Genetic or CD40L-Mediated Loss of Iκbα Is Associated with Resistance to the Dual SYK/JAK Inhibitor Cerdulatinib in DLBCL Cell Lines

Abnormal upregulation of NFκB activity is observed in a variety of B cell malignancies, resulting in proliferative and survival signals that contribute to tumor progression. Under normal resting conditions, NFκB is negatively regulated principally via its physical association with IκB (inhibitor of NFκB) family members, thereby inhibiting nuclear transport or access to DNA. In B cells, NFκB is typically activated via various external stimuli (e.g., ligation of the B cell antigen receptor (BCR), toll-like receptors, cytokine receptors, CD40), leading to IκB kinase complex-dependent phosphorylation of IκB members and targeting them for ubiquitination and degradation. In some cases, the need for external stimuli is diminished or completely circumvented by mutations to critical regulators of NFκB, as has been described in the context of activating mutations to CD79A/B, MYD88, and CARD11, as well as inactivation of negative regulators such as A20 and IκB family members (reviewed by Staudt, 2010). Each of these mutations has been observed clinically in patients with B cell malignancies (Wilson et al, 2012; Norenberg et al, 2015; Mansouri et al, 2015), and can impact the anti-tumor activity of selective BCR pathway inhibition (Davis et al, 2010; Wilson et al, 2012) in part via induction of autocrine cytokine stimulation leading to JAK/STAT-dependent up-regulation of MCL1 (Lam et al, 2008).

We previously reported that cerdulatinib, a small molecule kinase inhibitor that dually targets SYK and the JAK family members JAK1, JAK3, and TYK2, maintained anti-tumor activity in DLBCL cell lines bearing mutations to CARD11, MYD88, and A20 (Ma et al, 2015). The majority of DLBCL cell lines exhibit various degrees of reliance on SYK and JAK signaling for survival, however in a screen of 15 DLBCL cell lines we found 3 that were completely resistant to cerdulatinib and are described here. In one of the cerdulatinib-resistant cell lines, RCK8, next generation sequencing revealed bi-allelic inactivation of the IκBα gene. One allele carries a frameshift mutation in exon 1 resulting in the generation of a premature stop codon, and the second allele is a nonsense mutation in exon 3 at Gln154, also leading to a stop codon. In accord with a previous report (Kalaitzidis et al, 2002), the cell line lacks IκBα protein expression. We therefore proceeded to explore the possibility that loss of IκBα was responsible for resistance to cerdulatinib.

Consistent with the loss of IκBα, the RCK8 cell line exhibited enhanced basal NFκB activity. Genetic re-introduction of wild type IκBα led to rapid suppression of NFκB, and ultimately cell cycle arrest and cell death, indicating that the cell line was dependent upon loss of this gene for survival. Associated with suppression of NFκB was decreased phosphorylation of cellular pAKT S473 and pERK Y202, but not of pSTAT3 Y705. We then attempted to knock down IκBα in cerdulatinib-sensitive cell lines using siRNA to determine if resistance to SYK/JAK inhibition could be generated. None of the DLBCL cell lines tested (n=4) could tolerate IκBα gene knock down, suggesting an additional mutation in RCK8 enables survival under conditions of homozygous loss of IκBα. Ligation of CD40 leads to a transient down-regulation of IκBα at the protein level (Oeckinghaus and Ghosh, 2009). We therefore examined the effect of CD40L on multiple DLBCL cell lines and found that IκBα was maximally suppressed within 30-60 minutes post CD40 stimulation, returning to pre-treatment levels by 2-4 hours. In contrast, the impact on NFκB activation was much longer, and 5 of 7 cerdulatinib-sensitive cell lines tested were made resistant by incubation with CD40L. Associated with this resistance was not only induction of NFκB, but also pERK Y204, pAKT S473, and pSTAT3 Y705. Interestingly, whereas the CD40L-induced NFκB activation was not inhibited by cerdulatinib, the other signaling events were, despite the generation of resistance.

Loss of IkB family members has been described in the context of Hodgkin's lymphoma, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia (Cabannes et al, 1999; Norenberg et al, 2015; Mansouri et al, 2015). Here we demonstrate that loss of IκBα in multiple DLBCL cell lines generates resistance to cerdulatinib. We will be exploring the clinical relevance of these in vitro observations in cell lines as part of an ongoing phase II trial of cerdulatinib in patients with relapsed/refractory B cell malignancies.