Combination Strategies to Target Super Enhancer Transcriptional Activity by CDK9
and BRD4 Inhibition in Acute Myeloid Leukemia
Brigham L. Bahr, Kyle S. Maughan, Katherine K. Soh, Jeremiah J. Bearss, Wontak Kim, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven L. Warner, David Bearss
Tolero Pharmaceuticals, Inc., Lehi, UT
I. Abstract
III. Results
The super enhancer complex (SEC) is a group of transcrip9on regulatory proteins that coordinate the expression of gene9c programs which determine cell iden9ty and drive disease states, such as cancer. In acute myeloid leukemia (AML), SECs have been shown to turn on transcrip9onal programs that drive tumorigenesis and disease progression. The SEC is replete with poten9al therapeu9c targets that have been the focus of many drug development efforts; including cyclin-­‐dependent kinases (CDK), bromodomain proteins (BRD), histone deacetylases (HDAC), and histone methyltransferases (HMT). SEC-­‐regulated transcrip9on begins as CDK9/cyclin T1 is recruited from an inhibitory complex by BRD4 and brought to the transcrip9onal start site of genes. CDK9 phosphorylates RNA polymerase II, releasing it from the SEC and leading to transcrip9onal elonga9on and gene expression. Considering the close associa9on of CDK9 and BRD4, we hypothesized that the combina9on of CDK9 and BRD4 inhibitors would have synergis9c effects, par9cularly in AML, a disease largely driven by SEC func9on. Alvocidib is a potent CDK9 inhibitor with validated clinical ac9vity in AML from mul9ple Phase II studies in over 400 pa9ents. Addi9onally, BRD4 inhibitors have demonstrated early promise in clinical studies with a focus on AML. We found that CDK9 inhibitors combined with bromodomain inhibitors produced a synergis9c effect by inhibi9ng the SEC more effec9vely than either of these compounds alone. For example, cell viability studies with various combina9ons resulted in an increase in potency. This was observed with alvocidib combined with JQ-­‐1 (BRD4 inhibitor) in MV4-­‐11 AML cells. Furthermore, the combina9on of alvocidib with JQ-­‐1 completely abrogated SEC func9on, as measured by c-­‐myc expression through RT-­‐qPCR. Similar results were achieved with other combina9ons of CDK9 and BRD4 inhibitors. The alvocidib and JQ-­‐1 combina9on was also evaluated in an MV4-­‐11 mouse xenogra\ model. As single agents, alvocidib (2.5 mg/kg) exhibited a 44% tumor growth inhibi9on and JQ-­‐1 (25 mg/kg) a 1% growth inhibi9on. When these two doses were combined there was 100% tumor growth inhibi9on. These data, primarily focused on alvocidib and JQ-­‐1, suggest a strong ra9onal for combining CDK9 and BRD4 inhibitors as a treatment strategy for AML. Furthermore, these findings could be more broadly applied to addi9onal therapeu9c targets in the SEC, such as DOT1L and HDACs. These strategies yield synergis9c effects at inhibi9ng SEC func9on and are highly ac9ve in tumor growth studies of AML in vivo. Clinical studies u9lizing these combina9on strategies are the next steps to further explore this approach. Figure 2. Alvocidib reduces expression of MYC and MCL-­‐1. II. Background
» Alvocidib has been used to treat over 400 AML pa8ents in clinical trials employing single agent and combina8on regimens. » In clinical trials, an alvocidib-­‐containing regimen has demonstrated the highest CR rate of any therapeu8c agent or combina8on regimen to date in high risk AML pa8ents. » Alvocidib is a known CDK9 inhibitor. » A pivotal Phase III trial is planned this year to posi8on alvocidib for approval. Figure 1: Diagram showing the super enhancer complex in its transcrip8onally inac8ve and ac8ve forms. Figure 6. Combina8on of alvocidib and JQ1 completely inhibits tumor growth in MV4-­‐11 mouse xenograX. Athymic Nu/Nu mice were subcutaneously injected with MV4-­‐11 cells and treated with alvocidib (2.5mg/kg), JQ1 (25mg/kg), or a combina9on of the two. C: Quan9fica9on of western blots shows that the combina9on of alvocidib (0.1µM) and a BRD4 inhibitor (0.1µM) reduces MYC expression bejer than individual treatments. A: MV4-­‐11 cells treated with alvocidib for 2hr completely eliminates MYC expression. B: Alvocidib reduces MCL-­‐1 expression by 60% in MV4-­‐11 cells. Figure 3: Alvocidib combined with BRD4 inhibitors increases apoptosis in MV4-­‐11 cells. MV4-­‐11 