TRAF3 suppression encourages B cell recruitment and prolongs survival of microbiome-intact mice with ovarian cancer

Abstract

Background Ovarian cancer (OC) is known for exhibiting low response rates to immune checkpoint inhibitors that activate T cells. However, immunotherapies that activate B cells have not yet been extensively explored and may be a potential target, as B cells that secrete immunoglobulins have been associated with better outcomes in OC. Although the secretion of immunoglobulins is often mediated by the microbiome, it is still unclear what role they play in limiting the progression of OC. Methods We conducted an in-vivo CRISPR screen of immunodeficient (NSG) and immune-intact wild type (WT) C57/BL6 mice to identify tumor-derived immune-escape mechanisms in a BRAC1- and TP53-deficient murine ID8 OC cell line (designated ITB1). To confirm gene expression and signaling pathway activation in ITB1 cells, we employed western blot, qPCR, immunofluorescent staining, and flow cytometry. Flow cytometry was also used to identify immune cell populations in the peritoneum of ITB1-bearing mice. To determine the presence of IgA-coated bacteria in the peritoneum of ITB1-bearing mice and the ascites of OC patients, we employed 16S sequencing. Testing for differences was done by using Deseq2 test and two-way ANOVA test. Sequence variants (ASVs) were produced in Qiime2 and analyzed by microeco and phyloseq R packages. Results We identified tumor necrosis factor receptor-associated factor 3 (TRAF3) as a tumor-derived immune suppressive mediator in ITB1 cells. Knockout of TRAF3 (TRAF3KO) activated the type-I interferon pathway and increased MHC-I expression. TRAF3KO tumors exhibited a growth delay in WT mice vs. NSG mice, which was correlated with increased B cell infiltration and activation compared to ITB1 tumors. B cells were found to be involved in the progression of TRAF3KO tumors, and B-cell surface-bound and secreted IgA levels were significantly higher in the ascites of TRAF3KO tumors compared to ITB1. The presence of commensal microbiota was necessary for B-cell activation and for delaying the progression of TRAF3KO tumors in WT mice. Lastly, we observed unique profiles of IgA-coated bacteria in the ascites of OC-bearing mice or the ascites of OC patients. Conclusions TRAF3 is a tumor-derived immune-suppressive modulator that influences B-cell infiltration and activation, making it a potential target for enhancing anti-tumor B-cell responses in OC.

A novel pan-PI3K inhibitor KTC1101 synergizes with anti-PD-1 therapy by targeting tumor suppression and immune activation

“…qRT-PCR was performed as previously described [ 42 ]. RNA was isolated using Trizol and used to synthesize complementary DNA (cDNA) using a cDNA Synthesis Kit (GenStar), and RT-PCR was performed with aliquots of cDNA samples mixed with SYBR Green MasterMix (Applied Biosystems).…”

cAMP-PKA/EPAC signaling and cancer: the interplay in tumor microenvironment

“…Tumor necrosis factor receptor-associated factor 3 (TRAF3) plays a critical role in inhibiting B-cell survival via inhibiting CREB stability [ 294 ]. Loss-of-function mutations of TRAF3 are commonly found in multiple myeloma, ovarian cancer and B-cell lymphoma [ 295 , 296 ].…”