Graduation Date

Fall 12-20-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Jennifer D. Black

Second Advisor

Adrian R. Black

Third Advisor

Angie Rizzino

Fourth Advisor

Adam Karpf

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a dismal five-year survival rate of less than 10%. One major obstacle in PDAC treatment is acquired drug resistance. Pan-histone deacetylase inhibitors (pan-HDACi) are a relatively new class of anti-cancer drugs, with demonstrated ability to overcome drug resistance and re-sensitize PDAC tumors to Gemcitabine (Gem). These agents target HDACs, a family of 18 enzymes (divided into four classes, I-IV) that catalyze the deacetylation of histones and other cellular proteins and have multiple effects on cell growth, differentiation, survival, and tumorigenesis. Although pan-HDACi have shown significant efficacy in preclinical analysis, and some have been FDA approved for treatment of selected cancers, the results in clinical trials involving pancreatic cancer have been disappointing. Pan-HDACi’s are associated with serious side effects when administered at therapeutic doses for solid tumors, including PDAC, a limitation that is likely to account for the lack of clinical efficacy of these agents. The toxicity of pan-HDACi is thought to reflect their ability to inhibit multiple class I, II, and IV HDACs. With the goal of reducing this toxicity, selective HDAC inhibitors are being developed and are undergoing preclinical and clinical testing, with several of these agents receiving orphan drug status by the FDA. We hypothesized that targeted inhibition of a restricted number of clinically relevant HDACs would recapitulate the benefits of pan-HDACi, while eliminating or greatly reducing dose limiting toxicities.

To test this hypothesis, we initially compared the effects of pan-HDACi such as Panobinostat with those of selective inhibitors targeting HDACs 1, 2, 3, 4, 5 and/or 6, the HDAC proteins known to be overexpressed in PDAC. The HDAC inhibitor, Mocetinostat, was used to selectively target HDACs 1, 2, and 3, and LMK-235 was used to inhibit HDACs 4, 5, and 6. Mocetinostat and LMK-235 in combination demonstrated synergy in multiple PDAC cell lines. Mocetinostat/LMK-235 also synergized with Gem to suppress the growth/survival of PDAC cells, showing comparable synergy with that seen with the Panobinostat/Gemcitabine combination. siRNA technology was used to identify the specific HDAC(s), i.e., HDAC1, 2, 3, 4, 5, or 6, targeted by the combination that are required for the observed synergy. These experiments revealed that inhibition of only three HDAC proteins, HDACs 1, 2, and 6, is sufficient to recapitulate the antitumor effects of pan-HDACi in PDAC cells. Notably, targeted knockdown of HDACs 1, 2, and 6 demonstrated superior synergy with Gemcitabine than the FDA approved pan-HDACi Panobinostat, pointing to HDAC 1/2/6 inhibition and Gem as a promising combination for PDAC treatment.

This novel approach for PDAC treatment was tested using a combination of selective HDAC inhibitors: the HDAC 1 and 2 inhibitor, Romidepsin, and the HDAC6 inhibitors, ACY-1215 (Ricolinostat) and Nexturastat. Romidepsin is FDA approved for the treatment of cutaneous lymphoma and ACY-1215 is currently in phase I and phase II clinical trials for breast cancer and lymphoid malignancies. Synergy was observed with a combination of Romidepsin and ACY-1215/Gemcitabine, and this combination showed improved synergy compared with the Panobinostat/Gemcitabine or Mocetinostat/LMK-235/Gemcitabine combinations. AnnexinV-FITC and MTT viability assays demonstrated that the anti-tumor activity of the combination is partially due to an increase in apoptosis as well as a decrease in cellular proliferation. Studies in PDAC xenograft models show activity of the combination in vivo with no significant toxicity. Together, our results point to targeted inhibition of HDACs 1, 2, and 6 in combination with Gemcitabine as an effective treatment modality for PDAC, and support further research into this combination for the management of pancreatic cancer in patients.

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