ALBERT

Description: Abstract 62 Activating mutations of MyD88, particularly L265P, occur in about 30% of activated B cell-like diffuse large B cell lymphomas (ABC DLBCLs), the more malignant molecular subtype of DLBCL that responds poorly to standard chemotherapy. We found that the oncogenic signal of mutant MyD88 is transduced predominantly through the Interleukin-1 Receptor-Associated Kinase (IRAK) cascade consisting of IRAK4 and IRAK1, resulting in constitutive activation of classical NF-κB signaling. Importantly, using knockdown-rescue experiments, we found that the kinase activity of IRAK4, but not IRAK1, is required for the oncogenic effect of mutant MyD88 in the survival of ABC DLBCL cell lines. As such, we have proposed that inhibitors of IRAK4 kinase activity could have a therapeutic impact in lymphomas with MyD88 mutations. We have identified two potent small molecule IRAK4 inhibitors, ND-2158 and ND-2110, that are highly selective across a panel of more than 300 kinases. When administered to rodents, both compounds lead to 〉90% suppression of LPS-induced TNF-α release in serum. Both inhibitors are universally toxic towards ABC DLBCL but not GCB DLBCL cell lines, consistent with the highly specific mechanism of action. The molecules demonstrate good pharmacologic drug-like properties and are expected to have a suitable safety profile for clinical evaluation. Mechanistic studies indicate that both inhibitors potently abrogate IRAK4-mediated phosphorylation of IRAK1 and NF-κB activity resulting from the MyD88 (L265P) mutation in ABC DLBCL. Additionally, these agents suppress secretion of the pro-inflammatory cytokines IL-6 and IL-10 by ABC DLBCL cells. A second, parallel survival pathway in ABC DLBCL is engaged by “chronic active” B cell receptor signaling, which can be blocked by inhibiting Bruton's tyrosine kinase (BTK) either genetically or pharmacologically. Notably, the IRAK4 inhibitors strongly synergized with BTK knockdown in killing multiple ABC DLBCL cell lines. Our results provide a solid rationale for the further development of IRAK4 inhibitors for the therapy of ABC DLBCL and suggest that simultaneous inhibition of B cell receptor signaling may provide superior clinical responses. Disclosures: Romero: NIMBUS Discovery: Employment. Chaudhary:NIMBUS Discovery: Employment. Robinson:NIMBUS Discovery: Consultancy, Employment.

Description: 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. Disclosures: Chaudhary: Nimbus Discovery Inc.: Employment. Off Label Use: Exploratory inhibitor of IRAK4 for research purposes. Wood:Nimbus Discovery Inc.: Employment. Romero:Nimbus Discovery Inc.: Consultancy, Equity Ownership. Robinson:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Greenwood:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Shelley:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Morin:Nimbus Discovery Inc.: Consultancy. Kapeller:Nimbus Discovery Inc.: Employment. Westlin:Nimbus Discovery Inc.: Employment, Equity Ownership.

