Theses Doctoral

Delineating the function, efficacy, and mechanism of a novel preclinical agent for the treatment of pancreatic ductal adenocarcinoma

Eberle-Singh, Jaime

In 2018, it is estimated that 55,440 Americans will be diagnosed with pancreatic cancer and this figure is expected to continue to rise with increased life expectancy. Despite some measurable progress over the past few decades, pancreatic cancer remains one of the most lethal malignancies with five-year survival rate of 8.7%. Novel therapies, and their timely translation to the clinic, are urgently needed.

As part of an effort to identify and characterize novel therapeutic strategies for pancreatic ductal adenocarcinoma, we began a study of the role of Bmi1 in tumor maintenance and progression. While Bednar and colleagues showed that Bmi1 is critical for the development of pancreatic cancer, and that its pancreas-specific deletion impairs PanIN formation, we were interested in assessing its function in established tumors. During the course of this work, we acquired a novel compound, PTC596, developed by PTC Therapeutics as a post-translational inhibitor of BMI1. Treatment with PTC596 leads to hyperphosphorylated BMI1, and this modification is associated a loss of protein activity. We planned to study this compound, in vitro and in vivo, as a complement to genetic perturbations of Bmi1.

Initial characterizations of the effects of PTC596 on human and murine-derived pancreatic cancer cell lines revealed a potent anti-proliferative effect, accompanied by BMI1 hyperphosphorylation, and followed by polyploidy and cell death after prolonged treatment. Further analysis showed a clear G2/M arrest and elevated levels of phospho-histone H3. Bmi1 is known to play a role the cell cycle, but its inhibition in pancreatic cancer cell lines has been shown to induce G1 arrest.

We decided to further explore the mechanism of PTC596’s antiproliferative effects by carrying out RNA sequencing on Aspc1 cells treated with PTC596. We found that 8 of the ten most down-regulated genes were members of the tubulin family and began to study this compound’s effect on microtubules. Compelling results from a cell-free tubulin polymerization assay support inhibition of tubulin polymerization as the mechanism of action for PTC596. These data are further supported by evidence that PTC596 increases the fraction of free-tubulin in treated cells, as well as dramatically alters the cell’s microtubule network.

Given our laboratory’s interest in identifying novel therapies for pancreatic cancer, and the fact that PTC596 has already begun clinical trials, we continued to characterize this compound in vivo. We found PTC596 to have properties favorable for in vivo administration. PTC596 is orally available, has a plasma half-life of approximately 22 hours following oral administration, and accumulates in tumor tissue where it has an expected pharmacodynamic effect. Furthermore, it is well tolerated in vivo in combination with gemcitabine. We carried out a four-arm intervention study in tumor-bearing KPC mice, examining PTC596 alone and in combination with gemcitabine. We found that PTC596 synergizes with gemcitabine to significantly reduce tumor growth rates and provide a 3-fold extension of survival as compared to vehicle. These findings are, to our knowledge, the first evidence of in vivo synergy between a microtubule-destabilizing agent and gemcitabine for the treatment of pancreatic cancer. Importantly, this study identifies an alternative mechanism for PTC596 and implicates its efficacy in a novel treatment regimen for pancreatic ductal adenocarcinoma.


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More About This Work

Academic Units
Cellular, Molecular and Biomedical Studies
Thesis Advisors
Olive, Kenneth
Ph.D., Columbia University
Published Here
June 23, 2018