Braden, Dennis C.
(2024)
Dissecting the molecular mechanism of nitroalkene-mediated PARPi sensitization.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
This is the latest version of this item.
Abstract
PARP inhibitors (PARPi) represent a prime example of the fulfillment of the promise of targeted therapy in cancer. However, complex and heterogeneous resistance mechanisms limit their effectiveness. Multi-target agents present an opportunity to address PARPi resistance while avoiding many of the challenges posed by combination therapy with multiple agents. Electrophilic nitroalkenes represent one such option for multi-target agent therapy in cancer. Electrophilic nitroalkenes are endogenous products of the nitrite and nitric oxide-dependent metabolism of unsaturated fatty acids and post-translationally modify multiple protein thiols to mediate diverse anti-inflammatory signaling activities. In contrast to their canonical anti-inflammatory roles in benign cells, nitroalkenes exert anti-cancer effects in triple-negative breast cancer (TNBC) in vitro and in vivo, and sensitize TNBC cells to multiple antineoplastic DNA damaging agents including PARPi. Therefore, we sought to better characterize the molecular mechanisms driving nitroalkene-mediated PARPi sensitization in cancer.
Global proteomic analyses of the effects of nitro-alkylation on cancer cell signaling in U2OS osteosarcoma cells revealed that nitro-alkylation elicited adaptive changes in fatty acid metabolism, was distinct from global thiol alkylation, and was dynamically regulated by the cellular redox environment. A survey of novel nitroalkene targets observed in this proteomics study revealed that the emerging cancer therapeutic target RECQL4 was alkylated by the nitroalkene OA-NO2 at Cys1052. Nitro-alkylation of RECQL4 was further confirmed by click chemistry-based chemoproteomics, and nitro-alkylation of both RECQL4 Cys1052 and the previously validated nitroalkene target, RAD51 recombinase, were determined to be DNA damage-dependent using this approach.
Next, nitro-alkylation of RECQL4 was shown to inhibit RECQL4 helicase activity and prevent RECQL4 recruitment to DNA double-strand breaks. Consequently, nitro-alkylation of RECQL4 suppressed DSB end resection in a manner dependent on Cys1052 and selectively inhibited downstream homology-dependent DSB repair. Finally, nitroalkenes were observed to sensitize multiple high-grade serous ovarian cancer (HGSOC) cell lines to multiple PARPi and were revealed to be far less potent in a HGSOC cell line harboring a deep deletion in the gene encoding RECQL4. Taken together, the studies described here (1) identify OA-NO2 as a first in-class RECQL4 inhibitor, (2) supports reveal a previously unknown functional role for Cys1052 in regulating RECQL4 helicase activity, and (3) motivate further evaluation of small molecule nitroalkenes as PARPi-sensitizing agents in cancer.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
14 October 2024 |
| Date Type: |
Publication |
| Defense Date: |
10 June 2024 |
| Approval Date: |
14 October 2024 |
| Submission Date: |
22 July 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
165 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Pharmacology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
PARP, PARPi, nitroalkene, HGSOC, resistance, RECQL4, DSB, repair |
| Date Deposited: |
14 Oct 2024 15:46 |
| Last Modified: |
14 Oct 2024 15:46 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46717 |
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