Supplementary Information from <i>ERCC2</i> Helicase Domain Mutations Confer Nucleotide Excision Repair Deficiency and Drive Cisplatin Sensitivity in Muscle-Invasive Bladder Cancer
Authors: Li Qiang , Damish Alexis W. , Frazier Zoë , Liu David , Reznichenko Elizaveta , Kamburov Atanas , Bell Andrew , Zhao Huiyong , Jordan Emmet J. , Gao S. Paul , Ma Jennifer , Abbosh Philip H. , Bellmunt Joaquim , Plimack Elizabeth R. , Lazaro Jean-Bernard , Solit David B. , Bajorin Dean , Rosenberg Jonathan E. , D'Andrea Alan D. , Riaz Nadeem , Van Allen Eliezer M. , Iyer Gopa , Mouw Kent W. 
Published: April 2023

<p>Supplementary Figures and Methods // Supplementary Figure 1: The DDB2 proteo-probe assay measures cellular nucleotide excision repair (NER) capacity. Low magnification (20X) images of wild-type (WT) or mutant ERCC2-complemented XP cells before, 5 minutes after, and 150 minutes after UV exposure (see Figure 1 for high magnification [63X] images). DDB2 proteoprobe binding to UV-induced (6,4)-photoproducts is visualized using an anti-FLAG antibody (red). Nuclei are stained with DAPI (blue). ERCC2 expression is confirmed with an anti-Myc antibody (green). R683W is a common germline pathogenic allele, D312N is a common non-pathogenic germline allele, and G607A is a mutation identified in a cisplatin responsive tumor. Supplementary Figure 2: Sensitivity profiles for WT and mutant ERCC2-complemented cells for non-cisplatin agents. A. Carboplatin. B. Doxorubicin (Adriamycin) is a DNA intercalating agents that results in creation of double-strand DNA breaks. C. Rucaparib is a poly(ADP-ribose) polymerase inhibitor that demonstrates enhanced toxicity in homologous recombination (HR)-deficient cells. D. Ionizing radiation (IR). R683W is a common pathogenic germline allele in patients with the xeroderma pigmentosum complementation group D (XPD). D312N is a common, non-pathogenic germline ERCC2 allele in the general population. Y14C and G607A are helicase domain mutations identified in cisplatin responsive patients and are associated with NER loss and cisplatin sensitivity (Figure 2). Supplementary Figure 3: ERCC2 mutations identified in cisplatin non-responders are functionally deleterious. A. Cisplatin sensitivity curves for WT or mutant ERCC2-complemented cells. Whereas WT ERCC2 rescues cisplatin sensitivity, the sensitivity profiles for S44L and S246F, identified in two cisplatin non-responders from the FCCC cohort, are similar to mock-infected cells. B. The S44L and S246F mutants also fail to rescue sensitivity to UV sensitivity. C. Immunoblot showing expression of FLAG-tagged WT or mutant ERCC2. Supplementary Figure 4: Identification of an ERCC2 E86Q mutation in matched pre- and post-treatment tumor samples. The E86Q mutation was not identified in the pre-treatment sample by the standard mutation-calling algorithm due to the low number of reads, but was later identified by "force-calling" after the E86Q mutation was identified in the post-treatment sample. Correcting for tumor purity, the E86Q mutation was inferred to be clonal in both the pre- and post-treatment samples. Supplementary Figure 5: Phylogenetic tree showing relationship between pre- and post-chemotherapy tumors from a patient with an ERCC2 E86Q mutation. The pre- and post-chemotherapy tumors share several mutations, indicating a common ancestor. Shared mutations in other select cancer genes are noted. Supplementary Figure 6: CRISPR/Cas9 technology was used to create a KU19-19 cell line harboring an in-frame deletion at the endogenous ERCC2 allele. A. An sgRNA guide was designed to target the region near T484, a common site of ERCC2 mutations in MIBC tumors. B. Integrated Genomics Viewer (IGV) screenshot showing sequence of WT ERCC2 allele in KU19-19 parental cell line. C. The CRISPR-edited cell line KE1 harbors an in-frame deletion at T484. Supplementary Figure 7: An ERCC2 mutation confers an NER defect in a bladder cancer cell line. Low magnification (20X) images from a DDB2 proteoprobe assay comparing KU19-19 (WT ERCC2) and KE1 (mutant ERCC2) cell lines (see Figure 4 for high magnification [63X] images). Whereas the KU19-19 cell line efficiently resolves DDB2 foci, the foci persist in NER-deficient KE1 cells. Supplementary Figure 8: KU19-19 (WT ERCC2) and KE1 (mutant ERCC2) cell lines have similar sensitivity to the PARP inhibitor olaparib. Supplementary Figure 9: Overall survival for patients with ERCC2 wild-type (blue) versus mutant (red) tumors in the updated TCGA bladder tumor cohort (Robertson et al. Cell 2017; 171:1-17). There was no significant difference in survival between the groups (P=0.095). This figure was created using cBioPortal (Gao et al. Sci Signaling 2013; 6(269):pl1 and Cerami et al. Cancer Discov 2012; 2(5):401).</p>