ALBERT

Description: Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein–protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease.

Description: Abstract 3485 Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in the treatment of several B cell malignancies. Bruton's tyrosine kinase (Btk) plays a key role in promoting B cell proliferation and survival through participation in the BCR signaling pathway and represents a promising new drug target. AVL-292 is a covalent, highly selective, orally active small molecule inhibitor of Btk currently being evaluated in a Phase 1b clinical trial in relapsed, refractory B cell malignancies including Chronic Lymphocytic Leukemia (CLL) and non-Hodgkin lymphomas. AVL-292 forms a covalent bond with Cys481 in Btk and potently inhibits Btk in biochemical (IC50 〈 0.5nM) and cellular assays (EC50 1–10 nM) including anti-IgM stimulation of BCR signaling, B cell proliferation and activation. A quantitative pharmacodynamic assay to determine the level of AVL-292 bonded to Btk in vitro or in vivo was developed and this drug-target engagement by AVL-292 was shown to correlate directly with inhibition of Btk enzyme activity and substrate phosphorylation. To rationally determine the dose and dose frequency of AVL-292 most likely to benefit patients and to reduce the potential for sub-therapeutic dosing in initial oncology patient cohorts, AVL-292 was administered to healthy adult subjects in a double-blind, placebo controlled, single ascending dose study. This study assessed safety, pharmacokinetics, and quantitatively measured Btk protein levels and AVL-292-Btk engagement in freshly isolated peripheral B lymphocytes. In healthy human subjects, AVL-292 was found to be safe and well tolerated following oral administration at dose levels ranging from 0.5–7.0 mg/kg. AVL-292 plasma levels and pharmacodynamic measurement of Btk engagement was dose-proportional across cohorts. All subjects that received 1.0 mg/kg AVL-292 achieved 〉80% Btk engagement and mean peak plasma levels (Cmax 365 ng/mL) of AVL-292 were rapidly achieved (Tmax median 40 min). Subjects receiving 2.0 mg/kg AVL-292 had a mean peak plasma concentration of 542 ng/mL. All subjects demonstrated 〉84% Btk engagement at this dose, with 5 of 6 subjects achieving 〉98% drug-target engagement. Although AVL-292 plasma levels declined substantially by 8 hours, Btk engagement persisted throughout 24 hours, demonstrating that covalent inhibition of Btk with AVL-292 enables prolonged duration of activity without high levels of circulating drug. These results suggest that a once daily dosing schedule is sufficient for sustained Btk inhibition. Furthermore, the Btk protein level in circulating B lymphocytes from all study subjects was evaluated and the mean level was found to be 417.7 pg Btk/mg total protein. Interestingly, this finding in normal B cells correlates well with preclinical ex vivo analysis of Btk protein in primary CLL cells where comparable Btk protein levels were found. This suggests that the AVL-292 dose range and schedule identified for complete Btk engagement in this healthy volunteer trial is likely to inform appropriate dose selection in the subsequent phase 1b oncology study, allowing more rapid identification of a safe and clinically effective dose. Disclosures: Evans: Avila Therapeutics: Employment, Equity Ownership. Tester:Avila Therapeutics: Employment, Equity Ownership. Aslanian:Avila Therapeutics: Employment, Equity Ownership. Chaturvedi:Avila Therapeutics: Employment, Equity Ownership. Mazdiyasni:Avila Therapeutics: Employment, Equity Ownership. Sheets:Avila Therapeutics: Employment, Equity Ownership. Nacht:Avila Therapeutics: Employment, Equity Ownership. Stiede:Avila Therapeutics: Employment, Equity Ownership. Witowski:Avila Therapeutics: Employment, Equity Ownership. Lounsbury:Avila Therapeutics: Employment, Equity Ownership. Petter:Avila Therapeutics: Employment, Equity Ownership. Singh:Avila Therapeutics: Employment, Equity Ownership. Westlin:Avila Therapeutics: Employment, Equity Ownership.

