Combined Therapy With BRD4 Antagonist and JAK Inhibitor Is Synergistically Lethal Against Human Myeloproliferative Neoplasm (MPN) Cells
Authors: Valenta John A , Fiskus Warren , Manshouri Taghi , Sharma Sunil , Qi Jun , Schaub Leasha J , Shah Bhavin , Rodriguez Melissa , Devaraj Santhana , Iyer Swaminathan Padmanabhan , Verstovsek Srdan , Bradner James E , Bhalla Kapil N. 
Published: October 2019

Abstract
Following therapy with type I JAK kinase inhibitors that exert activity only against the active JAK kinase, a lack of complete clinical or molecular response due to incomplete elimination of the MPN clone strongly supports the rationale to develop novel combinations against advanced MPN. Recent studies have demonstrated that despite continuing JAK inhibitor treatment, persistent JAK2 phosphorylation and the resulting heterodimeric JAK2 signaling may be responsible for JAK inhibitor-insensitive MPN persisters and resistance to therapy. In the present studies, we determined the activity of the BET (bromodomain and extraterminal) protein BRD4 antagonists JQI or I-BET151 with or without JAK inhibitors SAR302503 (SAR) and ruxolitinib (Rux) against human MPN cells that are sensitive or exhibit resistance to JAK inhibitors. BRD4 binds to acetylated lysines on the histone proteins, helps assemble transcriptional co-regulators at the target gene promoters and enhancers, and regulates the expression of important oncogenes, e.g., c-MYC, CDK6, BCL-2 and IL7R. Treatment with the BET protein inhibitor JQ1 disrupts the binding of the bromodomain of BRD4 to acetyl lysines in the chromatin associated with the oncogenes, thereby depleting their expression and inducing apoptosis of human leukemia cells. Our studies here demonstrated that JQ1 (250 to 1000 nM) but not its inactive enantiomer (R-JQ1) dose-dependently induced apoptosis of the MPN HEL92.1.7 (HEL) and UKE1 cells, as well as of CD34+ primary MPN cells harvested from the peripheral blood of patients with advanced MF. This was associated with decreased binding of BRD4 and RNA Pol II, by ChIP-qPCR analyses, to the promoters of c-MYC, BCL-2, CDK6 and IL7 receptor (IL7R), concomitantly with marked attenuation of the c-MYC, BCL-2, CDK6 and IL7R, but induction of p21 mRNA expression in HEL and UKE1 cells. I-BET151 (100 to 2000 nM) also exerted similar effects in MPN cells. JQ1 treatment also depleted the protein levels of p-JAK2, p-STAT5, p-STAT3, PIM1, BCL-2, BCL-xL, MCL and CDK4/6, while inducing p21 protein levels in the MPN cells. Following engraftment of NOD/SCID mice with HEL cell xenograft, co-treatment with JQ1 (50 mg/kg/day, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, administered IP daily x 5 days, per week x 3 weeks) and SAR (100 mg/kg/day, PO daily x 5 days per week x 3 weeks) versus treatment with vehicle control, or JQ1 or SAR alone, resulted in significant in vivo attenuation of c-MYC and BCL-2 levels in the harvested MPN cells from the mice (p < 0.01). Importantly, JAK inhibitor (SAR and Rux)-resistant HEL cells (> 10-fold resistant to Rux) created under the selection pressure of a continuous exposure to JAK inhibitor (HEL/JIR) (Clin Cancer Res 2011;17:7347) remained sensitive to JQ1-induced apoptosis. JQ1 was also equally effective in inducing apoptosis of primary MPN cells harvested from patients pre- and post-JAK kinase inhibitor therapy. JQ1 treatment attenuated the levels of c-MYC, p-STAT5, PIM1, CDK6 and BCL-2 in HEL/JIR cells. Co-treatment with JQ1 or I-BET151 and the JAK kinase inhibitor SAR or Rux synergistically induced apoptosis of the cultured HEL and UKE1 cells, with concomitant greater attenuation of the p-JAK2, p-STAT5, p-STAT3, PIM1, BCL-2, BCL-xL, MCL and CDK4/6 levels. Collectively, these findings demonstrate that BRD4 antagonists exhibit potent activity against cultured and primary human MPN cells expressing JAK2V617F. These findings also highlight the novel and synergistic activity of the combination of BRD4 antagonist and JAK inhibitor against human MPN cells, and support the rationale for in vivo testing of the combination against JAK inhibitor-insensitive MPN persisters that exhibit resistance to the first generation of JAK inhibitors.

Disclosures:
No relevant conflicts of interest to declare.