Description: Recent work in CLL and other lymphoproliferative disorders has established the importance of somatic mutations in the pathogenesis and prognosis of disease. MYD88 L265P mutations occur in approximately 30% of ABC diffuse large B cell lymphomas (ABC DLBCL) and 90% of Waldenstrom’s macroglobulinemia (WM) cases and have been shown to be an activating mutation in both cancers. MYD88 is an important adaptor protein for IL-1 and many Toll-like receptors, which signal via dimerization followed by formation of a complex of MYD88 with IRAK4 and IRAK1, leading to activation of IRAK4 kinase activity, IRAK1 phosphorylation and downstream activation of NFκB. Previous work in CLL has demonstrated that MYD88 L265P mutations occur in a smaller subset of patients (2.9-6.25%) and although MYD88 mutation is thought to be a CLL driver mutation, its role in CLL is not clearly understood. We previously reported whole exome sequencing results from a cohort of 160 CLL patients, 10 of whom have MYD88 L265P mutation (6.25%). Eight of ten patients with MYD88 L265P mutation had mutated IGHV, consistent with prior reports of this association. However, patients with MYD88 L265P mutation did not have a younger age at diagnosis, possibly due to the overall younger median age of our patient cohort (54, range 34-77). RNA gene expression profiling of 146 CLL and 20 normal B cell samples revealed that normalized median expression of IRAK1, IRAK4 and NFKB1 appear lower in CLL samples versus normal B cells (IRAK1 p = 0.0001, IRAK4 p = 0.01, NFKB1 p = 0.0005). However, median expression levels of MYD88 appear higher in CLL samples versus normal B cells (p = 0.006). Although MYD88 L265P mutation has been shown to activate BTK in WM, mean BTK protein levels measured by flow cytometry were similar between CLL patients with and without MYD88 mutation (mean % cells positive for BTK, 98.3% in MYD88 L265P mutants vs. 98.16% in wild-type [WT], 3 patients/group), as were levels of phosphorylated BTK (mean % cells positive, 13.28% in MYD88 L265P vs. 14.35% in WT). Chronic BTK-mediated B cell receptor signaling is a hallmark of CLL and studies with the BTK inhibitor ibrutinib (PCI-32765) have demonstrated encouraging therapeutic results in vitro and in vivo. ND-2110 and ND-2158 are potent, highly selective IRAK4 kinase inhibitors which have single agent activity in vitro in ABC DLBCL cell lines as well as synergistic effects with BTK inhibitors. Given these findings, ND-2110 and ND-2158 were ideal candidates to test in CLL. As such, CLL samples with and without MYD88 L265P mutation (3 per group) were cultured in vitro in the presence of 5μM ND-2110 or ND-2158 as single agents or in combination with 2.5μM ibrutinib for 72 hours. A second, higher dose combination was also tested, using 25μM ND-2110 or ND-2158 and 3.75μM ibrutinib. Viability was assessed using Cell TiterGlo and normalized to DMSO treated cells. A general linear model was used to assess the association of % viability with MYD88 L265P mutation, IgM stimulation and combinations of IRAK4 inhibitor compounds and ibrutinib. MYD88 L265P was associated with higher viability, hence less cell killing (p = 0.002 5μM ND-2110 & 2.5μM ibrutinib; p 〈 0.0001 5μM ND-2158 & 2.5μM ibrutinib; p = 0.005 25μM ND-2158 & 3.75μM ibrutinib). Cells treated with 25μM ND-2158 + 3.75μM ibrutinib had significantly lower viability compared to cells treated with 25μM ND-2158 alone (p 〈 0.0001). IgM stimulation also resulted in lower viability compared to no stimulation, suggesting sensitization (p = 0.002 25μM ND-2100 + 3.75μM ibrutinib; p 〈 0.0001 all other combinations). Ibrutinib appeared to have less single agent activity in MYD88 L265P mutants, while the activity of single agent ND-2110 and ND-2158 was more equalized between groups. These preliminary results are currently being confirmed in additional patients. To further characterize the effects of these drugs, we have established a model system in which CLL cells are co-cultured with NIH3T3 cells expressing CD40L, with and without CpG stimulation. Preliminary results show that in one WT MYD88 CLL patient, these conditions resulted in significant induction of Ki67 expression, which was reduced by combination of either ND-2110 or 2158 with ibrutinib. These results suggest that IRAK4 inhibitors in combination with ibrutinib have interesting activity in CLL, which may differ based on MYD88 L265P mutation; experiments are in progress in additional patients to further characterize this effect. Disclosures: Chaudhary: NIMBUS Discovery: Employment. Romero:NIMBUS Discovery: Consultancy, Equity Ownership. Robinson:NIMBUS Discovery: Consultancy. Westlin:NIMBUS Discovery: Employment. Brown:Pharmacyclics: Consultancy; Genentech: Consultancy; Celgene: Consultancy, Research Funding; Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding.