Novel mechanistic insights for IRAK1 and IRAK4 signaling provide a framework for the development of dual IRAK1 and IRAK4 protacs for MYD88 mutated lymphomas. Free

Background: MYD88 mutations are found in patients with various B-cell cancers including Waldenstrom's Macroglobulinemia (WM; 95-97%), ABC Subtype of Diffuse B-cell Lymphoma (30-40%), Primary Central Nervous Lymphoma (80%), Marginal Zone Lymphoma (10%) and Chronic Lymphocytic Leukemia (5-10%). Both BTK and IRAK1/IRAK4 are triggered by mutated MYD88 (Yang et al, BLOOD 2013). While BTK is dependent on HCK for its activation, IRAK1 and IRAK4 remain independent of HCK and BTK (Yang et al, BLOOD 2016). Consistent with these benchtop observations, both IRAK1 and IRAK4 remained active and supported survival of primary bone marrow WM cells in patients who received 6 months of treatment with the BTK-inhibitor ibrutinib on a clinical trial (NCT01614821). IRAK1 and IRAK4 may therefore contribute to the intrinsic resistance of BTK-inhibitors. However, the relative impact of scaffold vs. kinase function on pro-survival signaling for either IRAK1 or IRAK4 remains unclear and is critical to understanding the development of kinase inhibitors vs. PROTACs for development of therapeutics targeting MYD88 mutated lymphomas.

Methods: We performed knockdown (KD) experiments using an inducible lentiviral short hairpin RNA expression vector tet-pLKO-puro containing a tetracycline-regulated expression cassette targeting IRAK1 or IRAK4 in MYD88 mutated BCWM.1 and TMD8 cells with puromycin selection. KD experiments and replacement with either wild-type or kinase dead IRAK1 or IRAK4 was accomplished by lentiviral transduction with a pLVX-EF1α-IRES-Puro vector. Immunoblotting was used to confirm protein expression and to evaluate downstream signaling. The CellTiter-Glo Luminescent Cell Viability Assay was used to assess proliferation and apoptosis analysis was performed using Annexin V/Propidium Iodide staining.

Results: KD of IRAK1 or IRAK4 resulted in decreased in proliferation and survival of MYD88 mutated lymphoma cells as well as reduction in p-NFKB-p65 and p-IKBa, though the effects were more pronounced with the KD of IRAK1. Unexpectedly, p-ERK signaling was greatly augmented following KD of either IRAK1 or IRAK4 which could be blocked by the BTK-inhibitor ibrutinib. Treatment of IRAK1 or IRAK4 KD BCWM.1 or TMD8 cells with ibrutinib also triggered greatly increased apoptosis. To clarify kinase vs. scaffold functions that contributed to the above findings, replacement experiments were performed following KD of either IRAK1 or IRAK4. Replacement with wild-type versus kinase dead IRAK1 or IRAK4 more fully reconstituted NFKB-p65 activity. Conversely, replacement with either wild-type or kinase dead IRAK1 or IRAK4 abolished p-ERK activity consistent with a scaffold function for the suppression of ERK activation following KD of either IRAK1 or IRAK4. Given the importance of both kinase and scaffold function for both IRAK1 and IRAK4, we utilized a novel bifunctional proteolysis targeting chimera (PROTAC) JH-XIII-05 that we recently developed and characterized [Hatcher et al, Blood 2024; 144 (Suppl 1): 4359]. JH-XIII-05 exhibited potent and highly selective kinase inhibition and degradation of IRAK1 and IRAK4 and showed superior anti-proliferative and apoptotic activity versus its non-degrading analogue JH-XI-82-01. Importantly, combination treatment of MYD88 mutated lymphoma cells with JH-XIII-05 and ibrutinib abrogated NFKB and ERK activity and showed synergistic activity at most doses.Conclusions: Both IRAK1 and IRAK4 contribute to NFKB pro-survival signaling in MYD88 mutated lymphoma cells which is dependent on their respective kinase activity. Loss of IRAK1 or IRAK4 scaffold triggered BTK mediated ERK activation consistent with a previously unrecognized stress response to blocking IRAK1 and IRAK4 NFKB survival signaling in MYD88 mutated lymphomas. Use of the bifunctional PROTAC JH-XIII-05 blocked the kinase activity and degraded both IRAK1 and IRAK4 and showed synergistic killing of MYD88 mutated lymphoma cells. Our studies provide novel mechanistic insights for IRAK1 and IRAK4 signaling and a framework for the development of dual IRAK1 and IRAK4 PROTACS for MYD88 mutated lymphomas.