Identification of the Epigenetic Reader BRD4 As a Novel Therapeutic Target in JAK2 V617F+ MPN Cells
Authors: Keller Alexandra , Peter Barbara , Zuber Johannes , Staber Philipp Bernhard , Bettelheim Peter , Valent Peter , Hadzijusufovic Emir 
Published: October 2019

Abstract
Myeloproliferative neoplasms (MPN) are characterized by clonal expansion and accumulation of erythrocytes, platelets, and myeloid cells in the bone marrow (BM) and other organs. Classical MPN are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2 V617F mutation is frequently detected in neoplastic cells in patients with MPN. Although MPN are chronic and indolent diseases in most patients, fatal progression may occur. So far, the only curative approach for these patients is hematopoietic stem cell transplantation. Therefore, current research is evaluating new therapeutic targets and the effects of various targeted drugs. The epigenetic reader bromodomain-containing protein 4 (BRD4) has recently been identified as a promising target in acute myeloid leukemia. In the present study, we investigated the potential value of BRD4 as a molecular target in MPN. We employed two JAK2 V617F+ cell lines, SET-2 and HEL, as well as BM samples obtained from 18 MPN patients (ET: n=7; PV: n=7; PMF: n=4). Three BRD4 inhibitors were applied: JQ1, BI2536, and BI6727. As assessed by qPCR, primary MPN cells as well as SET-2 cells and HEL cells were found to express BRD4 mRNA. In 3 H-thymidine uptake experiments, all three BRD4 blockers were found to suppress the proliferation of the two MPN cell lines and of primary MPN cells in 8/8 patients tested. The effects of these drugs were dose-dependent, with the following IC50 values obtained in SET-2 cells: JQ1, 50-100 nM; BI2536, 20-40 nM; BI6727, 50-75 nM; and in HEL cells: JQ1, 100-500 nM; BI2536, 20-40 nM; BI6727, 30-50 nM. In primary MPN cells, all three agents tested produced IC50 values between 500 and 1000 nM. In normal BM cells, JQ1 did not produce a reasonable IC50 value (>5000 nM). In one patient sample (PMF), we analyzed the effect of JQ1 on the percentage of putative (neoplastic) stem cells (CD34+/CD38-). In this experiment, exposure to JQ1 was followed by a decrease in the percentage of CD34+/CD38- cells compared to control medium (control: 0.16% vs JQ1: 0.045%). To confirm the role of BRD4 as a potential target in MPN cells, we performed target-knockdown experiments in SET-2 cells and HEL cells using two different BRD4 shRNAs (#602 and #1817) and a random shRNA as control. In these experiments, the shRNA-induced knockdown of BRD4 was found to block proliferation in SET2 cells and HEL cells when compared to untransfected cells or random shRNA-transduced cells. In a next step, we examined the mechanism of drug-induced growth inhibition. In cell cycle experiments, BI2536 and BI6727 were found to induce a G2/M phase arrest in both cell lines. By contrast, JQ1 induced a G1 arrest in HEL cells, but did not show a significant effect on cell cycle progression in SET-2 cells. We also asked whether BRD4 inhibition is associated with induction of apoptosis in MPN cells. All three BRD4 blockers induced apoptosis in SET-2 cells and HEL cells at relatively high concentrations after 48 hours, with ED50 values of >5 µM for JQ1 and 0.5-5.0 µM for BI2536 and BI6727. Finally, we asked whether exposure to BRD4 inhibitors is associated with modulation of BRD4 mRNA or MYC mRNA expression. As assessed by qPCR, JQ1, BI2536, and BI6727 were found to downregulate BRD4 mRNA levels as well as MYC mRNA levels in SET-2 cells and HEL cells. In conclusion, our data show that BRD4 is expressed in JAK2 V617F+ MPN cells and that BRD4 inhibition is associated with decreased proliferation and survival of neoplastic cells. The clinical value of BRD4 as a novel target in MPN cells remains to be determined.

Disclosures
Zuber: Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Staber:Genactis: Research Funding; Morphosys: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda-Millenium: Research Funding; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Valent:Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.