involvement was detected on ultrasound, based on mass effects and echogenic changes. The CBC showed a moderate leukocytosis (38.4 × 10 9 cells/L, reference interval [RI] 4.8-13.9 × 10 9 cells/L) characterized by a moderate lymphocytosis (28.4 × 10 9 cells/L, RI 1.0-4.6 × 10 9 cells/L). A mild hyperproteinemia (7.5 g/dL, RI 5.6-7.3 g/dL) was also determined by refractometry; however, no significant abnormalities were observed on the blood chemistry panel. Review of the blood smear confirmed the lymphocytosis, which was characterized by a homogeneous population of intermediate lymphocytes (98%) with the remainder being small and
Non-Hodgkin lymphoma (NHL) is a heterogeneous group of blood cancers arising in lymphoid tissues that commonly effects both humans and dogs. Protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes the symmetric di-methylation of arginine residues, is frequently overexpressed and dysregulated in both human solid and hematologic malignancies. In human lymphoma, PRMT5 is a known driver of malignant transformation and oncogenesis, however, the expression and role of PRMT5 in canine lymphoma has not been explored. To explore canine lymphoma as a useful comparison to human lymphoma while validating PRMT5 as a rational therapeutic target in both, we characterized expression patterns of PRMT5 in canine lymphoma tissue microarrays, primary lymphoid biopsies, and canine lymphoma-derived cell lines. The inhibition of PRMT5 led to growth suppression and induction of apoptosis, while selectively decreasing global marks of symmetric dimethylarginine (SDMA) and histone H4 arginine 3 symmetric dimethylation. We performed ATAC-sequencing and gene expression microarrays with pathway enrichment analysis to characterize genome-wide changes in chromatin accessibility and whole-transcriptome changes in canine lymphoma cells lines upon PRMT5 inhibition. This work validates PRMT5 as a promising therapeutic target for canine lymphoma and supports the continued use of the spontaneously occurring canine lymphoma model for the preclinical development of PRMT5 inhibitors for the treatment of human NHL.
Mantle cell lymphoma (MCL) is an incurable B-cell malignancy with overall poor prognosis particularly for patients that progress on targeted therapies. Novel more durable treatment options are needed for patients with MCL. Protein Arginine Methyltransferase 5 (PRMT5) is overexpressed in MCL and plays an important oncogenic role in this disease via epigenetic and post-translational modification of cell cycle regulators, DNA repair genes, components of pro-survival pathways, and RNA splicing regulators. The mechanism of targeting PRMT5 in MCL remains incompletely characterized. Here we report on the anti-tumor activity of PRMT5 inhibition in MCL using integrated transcriptomics of in vitro and in vivo models of MCL. Treatment with a selective small-molecule inhibitor of PRMT5, PRT-382, led to growth arrest and cell death and provided a therapeutic benefit in MCL patient derived xenografts. Transcriptional reprograming upon PRMT5 inhibition led to restored regulatory activity of the cell cycle (p-RB/E2F), apoptotic cell death (p53-dependent/p53-independent), and activation of negative regulators of BCR-PI3K/AKT signaling (PHLDA3, PTPROt, and PIK3IP1). We propose pharmacologic inhibition of PRMT5 for patients with relapsed/refractory MCL and identify MTAP/CDKN2A deletion and WT TP53 as biomarkers that predict a favorable response. Selective targeting of PRMT5 has significant activity in preclinical models of MCL and warrants further investigation in clinical trials.
