Targeting BTK By a microRNA Mechanism in Chronic Lymphocytic Leukemia
Authors: Bottoni Arianna , Lara Rizzotto , Tzung-Huei Lai , Mantel Rose , Simith Lisa , El-Gamal Dalia , Johnson Amy J. , Lapalombella Rosa , Blachly James S. , Byrd John C. , Woyach Jennifer A. , Sampath Deepa 
Published: October 2019

Abstract
The B-Cell Receptor (BCR) pathway is overactive and plays a crucial role in the survival, proliferation and trafficking of chronic lymphocytic leukemia (CLL), the most common leukemia in adults. Brutons agammaglobulenemia tyrosine kinase (BTK) is a key downstream component of the BCR pathway and targeting BTK with ibrutinib, an orally bioavailable irreversible inhibitor of BTK has had remarkable success in relapsed CLL including in patients with high risk genetic features. However, therapy-induced point mutations affecting BTK p.C481, which convert ibrutinib from an irreversible inhibitor to a less potent reversible inhibitor, have emerged in a subset of patients with high risk genomic factors. Alternative strategies are therefore needed to prevent and treat ibrutinib-resistant disease for these high risk patients. Micro-RNAs (miRNAs) are small non coding RNAs that are aberrantly expressed in many cancers including CLL. We have previously shown that epigenetic silencing, due to the overexpression of HDACs in CLL, was responsible for the low expression of specific miRNAs in CLL.
In this study we identified a key set of microRNA (miR147b, miR-210, miR425, miR1253, miR4267 and miR4667-3p) that targeted BTK. Ectopic expression of each individual miRNA reduced BTK expression by 30-50% whereas a pool of these miRNA reduced BTK by 80%. When the expression of the BTK targeting microRNAs was compared in the CLL cells from 80 patients with a pool of healthy B-cells (5 donors) we found that CLL cells expressed lower levels of the BTK targeting microRNA compared to healthy B cells. Chromatin immunoprecipitation followed by deep sequencing as well as real time-PCR experiments identified that a gene repressive complex, consisting of HDAC1, HDAC2 and KDM1, became recruited to the promoters of these microRNA to silence their expression. Consequently, targeting the HDACs with the novel 2nd generation HDAC inhibitor Abexinostat induced the expression of BTK targeting miRNAs by 5-200 fold with reciprocal decreases in the levels of BTK protein. Similarly RNAi against HDAC1 induced the BTK targeting miRNA as well as decreased BTK in primary CLL cells as well as in Mec2, a CLL cell line. We then demonstrated that HDAC inhibition with Abexinostat synergized with ibrutinib to dually target BTK (where abexinostat decreased total BTK protein and ibrutinib inhibits kinase activity of remaining BTK to achieve a more complete inhibition of BTK signaling and synergistic cytotoxicity. Importantly, we also demonstrated that HDAC inhibitors retain the ability to induce the BTK targeting microRNA and reduce BTK protein in ibrutinib-resistant CLL cells that developed the mutant p.C481S BTK.
Together, our results show that the over-expression of HDACs in CLL causes epigenetic silencing of BTK targeting miRNAs leading to a higher expression of BTK in these patients. HDAC inhibition restores the expression of these miRNAs which in turn target BTK effecting a quantitative reduction. Since Abex is well toleratd in phase 1 clinical trials for heme malignancies, it makes an attractive therapeutic strategy to target BTK as part of a combination therapy as well as in Ibrutinib resistant patients.

Disclosures
Byrd: Acerta Pharma BV: Research Funding.