Abstract 1131: IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia

Abstract

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that occurs disproportionately in the elderly population. First line therapy is a regimen of anthracycline and cytarabine treatment or, more recently, demethylating agents. However, elderly AML patients are unable to tolerate aggressive chemotherapy regimens and there is an urgent need for targeted, tolerable therapies. To identify candidate pathways for targeted treatments, a microarray on transcripts for inflammatory signaling was performed on 588 primary patient AML samples. Non-hierarchical clustering revealed that Toll-like receptor (TLR) signaling transcripts are overexpressed in myelomonocytic and monocytic AML subtypes (M4 and M5 AML subtypes, respectively). Myeloid differentiation primary response 88 (MYD88) and downstream IL-1 receptor-associated kinases (IRAKs) were found to be overexpressed in these AML subtypes. MYD88 is an adaptor molecule for IL-1 and most TLR mediated signaling. To determine whether MYD88-dependent signaling is required for leukemogenesis, we derived a CreERT2-LoxP inducible system to delete Myd88 in a murine leukemia model. Because M4 and M5 AML subtypes are enriched for mutations in the mixed lineage leukemia (MLL) gene and overexpressed TLRs, MYD88, and IRAK2, an MLL-AF9 expressing construct was utilized to transform murine progenitor cells into leukemia cells. To study whether Myd88-dependent signaling is required for in vitro colony formation of the leukemic cells, Myd88 knockout (Myd88-/-) MLL-AF9 leukemic cells were subjected to colony-forming assays. Compared to Myd88 wild-type (Myd88fl/fl and Myd88+/+) leukemia cells, reduced colony-forming capacity was observed in the Myd88-/- leukemia cells. Further, reduced proliferation was observed in Myd88-/- cells compared to Myd88+/+ cells. Cell surface staining indicates Myd88-/- cells exhibit a loss of CD117, a marker of myeloid stemness, and a gain of CD11b, a marker of myeloid differentiation, when compared to Myd88+/+ cells, indicating that Myd88 deletion correlates with MLL-AF9 leukemia cell partial differentiation. In vivo transplantation of Myd88-/- cells delays leukemia development compared to Myd88+/+ cells. To determine whether IRAK1, a downstream kinase of MYD88, is also required to prevent partial differentiation, an IRAK1 shRNA knockdown cell line was generated and assayed for partial differentiation. In IRAK1KD MLL-AF9 cells, no partial differentiation was detected. Similarly, application of an IRAK1/4 inhibitor to Myd88+/+ MLL-AF9 cells failed to induce partial differentiation, despite suppressing proliferation. This indicates that MYD88-dependent signaling has functions independent of IRAK1 signaling that prevent partial differentiation in MLL-AF9 leukemia cells. Thus, MYD88 and IRAK1 signaling appear to mediate divergent functions, and both MYD88 and IRAK1 function appear to be required for MLL-AF9 leukemia. Citation Format: Joseph M. Cannova, Wei Wei, Rafael Gutierrez, Peter Breslin, Jiwang Zhang. IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1131.