(H-297) Chromatin Conformation Mediates Plasticity and Impacts Post-Chemotherapy Cancer Cell Fates.

Introduction:: Epigenetics and Chromatin Regulation

Non-genetic mechanisms in cancer cells are likely to play a larger role than their genetic mechanisms in chemoresistance. While oncogenic driver mutations can take generations to accumulate in cancer cells in response to chemotherapy, non-genetic mechanisms such as transcriptional adaptation occur within hours to activate or upregulate survival pathways. Short-term changes in transcriptional patterns result from changes in gene accessibility through alterations of chromatin conformation. Specifically, our models predict that cancer cells with a plasticity-fostering chromatin conformation have increased transcriptional malleability (higher average level of upregulation in survival pathways) and transcriptional heterogeneity (more variance in the level of upregulation between cells), allowing for cell survival gene expressions to cross a threshold that determines whether a cell will survive or die. Using live-cell chromatin imaging on clusters of colon cancer cells undergoing chemotherapy treatment and a compound library screen, we identified agents that alter plasticity-fostering chromatin conformations, which we term transcriptional plasticity repressors (TPRs). Combination treatment using a TPR and chemotherapy increased cancer cell death in vitro compared to chemotherapy alone. To further analyze whether targeted modulation of chromatin conformation can improve treatment efficacy and reduce tumor adaptation to chemotherapy, we utilized a PDX model of ovarian cancer and repeated our combination treatment. These studies reveal that tumors treated with low-dose chemotherapy and TPR had stagnated tumor growth compared to monotherapy with either vehicle, TPR, or chemotherapy. Our findings highlight a novel, targetable chemoevasion mechanism that can be combined with current treatments to further improve clinical outcomes.

Materials and Methods::

Cells were plated on glass bottom 35 mm petri dishes or 6 well plate plates to image under live-cell Partial-Wave Spectroscopic (PWS) microscopy. PWS measures the spectral interference resulting from internal light scattering structures within the cell to capture their mass density distribution. After acquiring spectrally resolved images, regions of interest (nuclei) were drawn and their average light scattering distributions were related to the statistical parameter of chromatin structure, packing scaling (D), via power law autocorrelation function.

Ovarian A2780 cells and colon HCT-116 cells were respectively cultured in RPMI 1640 and McCoy’s 5A medium supplemented with 10% Fetal Bovine Serum and 1% PenStrep. Both cells were cultured at 37°C and 5% CO2 between passage 5 and 25. Cells were given at least 48 hours to adhere before pharmacological treatment. Cell viability, cytotoxicity, and caspase 3/7 activity was measured with ApoTox-Glo™ Triplex Assay. HCT116 cells were seeded 1.5k per well on 96 well white/clear bottom plates and were given 48 hrs before treatment with 15 uM oxaliplatin. Fluorescence/Luminescence measurements were performed with Synergy Neo2 Fluorescence.

Patient-derived OVCAR10 tumors were implanted into 8 week old immunosuppressant CB-17 SCID mice on the right flank. Implanted tumors were let to be grown until 150-200mm^3 and randomized into four experimental groups (vehicle, TPR, Chemo, Combination). Cage was observed daily and tumor dimension, body weight were measured twice a week. Tumors were explanted at 28 days and immunohistochemistry was performed by the Northwestern pathology core and analyzed on image J.

Results, Conclusions, and Discussions:: Chemotherapy treatment increases chromatin packing scaling (D) in various cancer cell lines, even on their chemoresistant counterparts, at 48 hrs post-treatment. Time course study on oxaliplatin treated HCT 116 cells show a population shift in D starting at 24 hrs, which corresponds with caspase 3/7 activity and cell viability assays that show differences between the treated and untreated groups at 16 and 24 hrs. In the surviving cell clusters, post-treatment % increase in D is more prominent in cells that had an initially low average D than cells twitch an initially higher average D. These findings show that the majority of the low D cells did not readily adapt to chemotherapy in time to upregulate critical survival genes, leading to cell death. Screening 200+ epigenetic regulators with various mechanisms of action, we identified two transcriptional plasticity regulators (TPRs) that reduce plasticity-fostering chromatin conformations (D) in cells at a short time scale (< 1hr). In vitro studies show that the cancer cells treated with combination treatment (chemotherapy + TPR) show less resistance to chemotherapy and result in more cell death compared to those treated with chemotherapy alone. In vivo studies with patient-derived ovarian tumors implanted in immunosuppressant mice show inhibition of tumor growth with the combination treatment compared to chemotherapy alone, TPR alone, and vehicle control. Modeling the effective inhibition rate and adaptive inhibition rate of the treatments, we showed that the combination treatment significantly inhibits tumor growth and slows the adaptation rate to the chemotherapy. However, semi-quantification of immunohistochemistry staining on the tumor slides for biomarkers (KI67, p53, p16, HMGA2, CD133) fail to show significant differences between the experimental groups.

Acknowledgements (Optional): : NIH grants U54CA268084, R01CA228272, R01CA225002
NSF grant EFMA-1830961

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