Burkholderia mallei causes the highly contagious and debilitating zoonosis glanders, which infects via inhalation or percutaneous inoculation and often culminates in life-threatening pneumonia and sepsis. In humans, glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. No vaccine exists to protect against B. mallei, and there is concern regarding its use as a bioweapon. The authors previously identified the protein BpaB as a potential target for devising therapies due to its role in adherence to host cells and the formation of biofilms in vitro and its contribution to pathogenicity in a mouse model of glanders. In the present study, the authors developed an immunostaining approach to probe tissues of experimentally infected animals and demonstrated that BpaB is produced exclusively in vivo by wild-type B. mallei in target organs from mice and marmosets. They detected the expression of BpaB by B. mallei both extracellularly and within macrophages, neutrophils, and epithelial cells in respiratory tissues (7/10 marmoset; 2/2 mouse). The authors also noted the intracellular expression of BpaB by B. mallei in macrophages in the regional lymph nodes of mice (2/2 tissues) and MALT of marmosets (4/5 tissues). It is interesting that B. mallei bacteria infecting distal organs did not express BpaB (2/2 mice; 3/3 marmosets), suggesting that the protein is not necessary for bacterial fitness in these anatomic locations. These findings underscore the value of BpaB as a target for developing medical countermeasures and provide insight into its role in pathogenesis.
Mantle cell lymphoma (MCL) is an incurable B cell malignancy, comprising 5% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. The average overall survival of patients with MCL is 4-6 years and for the majority of patients who progress on targeted agents, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life. The type II protein arginine methyltransferase enzyme, PRMT5 is overexpressed and promotes growth and survival of MCL. Inhibition of PRMT5 with a novel, SAM-competitive class of inhibitors drives anti-tumor activity in MCL cell lines and patient derived xenograft (PDX) models derived from patients with relapse or refractory disease. Selective inhibition of PRMT5 with PRT-382 (Prelude Therapeutics) in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to target genes. By performing chromatin immunoprecipitation-sequencing (ChIP seq) analysis, we identified over 800 newly emerged FOXO1-bound genomic loci, including multiple pro-apoptotic BCL2 family proteins (BAX, BAK1, BIK, BBC3, BMF and NOXA1). FOXO1 localization and transcriptional differences were confirmed by ChIP PCR and RT-PCR respectively. Protein levels were measured with Western blotting. BAX was identified as the most common direct target of FOXO1-transriptional activity that was upregulated on both a transcript and protein level. This led us to hypothesize that PRMT5 inhibition could potentially drive a therapeutic vulnerability to the BCL-2 inhibitor venetoclax. Single agent and combination treatment with venetoclax and PRT382 was performed in nine MCL lines. Of the nine lines, four were considered relatively resistant to PRT-382 and five resistant to venetoclax. Synergy scores, determined from MTS assays, showed significant levels of synergy in the majority of MCL lines tested. CCMCL1 and UPN1, BCL-2 negative MCL lines, and Maver1, which is highly resistant to PRMT5i, were the only cell lines to not show synergy. The cell line with the highest levels of synergy, Z-138, expressed high levels of BCL-2 and is ibrutinib resistant. Overall, there was a strong positive correlation between BCL-2 expression and synergy score (r= -0.8956, p=0.0064). The synergy seen was confirmed to be through the intrinsic apoptotic pathway based on caspase activity. To determine a mechanism of action, BAX and BAK1 were knocked down in four cell lines, three that displayed synergy and one that was resistant. This suggests that BAX expression is essential for synergy between PRMT5 and BCL2 inhibition to occur. Knock down of BAK1, the other effector of the BCL2 family of proteins, did not show protection suggesting that BAX is necessary and sufficient for this therapeutic synergy to occur. We also determined that p53 status did not correlate to the response seen (p=0.477), supporting that this mechanism is occurring through FOXO1 transcriptional regulation. In vivo evaluation in two preclinical MCL models showed therapeutic synergy with combination venetoclax/PRT382 treatment. Mice were treated with sub-therapeutic doses of venetoclax and/or PRT382 and disease burden was assessed weekly via flow cytometry. Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in the MCL in vivo models showed synergistic anti-tumor activity. Both PDX models showed an extension of life with combination treatment (P<0.001) and delayed disease progression (P<0.05). This data provides mechanistic rationale while demonstrating therapeutic synergy in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. Disclosures Zhang: Prelude Therapeutics: Current Employment. Vaddi: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: AstraZeneca: Research Funding; Janssen: Research Funding; Johnson and Johnson: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Eli Lilly: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Owkin: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Paik: Forkhead BioTherapeutics: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.
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