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

<b>Background/Objectives</b>: <i>TP53</i> 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 <i>TP53</i> 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 <i>TP53</i>-null status. <b>Methods</b>: 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. <b>Results</b>: COXA synergy displayed minimal <i>TP53</i> 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 <i>TP53</i>-null cells enhanced synergy between CF10, 5-FU, and OXA as S-phase drugs. <b>Conclusions</b>: Our results demonstrate that OXA synergizes with CF10 more effectively than with 5-FU through enhanced replication stress in both WT and <i>TP53</i>-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.