ROLE OF TRAF FAMILY OF PROTEINS IN MEDIATING CD40 STIMULATION-INDUCED DRUG RESISTANCE IN CHRONIC LYMPHOCYTIC LEUKAEMIA CELLS

Abstract

Despite remarkable advances in treatment options, chronic lymphocytic leukaemia (CLL) remains an incurable disease. Interaction of CLL cells with various components in the tumour microenvironment is believed to contribute to drug resistance in CLL. One of the critical microenvironment-mediated pro-survival signals involves the engagement of CD40 receptor on CLL cells by CD40 ligand on T cells, which leads to the activation of canonical and noncanonical NF-κB pathway, as well as several other signalling pathways. The initiation of CD40 signalling is primarily mediated by family members of the tumour necrosis factor receptor-associated factors (TRAFs). However, the mechanisms by which TRAFs mediate the CD40 signalling pathway resulting in CLL cell survival and drug resistance are not well characterised. Therefore, the present study aimed to uncover the immediate signalling events initiated upon CD40 ligation in primary CLL cells. Co-culture experiments used to mimic T cells effect on CLL cells showed that CD40 stimulation protected CLL cells from fludarabine- induced cell death. This protection was associated with overexpression of TRAF1 and post- transcriptional degradation of TRAF3 in primary CLL cells, suggesting the involvement of the TRAFs in the regulation of CD40 signalling. Using the in situ proximity ligation assay (PLA), it was shown that CD40 ligation induced rapid recruitment of TRAF2 and TRAF5 to CD40 receptor in primary CLL cells. RNA interference experiments showed that CD40 stimulation- mediated canonical activation of the NF-κB pathway was impaired in CLL-like HG3 cells with reduced expression of TRAF2, suggesting a role for TRAF2 in the activation of NF-κB. In contrast, cells transfected with TRAF5 siRNA showed normal NF-κB activation in response to CD40 ligation. Importantly, the current study identified for the first time a critical role of TRAF2 in mediating CD40 stimulation-induced resistance to fludarabine as knockdown of TRAF2, but not TRAF5, enhanced fludarabine-induced apoptosis in CD40-stimulated cells. Further studies are still required to identify the exact mechanisms by which TRAF2 modulates CD40 stimulation-mediated resistance to fludarabine. The findings from this study collectively suggest a role for TRAF2 protein in regulating CD40 stimulation-induced NF-κB activation and subsequent resistance to fludarabine treatment. Additional work is, however, still needed to validate the study findings.