The IRAK4 long isoform as widely upregulated in non-splicesome mutated acute myeloid leukemia and as altered by hypomethylating agent therapy.

6524 Background: IRAK4, a kinase effector of MyD88 signaling downstream of Toll-Like Receptor and IL-1 receptor pathways, has recently been shown to function independently of MyD88 in Myelodysplastic Syndrome (MDS) and AML leukemic cells. Our previous research demonstrated that AML leukemic stem and progenitor cells (LSPC) rely on signaling through IRAK4. Notably, AML LSPCs express a hypermorphic long splice isoform of IRAK4 (IRAK4-L) resulting from the inclusion of exon 4. IRAK4 inhibitors, currently in clinical trials for the treatment of MDS and AML, show improved efficacy in spliceosome mutants, which preferentially express IRAK4-L. However, in patients without known spliceosome mutations, the extent of IRAK4-L expression remains unclear. Additionally, the impact of IRAK4-L levels in Venetoclax and Azacitidine treatment is unknown in refractory or unfit AML. Methods: Umbilical cord vein blood stem cells expressing MLL-AF9 and NRAS G12D (CD34+MA9.NRAS) were maintained in vitro using supplemented media. Venetoclax and Azacitidine doses were incrementally increased in combination until cells tolerated co-treatment with up to 1 µM of each compound with minimal cell death. Patient-derived xenograft (PDX) samples were obtained from the Cincinnati Children’s Biobank and included AML samples from diverse genetic backgrounds, relapsed/refractory cases, and pediatric populations (ages 1-20). Cell lysates were normalized to total protein and analyzed using a chemiluminescent capillary-based immunoblotting system, which requires minimal lysate quantities. An IRAK4 C-terminus antibody was used to detect both the shorter and longer IRAK4 protein isoform. RNA sequencing was performed, and transcripts were normalized per million counts. IRAK4 transcript ratios were calculated by dividing the total long isoform transcripts by the short isoform transcript for each condition in duplicate. Results: Analysis of PDX and human AML cell lines revealed that 8/9 PDX preferentially expressed the IRAK4-L isoform (>70% of IRAK4-L), with 4/9 PDX samples and 5/6 previously uncharacterized cell lines producing it almost exclusively (>95% IRAK4-L). MA9.NRAS cells predominantly express IRAK4-L, but transiently shifted to IRAK4-S during treatment with AZA/VEN (27% IRAK4-S), an effect reversed upon recovery from treatment. Conclusions: IRAK4-L is widely expressed in AML patient-derived samples, including those with complex karyotypes and TP53 mutations, suggesting that IRAK4 inhibitors may be useful beyond splicing factor-mutant MDS/AML. Our models demonstrate that changes in IRAK4-L expression during treatment with hypomethylating agents may be useful as a biomarker to guide therapy strategies. Further research is needed to understand how prolonged treatment and other standard of care therapies affect IRAK4-L expression and sensitivity to IRAK4 inhibitors.