cells were seeded 1,000 cells per well in 384 well plates. BRD4 inhibitor was added at a single dose to cells for 24hr. Alvocidib was added at 24hr in a dose dilu9on. Cell Titer GLO and Caspase GLO were added and luminescence was measured at 48hr. Single doses of BRD4 inhibitors combined with alvocidib in a serial dilu9on demonstrated a synergis9c increase in caspase ac9vity, indica9ng that alvocidib combined with BRD4 inhibitors synergis9cally increases apoptosis in MV4-­‐11 cancer cells when compared with alvocidib or individual BRD4 inhibitors alone. Figure 5. Alvocidib increases associa8on of CDK9 and Hexim1 proteins in a 8me and dose dependent manner. MV4-­‐11 cells were treated in both a 9me and dose dependent manner to determine if alvocidib would increase associa9on of CDK9 and Hexim1, and thus prevent phosphoryla9on of RNA Pol II. A: 1µM alvocidib increases associa9on of CDK9 and Hexim1 in a 9me dependent manner. MV4-­‐11 cells were seeded and treated in iden9cal concentra9ons and doses. Cells were collected at given 9mes and subjected to high salt lysis buffer and immunoprecipita9on followed by western blogng. Representa9ve western blot is on the le\ and quan9fica9on of the blot is on the right. Figure 4. Alvocidib combined with BRD4 inhibitors reduces MYC expression in MV4-­‐11 cells be^er than individual treatments. MV4-­‐11 cells were seeded and treated (3hr) with alvocidib, a BRD4 inhibitor, a combina9on of alvocidib + BRD4 inhibitor, or no treatment. B: Body weight for all animals was comparable throughout the study. Vehicle and individual treatment mice weight gain could be ajributed to tumor growth. Weight gain in the mice given the combina9on treatment could be ajributed to the overall health and food intake of the mice. IV. Conclusions
B: Different doses of alvocidib alter CDK9 and Hexim1 associa9on at the half hour 9me point. A 500nM dose of alvocidib demonstrated an increased CDK9/Hexim1 associa9on. Representa9ve western blot on the le\ and quan9fica9on of the blot on the right. A: MV4-­‐11 cells were seeded (1 million cells) in 6-­‐well plates. A\er treatment, cells were analyzed by RT-­‐PCR. The combina9on of alvocidib and a BRD4 inhibitor reduced MYC expression bejer than individual treatments. RNA Pol II awaits ac9va9on from the recruitment of P-­‐TEFb by BRD4. Inac9ve P-­‐TEFb consists of the two proteins (CDK9 and CyclinT1) and is bound to Hexim1. Ac9ve P-­‐TEFb is uncoupled from Hexim1, and when aided by BRD4, phosphorylates RNA Pol II. This allows for transcrip9on of various oncogenes like MYC and MCL-­‐1. By combining alvocidib (CDK9 inhibitor) and BRD4 inhibitors, super enhancer transcrip9on is blocked, greatly reducing expression of oncogenes like MYC. A: Neither individual treatment was able to completely inhibit tumor growth a\er 19 days. The combina9on of alvocidib and JQ1 completely stopped tumor growth by day 8. B: MV4-­‐11 cells were seeded (3 million cells) in T75 flasks and treated for 3 hr. Cells were analyzed by western blogng which showed a decrease in MYC expression for all treated samples. Tolero Pharmaceuticals, Inc., 2975 W. Executive Parkway Suite #320 Lehi, UT 84043
C: 2hr treatment with different doses of alvocidib demonstrates a dose dependent increase in CDK9/Hexim1 associa9on. Representa9ve western blot on the le\ and quan9fica9on of the blot on the right. The following conclusions can be drawn from the above data: » Preliminary data suggests that alvocidib can reduce expression of MYC and MCL-­‐1. » Alvocidib/BRD4 inhibitor combina8ons synergis8cally increase caspase ac8vity in MV4-­‐11 cells indica8ng synergis8c apoptosis ac8vity compared to individual doses. » Western bloRng and RT-­‐PCR shows the combina8on of alvocidib and BRD4 inhibitors synergis8cally inhibit the expression of MYC. » Alvocidib increases the associa8on of CDK9 and Hexim 1, which prevents p-­‐TEFb from combining with BRD4 and ac8va8ng super-­‐enhancer transcrip8on. » Combina8on of alvocidib and JQ1 in a mouse xenograX study synergis8cally and completely eradicates tumor growth while not affec8ng body weight (overall health). This data supports the use of alvocidib as the backbone of future combina8on clinical studies in AML, specifically the combina8on of alvocidib and BRD4 inhibitors in clinical trials targe8ng AML.