A curative combination cancer therapy achieves high fractional cell killing through low cross-resistance and drug additivity

Abstract

Curative cancer therapies are uncommon and nearly always involve multi-drug combinations developed by experimentation in humans; unfortunately, the mechanistic basis for the success of such combinations has rarely been investigated in detail, obscuring lessons learned. Here, we use isobologram analysis to score pharmacological interaction, and clone tracing and CRISPR screening to measure cross-resistance among the five drugs comprising R-CHOP, a combination therapy that frequently cures Diffuse Large B-Cell Lymphomas. We find that drugs in R-CHOP exhibit very low cross-resistance but not synergistic interaction: together they achieve a greater fractional kill according to the null hypothesis for both the Loewe dose-additivity model and the Bliss effect-independence model. These data provide direct evidence for the 50 year old hypothesis that a curative cancer therapy can be constructed on the basis of independently effective drugs having non-overlapping mechanisms of resistance, without synergistic interaction, which has immediate significance for the design of new drug combinations.

Guided screen for synergistic three-drug combinations

“…A good rule-of-thumb while designing combinations is choosing synergistic combinations with non-overlapping toxicity. This design element is similar to choosing combinations with non-overlapping resistance mechanisms [1,2,8]. Since both heuristics are informed only by experimentation on single drugs, their solution does not require experimental screens of large number of combinations.…”

“…The treatment of many pathogens and tumors involve drug cocktails of three or more combinations. Such combinations are chosen primarily due to their non-overlapping resistance mechanisms [ 1 , 2 ] and the drug interaction (synergy/antagonism) phenotype [ 3 – 6 ]. Resistance is informed primarily by the mechanism of action of individual drugs.…”

MicroRNAs associated to single drug components of R-CHOP identifies diffuse large B-cell lymphoma patients with poor outcome and adds prognostic value to the international prognostic index

“…Evaluation of prognostic impact in this study is based on retrospective clinical cohorts where treatment conditions are not strictly unified regarding number of cycles, dosing [19,20] and individual dose reductions due to side-effects like neuropathy [36]. Taking this and the pharmacological information on no synergistic effect of R-CHOP into account [8], single drug screens were used rather than combinational assays to identify candidate miRNAs to be assessed in non-weighted models using either multivariate Cox regression or random survival forest models. The candidate miRNA originating from single drug screens are thus tested and evaluated for prognostic impacts in DLBCL patients receiving the full R-CHOP regimen, ensuring that only miRNAs with enough statistical power alone in univariate Coxregression analysis and in modelled combinations are included in the final miRNA signature/panel.…”

“…Since cyclophosphamide is a prodrug that requires hepatic activation to produce its active metabolite, the synthetic oxazaphophorine derivate mafosfamide was used in the dose-response assays. As the pharmacological effect of R-CHOP has limited cross-resistance rather than synergism [8], single drug screens were used instead of combinations in order to identify miRNAs that were specifically associated to response for the individual components of the treatment regimen. The cells were seeded 24 h prior to addition of the drug using cell line specific seeding concentrations to ensure exponential growth for 48 h (Table 1).…”

“…Although addition of rituximab to the regimen has improved treatment outcome of DLBCL substantially, up to 40% of patients have refractory disease or relapse after initial response to therapy due to drug-specific molecular resistance [5][6][7]. Study of the pharmacological principles underlying the R-CHOP regimen revealed no synergistic interaction but very low cross-resistance, showing a strong combination of independently effective drugs without overlapping mechanisms of resistance [8]. Thus, identification of biomarkers predictive for single drug components of R-CHOP is of great importance when attempting to improve clinical outcome.…”