Transcriptomic and Molecular Insights into the Anti-Leukemic Activity of the Imiqualine EAPB04303 in Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is a complex heterogeneous hematological malignancy of myeloid origin. AML is one of the most common leukemias in adults, accounting for the majority of acute leukemia-related deaths. Despite significant therapeutic advances, AML still associates with high relapse rates. Some molecular subtypes associate with poor prognosis and resistance to conventional chemotherapy. Toll-Like Receptors (TLRs) are innate immune sensors expressed on immune cells and AML blasts. Imiqualines, structural analogs of the TLR agonist Imiquimod, are promising immunomodulatory drugs with proven activities against AML. Our team previously demonstrated that the second generation imiqualine derivative, EAPB02303, is metabolized into the active compound EAPB04303, and exhibit potency against all tested subtypes of AML. Yet, AML with Nucleophosmin 1 (NPM1) mutation responds better to this metabolite.

To further investigate this differential response, we used the OCI-AML3, harboring NPM1 mutation (NPM1c) which occurs in 30% of AML cases, and OCI-AML2, expressing wild-type NPM1 (NPM1 WT). Both cell lines were treated with EAPB04303 and RNA-sequencing was performed to identify differentially regulated molecular and immune pathways. Key findings were validated by western blotting, RT-qPCR, or immunofluorescence. The in vivo efficacy of EAPB04303 was evaluated in a patient-derived xenograft (PDX) mouse model of AML.

We showed that EAPB04303 differentially modulated TLR signaling and nucleolar pathways in NPM1c AML. RNA-sequencing revealed significant enrichment in immune and inflammatory pathways in treated OCI-AML3, including upregulation of TLR signaling components. EAPB04303 increased MYD88 expression and promoted NF-κB (RELA/p65) nuclear translocation, resulting in an enhanced expression of NF-κB-dependent pro-inflammatory cytokines. Moreover, EAPB04303 disrupted nucleolar integrity and altered the expression of nucleolar and ribosomal proteins, including Nucleolin and RPL5, exclusively in OCI-AML3 cells. In vivo, EAPB04303 significantly prolonged the survival of PDX-engrafted mice compared to untreated controls, with a more pronounced benefit observed in the NPM1c AML cohort.

Collectively, our results explain the higher sensitivity of NPM1c AML cells to EAPB04303 through combined activation of TLR-MYD88-NF-κB signaling and disruption of nucleolar function, supporting a promising immunomodulatory therapeutic strategy of this metabolite in NPM1c AML.