Single Molecule Dual JAK2-BET Inhibition As an MPN Therapeutic
Authors: Lambert Que T. , Ember Stuart W. , Ayaz Muhammad , Lawrence Harshani R. , Berndt Norbert , Gunawan Steven , Lawrence Nicholas J. , Schönbrunn Ernst , Reuther Gary W. 
Published: October 2019

Abstract
The optimism of anti-JAK2 therapy for the treatment of MPNs is largely based on the ability of JAK inhibitors to improve MPN patient constitutional symptoms. Unfortunately, such inhibitors are generally unable to induce remission or even allele burden, suggesting anti-JAK2 based therapies need refinement and optimization. Combination therapies continue to be widely investigated, however, such approaches could have significant complications when translating to patients. These include the difficulty determining proper dosing of each drug in combination in patients, the cost of treatment, and issues with combining drugs developed by different pharmaceutical companies.
Recently, we and others have identified anti-BET bromodomain inhibitory properties of various kinase inhibitors, including the JAK2 inhibitors TG101209 and TG101308. Bromodomains are protein-protein interaction motifs that bind to acetylated lysine and, for example, can play roles in regulating gene expression by binding acetylated histones. These dual kinase-BET inhibitors bind to the acetylated lysine-binding pocket of BET bromodomains and thus inhibit the function of such domains. Importantly, cancer cells appear to be particularly sensitive to BET inhibitors compared to normal cells. Combining a BET inhibitor and a JAK2 inhibitor has been shown to be more effective against MPN cells than JAK2 inhibition alone.
Our recent identification of dual kinase-BET inhibitors allows for the rational design of drugs with polypharmacology to inhibit more than one class of targets. To this end, we have optimized small molecules for dual anti-JAK2 and anti-BET activity. MA2-014 is a chemical derivative of TG101209 that exhibits similar anti-JAK2 activity, but about ten-fold improved anti-BET activity than TG101209. In fact, the activity of MA2-014 to target BET domains is similar to the prototypical BET inhibitor JQ1. For example, the IC50s of MA2-014 and JQ1 against the second bromodomain of the BET family member BRD4 are each about 20 nM. MA2-014 retains comparable biochemical activity against JAK2 as TG101209 and ruxolitinib (IC50s of low single digit nM, ~0.5 to 3 nM). In MPN cells, however, the ability of MA2-014 to inhibit JAK2-V617F signaling in MPN cells, as measured by P-STAT5, is about ten-fold improved over TG101209, and is comparable to ruxolitinib. Likewise, the ability of MA2-014 to inhibit the expression of c-Myc, which is widely used as a biomarker for BET inhibition, is about ten-fold better than TG101209 in MPN cells. The IC50 for MA2-014 for growth of Uke1 MPN cells is about 200 nM, compared to 500 nM for TG101209. Taken together, these data suggest that MA2-014 is a dual JAK2-BET inhibitor that exhibits superior BET inhibitory activity with similar if not better cellular JAK2 inhibitory activity than TG101209. MA2-014 efficiently inhibited the erythropoietin independent erythroid colony formation of myeloid progenitors from MPN patients, which is a hallmark of these cells and is widely used to test MPN therapeutics. The IC50 of MA2-014 in this assay is 50 nM, essentially identical to ruxolitinib, the only FDA approved JAK2 inhibitor for MPNs. The IC50 of TG101209 to inhibit this colony formation of primary MPN cells is 200 nM, four times greater than MA2-014. Interestingly, JAK2-V617F-driven MPN model cells that are resistant to ruxolitinib retained sensitivity to MA2-014. The IC50s of ruxolitinib and MA2-014 against the growth of BaF3-JAK2-V617F cells are about 100 nM. However, the same cells that are resistant to ruxolitinib (IC50 >2000 nM) remain sensitive to MA2-014 (IC50 of 240 nM). Finally, in long-term culture assays, we have determined that JAK2-V617F driven MPN Uke1 cells are not able to become resistant to MA2-014 as readily as they do to TG101209 or ruxolitinib. These data suggest MA2-014 may be more resilient to drug resistance, the major hurdle in the clinical effectiveness of JAK2 inhibitors.
Collectively, our work demonstrates that rationally-designed polypharmacology may be a novel approach to develop effective therapeutics for cancer, especially diseases that are driven by aberrant kinase signaling and are also sensitive to BET inhibition, as exemplified by MPNs. The use of such an optimized polypharmacologic therapeutic may provide the benefits of combination therapy with fewer complications associated with clinical development.

Disclosures
No relevant conflicts of interest to declare.