# CF10 Displays Improved Synergy with Oxaliplatin in TP53-Null and Wild-Type CRC Cells from Increased Top1cc and Replication Stress

## TL;DR

This study shows that combining CF10 with Oxaliplatin is more effective than using 5-FU with Oxaliplatin for treating colorectal cancer, especially in cells with TP53 mutations.

## Contribution

The study reveals that CF10 + Oxaliplatin synergy is not affected by TP53 status, offering a new treatment strategy for CRC.

## Key findings

- CF10 + Oxaliplatin is highly synergistic and not dependent on TP53 expression.
- Oxaliplatin enhances CF10's DNA damage through Top1 cleavage complex and replication stress.
- COXA synergy is more potent than 5-FU + Oxaliplatin in both TP53-null and wild-type cells.

## Abstract

This study quantifies synergy between the nanoscale fluoropyrimidine polymer CF10 and Oxaliplatin (OXA), compared with 5-fluorouracil (5-FU), which is frequently combined with OXA in chemotherapy regimens for colorectal cancer (CRC). CF10 + OXA is highly synergistic and, unlike 5-FU, is not dependent on CRC TP53 expression, which is frequently mutated or absent in CRC tumors. OXA promotes CF10 cytotoxicity by enhancing its unique dual targeting of thymidylate synthase (TS) and DNA Topoisomerase 1 (Top1) to generate increased DNA damage. These findings support CF10 + OXA as a promising therapeutic strategy for CF10 clinical development, potentially reshaping the treatment for locally advanced and metastatic CRC.

Background/Objectives: TP53 mutation or deletion status is important for determining cellular responses to DNA-damaging drugs. Oxaliplatin (OXA) is combined with the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) in the FOLFOX regimen used to treat advanced colorectal cancer (CRC). However, the effects of TP53 deletion on 5-FU + OXA synergy are not well known. We investigated potential synergy between OXA and 5-FU and compared it with OXA synergy with a novel polymeric FP, CF10, in four cell lines harboring either wild-type (WT) or TP53-null status. Methods: Using CompuSyn and the highest single agent (HSA) models, we compared synergy between CF10 and OXA (COXA) and between 5-FU and OXA (FOXA). Cell cycle analysis was performed, as was Western blot quantification of canonical DNA damage pathway proteins. Likewise, immunofluorescent and confocal analysis allowed us to compare topoisomerase 1 cleavage complex and double-strand DNA break formation. Results: COXA synergy displayed minimal TP53 dependence with greatly improved potency compared to FOXA. COXA synergy resulted from OXA increasing: (i) Topoisomerase 1 (Top1) cleavage complex formation; (ii) DNA double-strand breaks (DSBs), and (iii) Checkpoint Kinase 1 and 2 (p-Chk1/2) phosphorylation, consistent with increased replication stress. Additionally, increased S-phase entry in TP53-null cells enhanced synergy between CF10, 5-FU, and OXA as S-phase drugs. Conclusions: Our results demonstrate that OXA synergizes with CF10 more effectively than with 5-FU through enhanced replication stress in both WT and TP53-null cells by causing greater Top1-mediated DNA double-strand breaks. Our studies provide a foundation for further testing of this combination in an orthotopic liver metastatic setting and eventual clinical development.