Abrogating Treatment Resistance By Mir-451 in FLT3-ITD-Positive Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a fatal malignant hematologic disorder. Although many patients with AML initially respond to standard chemotherapy and achieve complete remission (CR), the majority of patients relapse due to the development and clonal expansion of chemoresistant subclones. Albeit being a heterogenous disease, individual mutations are still scarcely considered in the current treatment regimen.

Approximately 30% of AML patients exhibit gain-of-function mutations in the FMS-like tyrosine kinase 3 receptor (FLT3-receptor) gene. Here, an internal tandem duplication (ITD) in the juxtamembrane region of the receptor is among the most frequently observed mutations. This usually entails a constitutive activation of FLT3 receptor downstream signaling pathways, leading to differential gene expression patterns and aberrant cell growth. Clinically, FLT3-ITD mutations confer adverse prognosis. Thus, many promising FLT3 signaling inhibitor-based therapies are currently utilized to improve patient survival. However, the phenomenon of treatment resistant subclones persists and constantly calls for new angles of treatment.

MicroRNAs, a group of small non-coding RNAs, regulate gene expression on a posttranscriptional level by a complementary binding to 3'UTRs of protein-coding mRNAs. They have been shown to influence healthy and malignant hematopoiesis and to predict treatment response in cancer. In addition, several candidates have been connected to treatment resistance or sensitivity in AML.

Within this study, we hypothesized that microRNAs are biological important players in the adverse treatment response and the development of chemoresistance in FLT3-ITD associated AML.

Initially, we were interested in the microRNA expression pattern in FLT3-ITD associated AML. Therefore, we performed a next generation sequencing screen in bone marrow mononuclear cells from healthy donors, AML patients with FLT3-wildtype and AML patients with FLT3-ITD mutation. Here, we identified miR-451 to be significantly lower expressed in AML compared to healthy individuals. Furthermore, it was nearly absent in FLT3-ITD associated AML. In addition, we compared our results to the AML patient cohort of "The Cancer Genome Atlas" (TCGA) and found a similar miR-451 expression distribution. In agreement with this, miR-451 expression increased during differentiation of primary human and mouse hematopoietic cells. Moreover, transduction of U937 as well as 32D cells with FLT3-ITD led to a decrease in miR-451 expression.

Downregulation of miR-451 has been shown for various cancerous diseases to correlate with an increased cancer persistence as well as recurrence, as one established target of miR-451 is multi drug resistance protein 1 (MDR1). MDR1 is an ATP dependent efflux pump that mediates chemoresistance by transporting drugs out of the cell. Today, only limited data are available on the miR-451-MDR1 axis in FLT3-ITD associated AML. Thus, we hypothesized that low miR-451 expression and high MDR1 activity is associated with treatment resistance.

Therefore, we showed an inverse correlation of miR-451 expression and MDR1 protein levels in various leukemic cells lines. Furthermore, we observed that a repression of FLT3-ITD signaling by both Quizartinib and Gilteritinib causes a miR-451 expression increment and a subsequent reduction of MDR1 protein levels. By luciferase activity assay, we could demonstrate a direct binding of miR-451 to the 3'UTR of MDR1. In order to investigate whether miR-451 alone is able to abrogate treatment resistance, we overexpressed miR-451 in both normal MV4;11 and treatment resistant MV4;11 cells (MV4;11-R). Subsequently, MDR1 protein levels were decreased and cells exhibited an enhanced sensitivity towards both FLT3-ITD inhibitor and chemotherapeutic agent-based treatment. Moreover, exogenous miR-451 was able to restore drug sensitivity in MV4;11-R cells comparable to that of normal MV4;11 cells. As proof of principle, MDR1 inhibitor Tariquidar mimicked miR-451-mediated effects and sensitized MV4;11-R cells to FLT3-ITD inhibitor Quizartinib.

In conclusion, we identified a novel mechanism by which FLT3-ITD is able to alter treatment response and confer a poor prognosis. Hence, not only inhibiting FLT3-ITD but mimicking the effects of negatively regulated downstream targets such as miR-451 presents as promising new avenue for FLT3-ITD AML treatment.