Description: The L265P somatic mutation in the Myeloid Differentiation Primary Response 88 (MYD88) gene is recurrently observed in CLL; although this mutation has been demonstrated to have functional effects in multiple hematologic malignancies, its role in CLL is largely unknown. To address this gap in knowledge, we examined the clinical and biological impact of MYD88 L265P mutations in CLL by analysis of gene expression, cell viability and Toll-like Receptor 9 (TLR9)-induced signaling and cytokine production. Out of 160 CLL patient samples subjected to whole-exome sequencing and previously reported by our group, 10 were found to harbor MYD88 L265P mutations, all of which possessed mutated immunoglobulin heavy chain variable (IGHV) regions (p = 0.006). While IGHV mutated patients are generally known to exhibit better prognosis compared to IGHV unmutated patients, the presence of MYD88 L265P within the IGHV mutated subset was associated with earlier age of disease onset (p = 0.04) and worse overall survival (OS; p = 0.00017), comparable to IGHV unmutated samples with wild-type (WT) MYD88. No association with the presence of chromosome 13q deletions (p = 0.26) or prior treatment at the time of sampling (p = 0.10) was observed. Gene expression microarray analysis restricted to the IGHV mutated subset (MYD88 WT: n = 76; MYD88 L265P: n=10) and conducted using a PAM-based approach demonstrated that MYD88 L265P mutation was associated with differential expression of 28 genes, whose expression was then examined across all CLL samples with available gene expression data (n = 150). Using Cox modeling, a composite gene signature score was determined for each patient, who were subsequently dichotomized based on median signature. This method was able to predict both OS and event free survival (EFS) in a univariable analysis (OS: p = 1.2E-06; EFS: p = 7.6E-13). Statistical significance was maintained when a multivariable analysis was conducted, adjusting for known CLL risk factors including age, IGHV status, ZAP70 expression, cytogenetics and prior treatment (p 〈 0.0001 for OS and EFS). The univariable (OS: p = 1.6E-05; EFS: p = 5.7E-10) and multivariable findings (p 〈 0.003 for OS and EFS) were further confirmed in an independent validation cohort (n = 87). To identify a more parsimonious gene set, we applied a L1 penalized proportional hazards model to the discovery and validation cohorts, separately. This approach identified 5 overlapping genes (BCAT1, BMP6, CHAD, IKZF2, and TRIO) between the two cohorts that appear to be the main drivers of the predictive signature. To inhibit MYD88 signaling in CLL cells, we treated MYD88 L265P and WT cells (n = 6/group, matched for clinical characteristics: IGHV, ZAP70, cytogenetic, and treatment status) in vitro with a highly-selective small molecule IRAK4 inhibitor, ND-2158 (Nimbus Therapeutics). ND-2158 significantly reduced cell viability in a dose dependent manner in both MYD88 WT and L265P primary CLL cells, either alone or in combination with a fixed concentration of the B-cell receptor (BCR) pathway inhibitor, ibrutinib. The TLR9 agonist CpG was used to stimulate signaling through the MYD88 pathway in vitro. ND-2158 inhibition of CpG-induced IRAK4 activation in CLL cells (n = 3/group, matched for clinical characteristics) blocked IRAK1 and IκBα degradation and led to a dose-dependent decrease in the ratio of phospho/total proteins. No significant differences were noted between MYD88 WT and L265P samples, consistent with our cell viability results. CLL-secreted levels of IL-6, IL-10 and CCL3 were measured in culture supernatants treated with ND-2158+/- CpG stimulation (n = 4/group, matched for clinical characteristics). CpG stimulated cytokine levels (p 〈 0.0001 for all cytokines+/- CpG) were significantly inhibited in a dose-dependent manner by ND-2158. Again, no significant differences were observed between MYD88 WT and L265P CLL with respect to cytokine production, either at baseline or in CpG-stimulated DMSO treated control cells. In conclusion, the differences in clinical outcome and gene expression observed between MYD88 WT and L265P IGHV mutated CLLs indicate a functional role for MYD88 L265P in CLL. The inferior clinical outcome in IGHV mutated CLL with L265P mutation suggests that MYD88 signaling may be a relevant target in CLL. ND-2158 inhibits signaling in the MYD88 pathway, suggesting potential therapeutic utility of IRAK4 inhibitors in CLL. Disclosures Chaudhary: Nimbus Therapeutics: Equity Ownership. Miao:Nimbus Therapeutics: Employment. Westlin:Nimbus Therapeutics: Employment.

