Evaluating synergy of belinostat and ribociclib in triple-negative breast cancer
Abstract
Purpose:
Triple Negative Breast Cancer (TNBC) has a poor prognosis due to higher rates of metastases and a lack of targeted treatments. Emerging targeted therapies currently being studied include Histone Deacetylase Inhibitors (HDACi) and Cyclin Dependent Kinase Inhibitors (CDKi). We hypothesize that a synergistic effect will occur in the treatment of TNBC when using Belinostat (an HDACi) and Ribociclib (a CDKi). We propose that the mechanism of synergy between Belinostat and Ribociclib is through the Retinoblastoma protein (Rb) pathway. Evidence suggests that HDACi increases levels of endogenous CDKi thereby increasing the level of active Rb which arrests cell cycle progression from the G1 to the S phase.
Methods:
In this study, we used five hormone receptor-positive (HR+) breast cancer cell lines and five TNBC cell lines. 1,000 cells of each line were plated per well and grown in 3-D culture in a 96 well plate. Cells incubated for 24 hours until spheroids formed. Six doses of Belinostat and Ribociclib from 0.316 to 31.6 micromolar and nine combination doses were added to the cells. After 72 hours of drug incubation, CellTiter GLO was used to determine cell viability by measuring ATP. Dose-response curves for the individual drugs were obtained and the Bliss Independence Equation was used to determine if synergy was achieved with the drug combinations. Synergy within a cell line was determined if at least 4 of the 9 combination doses in each line showed synergy through the Bliss Independence Equation.
In order to delve into the proposed mechanism, we looked at Rb levels of each cell line. 8,000 cells of each line were plated on their own 96 well plate. After spheroids formed, drugs were treated with the 50% Effective Concentration (EC50) dose of Belinostat and Ribociclib (both separately and in combination) or DMSO for 24 hours. Cells were then lysed and protein from each sample was collected. 15 nanograms of protein from each sample were run by gel electrophoresis. Western Blots were performed to view levels of Rb.
Results:
Dose-response curves were obtained for all ten cell lines. We found that synergy was achieved in one out of five HR+ cell lines (T47D cell line) and four out of five TNBC cell lines (HCC38, HCC70, HCC1806, and HCC1937 cell lines).
Based on the obtained EC50, the combination doses used to determine levels of Rb were 0.316 micromolar of Belinostat and 3.16 micromolar of Ribociclib. Rb levels were elevated with drug treatment in three of the five TNBC cell lines (HCC38, HCC1806, and MDA-MB 231) indeterminate in one cell line (HCC70) and nonexistent in the last (HCC1937). Genetic data shows that the HCC1937 cell line has a genetic mutation in the Rb gene thereby explaining the lack of the protein in our Western Blots. Rb levels were elevated with drug treatment in four of the five ER+ cell lines (MCF7, BT483, T47D, and HCC1428) and indeterminate in one cell line (CAMA-1).
Conclusion:
Cell lines that showed synergy and an increase in Rb were the T47D (ER+), HCC38 (TNBC), and HCC1806 (TNBC) cell lines. Even with a lack of Rb, synergy was still seen in the HCC1937 cell line (TNBC).
From our results, we can see synergy in TNBC cell lines. However, this synergy does not seem to correlate solely with a rise in Rb protein levels. Other mechanisms for synergy between Belinostat and Ribociclib may exist and need further exploration to find other potential biomarkers. From these experiments, we conclude that synergy is commonly seen in TNBC cells and that further clinical trials with Belinostat and Ribociblib warrant investigation.
Published in College of Pharmacy, Virtual Poster Session Spring 2020
Good work, Taylor. Looking back on your experience with the Pharma project, what was one thing you learned that might help you in your future career?
Dr. Witt,
I think the biggest takeaway from my project is the planning and time management that go into a successful project. My project didn’t come together without effort and my career won’t either. I feel that those general principles can apply to any part of my future career, but they still have left the biggest impact on me. Thanks for the question!
Thanks,
Taylor
Your poster looks great Taylor! Do you have any guesses about what could be causing the synergistic effect (other than the rise in Rb)?
Hey Kacey!
So the model that we proposed to explain synergy between belinostat and ribociclib is super oversimplified. The truth is that Rb isn’t just active or inactive, but rather there are many states in between. There are also many different interactions that occur with CDK, HDAC, and Rb that can also affect the way Rb functions so I still believe that it has to do with the more complex parts of the Rb pathway that we oversimplified in our model. I hope that answers your question!
Thanks,
Taylor
Interesting findings. How does one go about determining why synergy occurred (since it’s not solely by the mechanism you predicted)?
Great job!
Dr. Lim,
In order to determine the true mechanism of synergy, we would have to do many more Western Blots. We definitely oversimplified our prediction model and know that there are many other interactions between proteins within the Rb pathway. However, to make it easier to decipher, these two drugs will be used in a clinical trial soon where transcriptomics and single-cell analysis will help narrow down a possible mechanism for synergy. I hope that answers your questions!
Thanks,
Taylor
Well done, Taylor. Do you have any ideas why certain combinations of the drugs are synergistic for some TNBC lines, yet antagonistic in others. Can you use knowledge about the differences between those different lines to help decipher the mechanisms underlying synergy?
Dr. Keefe,
We know that there are differences in Rb expression between the different TNBC cell lines (for example the HCC 1937 cell line has a mutation in its Rb protein). We also know that our model for synergy within the Rb pathway is oversimplified as there are many other interactions between Rb, CDKs, and HDAC. So from what we have researched so far, we’re not sure what is causing the difference. However, these two drugs will be used in a clinical trial soon where transcriptomics and single-cell analysis will help narrow down a possible mechanism for synergy. I hope that answers your questions!
Thanks,
Taylor
Hi Taylor,
What an interesting study. Do you think this synergy could be clinically relevant? Are the doses/concentrations achievable in patients?
Dr. Barrows,
I think that this synergy could very well be clinically relevant. We think that if we had more data points/replicates that we would see a statistical difference. As for a clinical difference, we are only looking at synergy of efficacy between the two drugs. Many other types of synergy exist such as synergy of potency, synergy of time, and synergy of populations that would all come into play in a clinical situation. I hope that answers your question!
Thanks,
Taylor