MiR-155 Promotes FLT3-ITD-Induced Myeloproliferative Disease through Inhibition of Interferon Signaling

Introduction & Background: MicroRNA expression is dysregulated in many human cancers, including hematologic malignancies. Among hematologic malignancies, acute myeloid leukemia (AML) carries a particularly poor prognosis, leading to over 10,000 deaths each year. The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor. FLT3 internal tandem duplication (ITD) occurs in ~25% of all AML diagnoses, and confers a negative prognosis. MicroRNA expression has been shown to be dysregulated in FLT3-ITD+ AML, and miR-155 has been identified as the most highly overexpressed microRNA in this disease. MiR-155 is critical for the growth of FLT3-ITD+ cells in vitro, but the functional relevance of miR-155 in FLT3-ITD-mediated disease in vivo, and the downstream effects of miR-155 expression remain unclear.

Methods and Design: In this study, we crossed mice homozygous for the FLT3-ITD mutation with miR-155 knockout mice to determine the specific role of miR-155 in the context of FLT3-ITD. The FLT3-ITD mice develop a chronic myeloid malignancy that we evaluated between 4-6 months of age, and compared this phenotype between groups (FLT3-ITD 155+/+ vs FLT3-ITD 155-/-).

Results: FLT3-ITD miR-155-/- mice exhibited decreased myeloid expansion in the bone marrow, reduced splenomegaly, and decreased peripheral blood monocytosis and neutrophilia compared to their FLT3-ITD miR-155+/+ counterparts, indicating that miR-155 was playing a crucial role in promoting FLT3-ITD-mediated myeloproliferation. When examining the stem cell compartment of these animals, we found that miR-155 deficient animals had a reduced number of myeloid progenitors compared to their FLT3-ITD 155+/+ counterparts, suggesting miR-155 was critical for maintaining a robust myeloid progenitor pool in this disease. This phenotype was attributed to miR-155's role in promoting proliferation, but not survival, of the hematopoietic stem and progenitor cell (HSPC) and myeloid progenitor cell compartments in the bone marrow. RNA sequencing of the HSPC population in FLT3-ITD 155+/+ mice and FLT3-ITD 155-/- mice revealed that mice lacking miR-155 had an increased response to interferon, known to have a growth-suppressive effect on hematopoietic cells. We confirmed this finding through qPCR, where we found a number of interferon responsive genes were elevated in the HSPC and myeloid progenitor compartments of FLT3-ITD 155-/- mice compared to FLT3-ITD 155+/+ mice. We also observed increased protein levels of STAT1, a key transcription factor that activates interferon signaling, in the HSPCs and myeloid progenitors of FLT3-ITD 155-/- mice. These findings corresponded with human AML data from The Cancer Genome Atlas, where we found that FLT3-ITD+ AML samples had a decreased interferon signature compared to FLT3-WT AML samples. A number of putative miR-155 targets in the HSPCs are upregulated in the absence of miR-155, including PU.1, SHIP1, and CEBPB, all of which have previously been implicated as critical miR-155 targets in FLT3-ITD+ AML. Interestingly, CEBPB is known to regulate interferon responses.

Conclusions and Future Directions: Our study establishes miR-155 as a critical promoter of FLT3-ITD-mediated myeloproliferative disease in vivo, a finding we attributed to miR-155's inhibition of the interferon response in this context. Further work will determine which of miR-155's targets could be potentiating this effect. We will also focus on the importance of miR-155 in FLT3-ITD+ leukemogenesis, as FLT3-ITD mice need collaborating mutations to undergo leukemic transformation. These findings suggest that miR-155 inhibitors may warrant clinical consideration as therapeutics in FLT3-ITD+ AML.