Description: A member of the Tec family kinases, Bruton’s tyrosine kinase (Btk) modulates B-cell development and activation, and plays an important role in antibody production. Interestingly, Btk and Tec (the other Tec kinase family) regulate osteoclast (OC) differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, OCs derived from X-linked agammaglobulinemia (XLA) patients who harbor Btk null mutations have impaired function. Here we show that a potent and specific Btk inhibitor, CC-292 inhibits OC function in multiple myeloma (MM) patients. CC-292 is a highly selective, covalent Btk inhibitor. OC derived from MM patient monocytes were assayed with or without CC-292. Interestingly, OC function was significantly inhibited in the presence of CC-292 (100 nM and 1000 nM) as demonstrated by pit formation assay. However, mRNA expression for TRAP and Cathepsin K, two OC differentiation markers were increased in the presence of CC-292 suggesting that CC-292 inhibits OC function without inhibiting OC differentiation. OC sealing zone contributes to OC bone resorption function. Given the role of c-Src and Proline-rich tyrosine Kinase 2 (Pyk2) signaling in sealing zone formation and OC function we next evaluated CC-292’s effect on Pyk2 and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, CC-292 (100 nM) inhibited c-Src total protein, c-Src phosphorylation and Pyk2 phosphorylation. Furthermore, CC-292 (100 nM) inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OC derived from MM patient monocytes with resultant inhibition of OC sealing zone formation. However, at the same low doses (100 nM) CC-292 did not show any direct in vitro effect against MM cell viability. Because carfilzomib, a proteasome inhibitor that binds irreversibly to its target, has potent anti-MM activity and also inhibits OC resorptive activity, we studied CC-292 in combination with carfilzomib. Our data suggests that carfilzomib (1.25 nM) has no impact on OC sealing zone formation but inhibits OC differentiation. CC-292 (100 nM) in combination with carfilzomib (1.25 nM) inhibited not only sealing zone formation but also OC differentiation, resulting in stronger suppression of OC function than carfilzomib alone. The combination of CC-292 (30mg/kg p.o. for 5 days per week for 6 weeks) and carfilzomib (3 mg/kg i.v. x 2 days per week for 4 weeks and 2 mg/kg i.v. x 2 days per week for 2 weeks) significantly inhibited tumor burden and myeloma cell numbers in a diffuse NOD-SCID MM model. The calvarial cells derived from these mice treated with CC-292 alone, carfilzomib alone or the combination showed higher osteocalcin mRNA (osteoblast differentiation marker) expression. A specific bone resorption marker, carboxy-terminal telopeptide collagen crosslinks (CTX) in mouse serum was significantly inhibited in CC-292 and CC-292 in combination with carfilzomib treatment groups in comparison with control mice. Furthermore, 3D microCT reconstructions showed increase in cancellous bone volume in lumbar vertebrae in mice treated with CC-292 or carfilzomib, while the combination treatment resulted in an increase in cancellous bone volume in an additive manner. These data demonstrate that the novel BTK inhibitor CC-292 inhibits OC function through inhibition of OC sealing zone formation. Moreover, CC-292 in combination with carfilzomib augments effects against the bone microenvironment with resultant anti-MM activity. Disclosures: Arastu-Kapur: Onyx Pharmaceuticals, Inc.: Employment. Evans:Celgene Avilomics Research: Employment, Equity Ownership. Singh:Celgene Avilomics Research: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals, Inc.: Employment. Westlin:Celgene Avilomics Research: Employment, Equity Ownership. Raje:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Amgen: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding.

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.