OBJECTIVE: Despite advancement in targeted therapeutics, women’s cancers remain particularly deadly. The response to Poly (ADP-ribose) Polymerase (PARP) inhibitors, a prominent targeted therapy indicated for use in these cancers, is largely dictated by the cellular status of homologous recombination (HR) and varies among patients possibly due to intrinsic and acquired mechanisms of resistance. A recent effort to identify targetable pathways to enhance toxicity of PARPi identified the Snf2-like chromatin remodeling enzyme, Amplified in Liver Cancer 1 (ALC1), as a key determinant of PARPi sensitivity. While this discovery proposes a link between chromatin re-modeling and PARPi sensitivity, the mechanism underlying the relationship remains unclear. This study aims to validate ALC1 as a determinant of PARPi sensitivity, characterize the phenotype of ALC1 loss, investigate mechanisms of synthetic lethality and identify possibilities for future studies and clinical implementation.
METHODS: UWB1.289, SUM149PT, DLD1, U-2 OS, and hTERT-RPE1 cells were cultured and used to validate ALC1-mediated PARPi sensitivity in a variety of viability assays. ALC1 depleted cells were complemented with various functional mutants to assess protein domains that were essential to PARPi-resistance. A Dual CRISPR-Cas9 system was implemented to screen BRCA1 complemented UWB1.289 cells with a sgRNA library targeting a multitude of DNA-repair associated genes in order to assess synthetic lethal/resistant relationships across different DNA-repair pathways. A MNase-sensitivity assay was developed and optimized to assess the global condensation of chromatin with combinational loss of ALC1 and PARP1. The 265-FokI system was employed to quantify the DNA damage response (DDR) to singular induced double strand breaks. Clonogenic assays were used to determine synergy with ionizing radiation.
RESULTS: The loss of ALC1 significantly and selectively hypersensitized both BRCA1 and BRCA2-deficient cells to PARPi. In ALC1 depleted cells, the addition of ALC1 cDNA was able to rescue cells from PARPi hypersensitivity while cDNA with mutations in either the macro-domain or ATPase active domain remained sensitive. Screening DNA-repair pathways to assess synergy with PARPi and ALC1 loss in HR-proficient settings revealed that the loss of several HR and Alt-EJ genes selectively re-sensitized cells to PARPi. Interestingly, the loss of BER genes, including several glycosylases, were epistatic or resulted in a protective effect to PARPi. The reduction of NHEJ, NER, MMR or RER did not significantly alter cellular response to PARPi. Further, nucleosome relaxation was significantly inhibited in cells treated with PARPi, ALC1 loss or in combination via MNase assay. The recruitment of repair proteins to DSBs was significantly inhibited by PARPi as assessed by 265-FokI immunofluorescence. The addition of PARPi in the setting of ALC1 loss significantly increased the cytotoxicity of ionizing radiation. Analyzing TCGA data collected from patients’ tumors, ALC1 may be overexpressed in many cancers and alterations in ALC1 may predict patient responses to traditional and targeted cancer therapies.
CONCLUSION: Using various models of BRCA1/2 deficiency we were able to validate the ability of ALC1 depletion to hypersensitize HR-deficient cells to PARPi. We provided insight into the mechanisms by which this phenomenon may be taking place, including chromatin compaction and the inhibition of DNA-damage repair. In addition, we provided therapeutic rationale that ALC1 may be targeted with PARP1 in synergy with other DNA-damaging agents. Overall, we believe that ALC1 is a prominent, viable and novel target of inducing PARP inhibitor sensitivity, that may help improve outcomes for patients with PARP inhibitor-resistant HR-deficient cancers. / 2022-06-04T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/41129 |
| Date | 04 June 2020 |
| Creators | Deraska, Peter |
| Contributors | Garcia-Diaz, Fernando |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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