Inhibition of Both IRAK1 and IRAK4 Is Required for Complete Suppression of NF-Kb Signaling across Multiple Receptor-Mediated Pathways in MDS and AML

There is increasing interest in the role of the IRAK kinases as targets in the treatment of MDS and AML (reviewed in J Bennett and DT Starczynowski, Curr Opin Hematol 2022). IRAK1 and IRAK4 lie downstream of multiple receptors that stimulate the canonical NF-κB signaling pathway upstream of TRAF6 via the myddosome complex. While TRAF6 is necessary to preserve the tonic NF-κB signaling that is required for hematopoietic stem cell (HSC) homeostasis (J Fang et. al. Cell Reports 2018), overactivity of the NF-κB signaling pathway has been implicated in both MDS and AML (reviewed in MCJ Bosman et. al., Crit Rev Oncol/Hematol 2016; JJ Trowbridge and DT Starczynowski, J Exp Med 2021). The requirement for both IRAK1 and IRAK4 in this pathway has been explored using both genetic and pharmacologic technologies. The use of genetic loss-of-function approaches revealed that deletion and/or inhibition of IRAK4 results in a compensatory increase in IRAK1 protein and activation (data unpublished), suggesting that high potency antagonism of both kinases will be required for optimal inhibition of NF-κB-mediated transcriptional responses in disease-propagating MDS and AML cells. In this report, we use a pharmacological approach in which a series of IRAK inhibitors of varying relative potency at IRAK1:IRAK4 were examined for both inhibition of NF-κB activation in AML cells and inhibition of leukemic progenitor cell function in vitro. We find that potency and efficacy for antagonism at NF-κB requires effective inhibition of both IRAK1 and IRAK4. We also find that the relative potency of NF-κB inhibition correlates with suppression of leukemic progenitor cell function in vitro, providing pharmacological validation that inhibition of both IRAK1 and IRAK4 are necessary for optimal inhibition of NF-κB and effect on leukemia progenitor cell function.

We utilized an NF-κB reporter system expressed in human AML cells (THP1) to measure NF-κB dependent activation in response to a variety of IRAK4, IRAK1, or IRAK1/4 antagonists. The cells are highly responsive to both TLR agonists as well as to IL-1b, which allows for measurement of IRAK-mediated antagonism of multiple receptor-mediated pathways. Using the IRAK4-selective antagonists PF-06650833 and BAY 1834845 we find that both compounds fully suppress signaling through TLR2 (IC₅₀vs. Pam3CSK4 = 7.2 and 150 nM, respectively). The IRAK4 antagonists inhibit signaling through the IL-1R with similar relative potency (IC₅₀vs. IL-1b = 5.7 and 81 nM), but do not fully suppress signaling through this receptor (Span = 75% and 59%, respectively). Interestingly, the IRAK1 selective covalent inhibitor JH-X-119-01 does not inhibit signaling against either the TLR or the IL-1 receptor agonist. Together these data imply that neither IRAK4 nor IRAK1 inhibition alone is sufficient to fully inhibit NF-κB-mediated signaling through multiple receptor mediated pathways.

For this reason, we then examined the activity of a series of IRAK4/IRAK1 inhibitors in AML cells. This allowed us to study the effect of adding in additional inhibitory activity at IRAK1 on the background of high potency IRAK4 inhibitors. Using two reference compounds with varying IRAK1:IRAK4 potency, we then compared the relative ability to inhibit TLR-agonist or IL-1b-agonist stimulated NF-κB activity in AML. KME-2780 has an IRAK1:IRAK4 potency ratio of 36 whereas KME-3859 has an IRAK1:IRAK4 potency ratio of 100. Unlike what we observed with the IRAK4-selective antagonists, both compounds can completely suppress NF-κB signaling through IL-1b, with relative potencies apparently determined by their activity at IRAK1: (IC₅₀vs. Pam3CSK4 = 6.3 and 73.8 nM for KME- 2780 and KME-3859, respectively); (IC₅₀vs. IL-1b = 9.3 and 227 nM for KME- 2780 and KME-3859, respectively).

Finally, we examined the correlation between potency in the biochemical kinase assay at IRAK1 or IRAK4, activity in the NF-κB reporter assay, and leukemic progenitor cell activity in the colony forming assay for a series of KME compounds in AML and MDS cells. We find a correlation between kinase activity for both IRAK1 and IRAK4 and NF-κB activity that extends to the leukemia colony forming assays. This suggests that NF-κB signaling contributes to leukemia progenitor cell function and that optimal inhibition requires potent antagonism of both IRAK1 and IRAK4 in the setting of MDS and AML.

Rosenbaum:Radius Health: Consultancy, Other: consultant ; Enable Injections: Current equity holder in private company; Airway Therapeutics: Current holder of stock options in a privately-held company, Patents & Royalties: Patents, no royalties. Luedtke:Kurome Therapeutics: Current Employment. Starczynowski:Treeline Biosciences: Research Funding; Tolero Therapeutics: Research Funding; Kymera Therapeutics: Consultancy; Kurome Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Captor Therapeutics: Consultancy; Sumitomo Pharma Oncology, Inc: Research Funding.