Claire Hinterschied – Microbiology

FOXO1 Plays a Crucial Role in Mediating Apoptosis in PRMT5-Inhibited Mantle Cell Lymphoma

“Mantle Cell Lymphoma (MCL) is a cancerous B-cell malignancy which constitutes 3-6% of all Non-Hodgkins Lymphomas diagnosed annually. MCL is associated with poor prognosis due to the late median age of diagnosis, high incidence of relapse, and resistance to chemotherapies. The average survival of patients with MCL is 5 years, and for those who relapse on targeted therapy, 3-8 months. MCL is currently incurable.
A key driver of MCL pathogenesis is an enzyme called Protein Arginine Methyltransferase-5 (PRMT5), which can post-translationally modify proteins by methylating arginine residues. This allows PRMT5 to alter histones and silence genes that regulate cell growth and proliferation, supporting tumorigenesis. Inhibiting PRMT5 significantly delays disease progression and reduces tumor burden in pre-clinical MCL models. In order to better understand these effects, we explored the activity of the tumor suppressive transcription factor FOXO1, an indirect target of PRMT5 through the PI3K/AKT pathway. We hypothesized that FOXO1 promotes cell death in PRMT5 inhibited cells through reactivation of pro-apoptotic target genes.
FOXO1 activation status and localization was determined through nuclear and cytoplasmic fractionation with western blotting on control and PRMT5 inhibited cells. Target genes of FOXO1 were determined by using ChIP sequencing on CCMCL1, a MCL cell line, with or without PRMT5 inhibition. Genes of interest were confirmed as FOXO1 targets with ChIP qPCR and mRNA qPCR. Protein level changes were measured through western blotting. The importance of FOXO1 and pro-apoptotic target genes were explored with pharmacologic and shRNA inhibition. Cell viability was tested through Annexin V/PI staining and flow cytometry.
We show that FOXO1, a transcription factor associated with upregulating tumor-suppressor genes, is a critical component of mediating intrinsic apoptosis in MCL. PRMT5 inhibition causes dephosphorylation (activation) and subsequent nuclear localization of FOXO1. Of the gene targets of FOXO1, the pro-apoptotic BCL-2 family genes were identified as possible mediators of cell death. These genes were up-regulated on a transcript and protein level. Inhibition of the BCL-2 family members showed partial rescue of cell viability. FOXO1 inhibition did not provide rescue, raising questions about the other target genes of FOXO1 and how this protein plays a dual biological role.”