TRAF1 S146 is constitutively phosphorylated in OP9-cultured primary chronic lymphocytic leukemia cells by PKN1/2

Abstract

TNF receptor–associated factor 1 (TRAF1) is a prosurvival signaling adaptor that contributes to NF-κB activation downstream of a subset of TNF receptor superfamily members. TRAF1 is overexpressed in many cancers of mature B cells, including chronic lymphocytic leukemia (CLL). Previous studies have established that TRAF1 S146 is a target of phosphorylation by the kinase PKN1 and that PKN1 is required to prevent cellular inhibitor of apoptosis protein (cIAP)–dependent degradation of TRAF1 in the CD40 signaling complex. The kinase inhibitor OTSSP167 inhibits PKN1 in the nanomolar range and its addition to primary CLL cells was previously shown to induce dose-dependent loss of TRAF1 and concomitant increases in activated caspase 3 and cell death. These studies identified PKN1 as a target for therapy of CLL. To identify more potent and specific PKN1 inhibitors for therapy of B-cell cancers, it is important to measure a direct target of PKN1, such as phospho-TRAF1. To this end, here we use overexpression of an S146A mutant of human TRAF1 in HEK293 cells to validate a recently generated TRAF1 phospho (p)S146-specific antibody and to confirm that this phosphorylation is lost upon treatment with OTSSP167. Using CRISPR/Cas9 knockout in RAJI cells, we also show that both PKN1 and the closely related family member PKN2 can phosphorylate TRAF1 S146. We further show that TRAF1 S146 is constitutively phosphorylated in OP9 cultured primary human CLL cells, including those with p53 mutations, and that this phosphorylation is sensitive to inhibition with OTSSP167. These findings provide support the development of more potent PKN1/2 inhibitors for CLL.