GSK3745417 is currently in phase 1 development targeting solid tumor indications. Recent literature has suggested a role for STING (Stimulator of Interferon Genes) activation induced cytotoxicity in AML cells (Baba et al, Cell Death Dis, 2021; 12(4):322.), indicating potential for therapeutic benefit in AML/MDS. To determine the implications of STING activation on tumor cells directly, GSK3745417 treatment was assessed in a screen against a panel of 93 tumor cell lines from various tumor origin. GSK3745417 treatment resulted in cytokine production across cell lines but cell growth inhibition in only 3 of the cell lines, including the only 2 AML cell lines in the panel. GSK3745417 was subsequently tested in an expanded panel of 13 AML cell lines. The data confirmed the cell growth inhibitory effect of GSK3745417 in 11/13 AML cell lines. Overall, growth inhibition was more potent than IFNβ induction, indicating a distinct growth inhibition/cytotoxicity mechanism in AML cells upon STING activation in addition to the potential to induce anti-AML immune response through type 1 IFN induction (Curran et al, Cell Rep, 2016;15(11):2357-66.). Further study demonstrated caspase induction in 7 out of 11 responding cell lines upon GSK3745417 treatment which was blocked by apoptosis inhibitor QVD. This supports the hypothesis that GSK3745417 induced cytotoxicity in AML cells is driven through an apoptosis mechanism. GSK3745417 also blocked colony formation in primary AML samples from 5 donors. GSK3745417 is able to induce growth inhibition with transient compound incubation in vitro in as little as 30 minutes to 1 hour. Taken together, this data suggests a role for GSK3745417 as a therapeutic in AML/MDS indications through dual mechanisms of direct tumor cell killing and immune activation at a clinically relevant dose. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol.

Hetero-trimeric CARMA/BCL10/MALT1 CBM complexes play important roles in B and T cell antigen receptor signaling and other pathways. Previous studies have suggested that MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a cysteine protease to cleave and inactivate downstream signaling molecules such as A20 and CYLD. However, little is known about the relative importance of these two distinct activities in orchestrating immune responses. Utilizing mice homozygous for either null or protease-dead C461A mutations in MALT1, we found that some but not all MALT1 functions were dependent upon its protease activity. Although protease-dead mice had normal follicular B cell and T cell populations in the spleen, defects were observed in the generation of splenic marginal zone and peritoneal B1 B cells. Ex vivo, B cell receptor-stimulated proliferation was normal, but CD4+ and CD8+ T cells displayed decreased activation-induced proliferation and IL-2 production. C-type lectin receptor stimulation of cytokine production by dendritic cells was also found to be partially dependent upon protease activity. In vivo, protease-dead mice displayed severely reduced basal immunoglobulin levels, and defective T-dependent and T-independent immune responses. Our findings suggest that pharmacologic inhibition of MALT1 protease activity may be a useful therapeutic approach for diseases linked to uncontrolled immune cell activation.

Loss of intestinal epithelial barrier integrity in inflammatory bowel disease (IBD) leads to aberrant interaction between commensal bacteria and mucosal immune cells. This triggers inflammation via activation of pattern recognition receptor (PRR) signaling pathways. Which PRR signaling complexes are critical in mediating disease pathogenesis remains to be elucidated. Our recent studies have shown that selective and potent inhibitors of RIP2 kinase, the signaling partner of NOD1 and NOD2, can dramatically reduce murine TNBS-induced colitis and the spontaneous release of proinflammatory cytokines in cultured intestinal biopsies from IBD patients. The striking magnitude of the inhibition observed led us to interrogate the effect of these inhibitors on the modulation of crosstalk between NOD1/2 and other PRRs. Using human PBMCs we defined conditions for observing synergistic cytokine release (TNF-α, IL-6) in response to combinations of NOD2 and either TLR2, TLR4, or TLR5 ligands. In each instance, selective inhibition of RIP2 kinase activity robustly blocked synergistic cytokine release, but had little or no effect on TLR ligand alone induced cytokine production. These results suggest that RIP2 kinase inhibitors are likely to have profound anti-inflammatory effects in situations where multiple PRRs are being stimulated simultaneously, and illustrate the potential of RIP2 inhibitors as therapeutics for the treatment of human inflammatory diseases.

NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.