Histone Deacetylase Inhibitors Induce microRNAs Targeting BTK in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a highly heterogeneous disease primarily affecting the elderly. Despite considerable progress in understanding the biology of AML, the prognosis of the majority of patients still remains poor due to lack of progress in improving therapy over the past 3 decades, thus creating the need to identify new pharmacological strategies.

Bruton's Tyrosine Kinase (BTK) has been demonstrated to be functionally important in a variety of hematological malignancies and several groups have recently shown that BTK is highly expressed and constitutively active in AML cell lines and in a subset of AML patients. Moreover, treatment with ibrutinib, a covalent irreversible inhibitor of BTK, can reduce proliferation, blast adhesion to bone marrow stromal cells and migration of both AML cell lines and primary AML blasts. Validation of this target also occurred with shRNA experiments confirming the specificity of BTK for growth inhibition of AML.

Dysregulation of microRNA (miRNA) expression contributes to the pathogenesis of numerous human malignancies including AML, where multiple miRNAs have been associated with specific cytogenetic and molecular subsets of the disease.

Using a target prediction algorithm we identified several miRNAs as potential regulators of BTK (miR-147b, miR-210-3p, miR-425-5p, miR-1253, miR-4269, miR-4667-3p). We performed 3′UTR luciferase reporter assays in the AML cell line OCI-AML3 and found that relative luciferase activities were significantly decreased (20-30%) in cells transfected with miR-147b, miR-425-5p, miR-4269 and miR-4667-3p mimics. Flow cytometry and western blotting analysis confirmed that BTK protein expression was significantly down-regulated by all the predicted miRs except miR-1253.

Previous data from our lab have shown that histone deacetylase (HDAC) 1 and 2 become recruited to multiple microRNA promoters, including the ones targeting BTK. To determine the consequence of HDAC inhibition on BTK signaling, we exposed primary blasts from 14 AML patients to 10nM of the pan-deacetylase inhibitor panobinostat (LBH589) for 48h. Our results showed that panobinostat induced a 2-23 fold increase in the expression miR-147b, miR-210-3p, miR-425-5p, and miR-4667-3p in most of the patients and a time-dependent depletion of total BTK protein and downstream BTK signaling (phospho- and total ERK and phospho- and total AKT) in about half of them. BTK protein at 24h directly correlated with BTK and HDAC1 mRNA expression at 24h and miR-210 induction at 24h inversely correlated with BTK mRNA levels at 24h. Moreover, 2 days of incubation with 10nM panobinostat is able to induce robust cell death in AML primary samples (9.6-92% apoptosis).

We hypothesized that HDAC inhibitors would cooperate with kinase inhibitors of BTK to synergistically abolish BTK signaling and induce death in AML cells. Therefore, we treated OCI-AML3 and MV4-11 cell lines with 0.40 µM abexinostat, 1 µM ibrutinib for 2h alone followed by washout, or a combination of 0.40 µM abexinostat with 1 µM ibrutinib for 2h, and viability was measured at 48h. The combination of abexinostat and ibrutinib induced greater than additive cytotoxicity compared to either abexinostat or ibrutinib alone. Exposure to ibrutinib alone for 2h followed by washout inhibited the kinase activity of BTK (measured by the auto-phosphorylation on Y223) but did not affect total BTK protein. However, the combination of abexinostat and ibrutinib showed both loss of BTK kinase and decrease in total BTK protein, in addition to attenuation of BTK signaling, thus confirming the dual targeting of BTK via independent mechanisms.

Our results provide the rationale for the clinical evaluation of ibrutininb in combination with histone deacetylase inhibitors in AML patients.