Synergistic Blockade Of Activated B Cell-Like DLBCL Proliferation With a Selective Inhibitor Of IRAK4 In Combination With Inhibition Of The B-Cell Receptor Signaling Network

Toll-Like Receptor (TLR) and IL-1 signaling is mediated by the adaptor protein MyD88 through IRAK4 activation. TLR and IL-1 family ligands activate NFkB through this pathway and stimulate proliferation and cell survival, as well as induce cytokine and chemokine production that can amplify tumor cell survival. The gain-of-function L265P mutation in MyD88 occurs in ∼30% of patients with activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) and ∼90% of Waldenström’s macroglobulinemia. Therefore, inhibition of IRAK4 may be therapeutically relevant in hematologic malignancies containing MyD88 mutations. Recent clinical results with kinase inhibitors strongly support a role for signaling through the B-cell receptor (BCR) pathway in the progression of hematological malignancies including ABC-DLBCL. We were interested to understand the potential utility of selective IRAK4 inhibitors in combination with inhibition of the BCR signaling networks. We have reported previously the identification and characterization of potent and selective IRAK4 inhibitors that are effective in blocking inflammatory signaling in immune cells and demonstrate efficacy in vivo in models of autoimmune disease. ND-2158, a potent (Ki of 1.2 nM) and highly selective IRAK4 inhibitor has been shown to be effective in reducing the proliferation of ABC-DLBCL cell lines. ND-2158 does not decrease cell viability for other cell lines that lack the MyD88 mutation including a germinal center-like DLBCL cell line, BJAB, suggesting that the anti-proliferative effects in ABC-DLBCL cells relate in part to the activating MyD88 mutation. Complete cross-over dose-response proliferation studies of the ABC-DLBCL cell line, OCI-LY10, were conducted using ND-2158 in combination with blockade of key BCR signaling network nodes, using inhibitors of either Btk (ibrutinib), PI3Kdelta (GS-1101), or Syk (P505-15). Isobologram analysis using the Chou-Talalay method revealed that ND-2158 was able to synergistically block cell proliferation in combination with ibrutinib, P505-15, or GS-1101. Interestingly, we find that blockade of SYK, PI3Kdelta, or BTK signaling enhances the potency of ND-2158 in ABC-DLBCL cells. The IC₅₀ values observed in this context are comparable to the potency of ND-2158 when used as a single agent to inhibit inflammatory signaling in immune cells that are not dependent on BCR signaling. The cell proliferation blockade IC₅₀for ND-2158 shifted from an average value of ∼7 μM to 0.19, 0.05, or 0.15 μM, when combined with the IC₅₀ concentrations of the inhibitors of BTK, PI3Kdelta or SYK kinases, respectively. These results suggest that inhibition of both BCR signaling pathways that are amplified in ABC-DLBCL, and IRAK4 signaling activated through MyD88 mutations, are required for a more complete blockade of ABC-DLBCL proliferation. Moreover, we explored ND-2158 combination with lenalidomide, known to be synergistic with BCR and NFkB pathway inhibitors. In contrast to combinations with BCR signaling inhibition, studies with lenalidomide failed to demonstrate an additive or synergistic activity when combined with IRAK4 inhibition in ABC-DLBCL cell lines. Therefore, we conclude that IRAK4 activation, as well as aberrant BCR signaling, are likely to contribute to the proliferative capacity of ABC-DLBCL. We propose that combinatorial therapeutic approaches, including inhibition of IRAK4, may provide benefit for patients with ABC-DLBCL.