TOP1 Mutations and Cross-Resistance to Antibody-Drug Conjugates in Patients with Metastatic Breast Cancer

Abstract

Purpose: Antibody-drug conjugates (ADC) harboring topoisomerase I (TOP1) inhibitor payloads have improved survival for patients with metastatic breast cancer. However, knowledge of ADC resistance mechanisms and potential impact on the sequential use of ADCs is limited. In this study, we report the incidence and characterization of TOP1 mutations arising in the setting of ADC resistance in metastatic breast cancer.

Experimental design: Patients with metastatic breast cancer treated with ADCs with available posttreatment plasma-based genotyping were included. TOP1 mutation incidence, mutant allele frequency, and functional characterization were assessed, and incidence was compared with that in patients with metastatic breast cancer not receiving ADC treatment and in The Cancer Genome Atlas.

Results: Plasma-based genotyping identified distinct TOP1 mutations (S57C, R364H, W401C, and G359E) in 12.9% of patients (4/31) at the time of disease progression on ADC, compared with 0.7% (3/420) in non-ADC-treated patients with metastatic breast cancer and 0.5% in The Cancer Genome Atlas. The appearance of mutations was associated with clinical cross-resistance, as median duration on the first ADC was 455 versus 52 days for the second ADC. The functional characterization of three novel TOP1-mutant proteins demonstrated that all exhibited reduced enzymatic activity, attenuated covalent DNA binding, and resistance to TOP1 inhibitor ADC payloads SN38 and deruxtecan.

Conclusions: We describe the recurrent emergence of functionally altered, resistance-associated TOP1 mutations in vivo under selective pressure from ADCs and the potential impact on mediating cross-resistance to sequential ADCs. TOP1 mutation may represent a biomarker of resistance in this setting, and additional work is needed to optimize biomarkers and ADC payload design to improve outcomes for the sequential use of ADCs. See related commentary by Gwin and Hurvitz, p. 1824.

Figure 1.

Prevalence, clinical course, and quantitation associated with TOP1 somatic mutations. A,TOP1 mutation percentages associated with (i) MBC after ADC therapy, (ii) MBC and no prior ADC therapy, and (iii) primary tumors in the TCGA database. B, Summary of time on treatment for four patients in the MGH cohort who received sequential ADCs for MBC, including time on ADC1, time on ADC2, and the identity of the TOP1 mutation. C, Clinical course and MAF assessed in plasma for four patients with TOP1 mutations. Day 0 is the day of diagnosis of MBC. Time on treatment with each ADC is outlined in color. The MAF of each TOP1 mutation and a reference mutation identified in each patient is indicated on the Y-axis. Samples represented with circles are values from ddPCR data, whereas those represented by triangles reflect values from GuardantOMNI testing. All samples represent GuardantOMNI samples except the first timepoint of MGH1, which was a tumor (asterisk). MGH3 received a non-TOP1 inhibitor ADC for ADC2 (asterisk). Chemo, chemotherapy; MBC, metastatic breast cancer; PARPi, PARP inhibitor; TCGA, The Cancer Genome Atlas.

Figure 2.

Location and conservation of identified TOP1-mutant amino acids. A, Alignment of TOP1 amino acid sequence regions harboring identified mutations. Mutation-associated amino acids across species are indicated in red, and alterations are shown below. B, Schematic crystal structure of DNA-bound human TOP1 protein/inhibitor (topotecan) complex. Mutated amino acid residues (purple) are highlighted by red circles, and topotecan is shown in orange. The three mutations within TOP1 core domain (residues 214–635) are highlighted. S57C, which is located within an unstructured NH2-terminal domain, is not shown.

Figure 3.

Altered enzymatic activity, DNA binding, and TOP1 inhibitor resistance of TOP1-mutant proteins. A, IP–Western blot of Flag-tagged TOP1 after anti-FLAG IP from CRISPR/Cas9-TOP1 HCC1806 cells reconstituted with WT or mutant TOP1. B, TOP1-mediated plasmid relaxation. Supercoiled pHOT1 plasmid was incubated for 30 minutes with the indicated amount of TOP1-Flag IP extracts. C, Quantification of percent relaxed DNA from B, n = 3 experimental repeats. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. P values were calculated by two-way ANOVA followed by a multiple comparison test (Fisher LSD test). D, Western blots of Flag-tagged ectopic TOP1 and total TOP1 in parental HCC1806 TNBC cells. E, Summary of dose–response analysis following SN38 treatment (0.18–0.8 nmol/L, 3 days) of cells from D, n = 5 experimental repeats. P values were calculated by multiple unpaired t tests. F, Western blots of Flag-tagged ectopic TOP1 and total TOP1 in CRISPR/Cas9-TOP1 HCC1806 cell pools (left side). Dot blots showing G359E and W401C exhibit the most reduction of SN38-stabilized TOP1 DNA binding, as assessed by the RADAR assay (right side; see “Materials and Methods”). dsDNA, double-strand DNA; ns, nonsignificant.