Novel topoisomerase I (Top1) inhibitors are in clinical development to circumvent the drawbacks of camptothecins (CPT). Here, we report molecular investigations into LMP-400, an indenoisoquinoline Top1 inhibitor in phase 1 clinical trial, by itself and in combination with the cell-cycle checkpoint inhibitor AZD7762. We examined drug effects on DNA replication and killing of cancer cells and found that LMP-400 showed synergistic antiproliferative activity when combined with AZD7762 in human colon carcinoma cells. Inhibition of S-phase progression and bromodeoxyuridine incorporation were similarly induced by LMP-400 and CPT and were abrogated by AZD7762. Replication studied by single DNA molecule analyses and immunofluorescence microscopy (molecular combing) showed rapid inhibition of fork progression in response to LMP-400 treatment with subsequent recapitulation after AZD7762 addition. AZD7762 inhibited both the activation/autophosphosphorylation of Chk1 and Chk2 at nanomolar concentrations in LMP-400-treated cells. This potent dual inhibition of Chk1 and Chk2 by AZD7762 was below the drug concentrations required to abrogate cell-cycle inhibition and produce synergism with LMP-400. Also, the synergism was independent of Chk2 both in Chk2-complemented cells and Chk2 knockout cells, suggesting additional mechanisms for cell-cycle abrogation by AZD7762. Together, our findings show a rationale for combining cell-cycle checkpoint inhibitors with the novel non-CPT indenoisoquinoline Top1 inhibitors.