Article Figures & Data
Figures
- Figure 1.
The treatment history and genomic landscape of a metastatic carcinoma with an extreme outlier response to combination therapy. A, schematic representation of the treatment history of the index responder, a patient with metastatic small-cell carcinoma of the ureter (PR, partial response; CR, complete response; NED, no evidence of disease). B, CT images of the index patient before surgery of the recurrent tumor, before combined AZD7762 and irinotecan therapy, and 1 month after combined treatment (left, middle, and right, respectively). C, somatic abnormalities in the responder's genome (from outside to inside) included a heavy burden of CNAs; mutations at approximately 10-Mb resolution; regulatory, synonymous, missense, nonsense, and frameshift insertions and deletions (gray, black, orange, red, and green); and intra- and interchromosomal rearrangements (light and dark blue). D, the allelic fraction of mutations is shown in genes identified by WGS of the post–etoposide/cisplatin tumor and also covered by the IMPACT assay and resequenced in the treatment-naïve primary tumor (blue and gray, respectively).
- Figure 2.
D-loop and adjacent mutations in RAD50. A, DNA copy number segmentation inferred from WGS of the index case indicates a focal heterozygous loss spanning the RAD50 locus on 5q31.1, deleting the wild-type allele (as indicated by the sequence logo representing the allelic frequency), retaining only RAD50L1237F. B, the RAD50L1237F mutation (red) is present in the D-loop of the ATPase domain near the Walker B motif (top and bottom, schematic of RAD50 protein at multiple scales). Directly adjacent appear a cluster of mutations in diverse malignancies (black). C, conservation of the RAD50 D-loop motif and adjacent sequence is indicated across 9 organisms in which the mutated leucine and adjacent aspartate residues are highlighted in green and yellow, respectively, along with the positions of other mutations. The mutation position for human and yeast (in brackets) is given and indicated by arrowheads (Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster; At, Arabidopsis thaliana; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Ec, Escherichia coli; Pf, Pyrococcus furiosus; T4, Bacteriophage T4). D, the three-dimensional structure of the Rad50 dimer indicating the position of the affected subunit with mutations colored in red. Inset, position of mutant residues within close proximity to bound ATP.
- Figure 3.
RAD50 hypomorphism attenuates ATM signaling, synergizing with checkpoint inhibition to confer chemotherapy sensitivity. A, although the Rad50L1240F protein level is reduced, the Mre11 complex is intact in rad50L1240F cells as well as those harboring similar D-loop or adjacent mutations. The Mre11–Rad50 interaction was assessed by coimmunoprecipitation with Rad50 or Mre11 antibodies (Rad50-IP or Mre11-IP) and Western blot analysis (anti-Rad50 or anti-Mre11) from yeast extracts of the indicated genotypes. Preimmune antibodies (PI) were included as a negative control. Rad50L1240R abundance was too low to rigorously determine whether complex formation was disrupted. B, Mec1 (yeast ortholog of human ATR) deficiency dramatically potentiates the DNA-damage sensitivity of rad50L1240F-mutant cells, an affect that could not be rescued by Sae2 deletion. Mec1-proficient (MEC1 WT, top 8 strains) or Mec1-deficient (mec1Δ, which also contains the sml1Δ suppressor, bottom 14 strains) cells of the indicated genotypes were 1/5 serially diluted and spotted on plates with or without the indicated concentrations of camptothecin (CPT) and grown for 3 days. C, although Rad53 is phosphorylated (P-Rad53) upon methyl methanesulfonate (MMS) treatment in Rad50-wild-type cells with (+) or without Mec1 or Sae2 (Δ), these levels are attenuated in rad50L1240F mec1Δ sae2Δ triple-mutant cells or those with adjacent mutations, indicating that Tel1 (ATM) is not activated in rad50-mutant cells. D, graphical summary of the impact of the indicated rad50 alleles on Rad50 levels (blue); the integrity of the Mre11 complex; on camptothecin sensitivity in checkpoint-proficient (MEC1) and checkpoint-compromised (mec1Δ) cells (green), the ability of the Tel1 checkpoint to rescue cell survival (gray/black); Rad53 phosphorylation (blue) upon methyl methanesulfonate treatment; telomere length (red). E, a model of sensitivity to DNA-damaging agents such as irinotecan driven by the synthetic lethality between simultaneous genetic and pharmacologic perturbation of both axes (ATM and ATR) of the DDR by RAD50 hypomorphism and checkpoint inhibition, respectively.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Materials - PDF file - 321KB, A detailed description of additional methods utilized (and associated references) for sequencing and analysis as well as an explanatory note regarding the TM pathway in yeast.
- Supplementary Figures 1 - 10 - PDF file - 470KB, All supplementary figures including tumor histology, RAD50 immunohistochemistry, candidate mutations and their clonality, and supporting yeast and MEF experimental data.
- Supplementary Tables 1 - 6 - PDF file - 2461KB, A complete listing of somatic mutations and rearrangements detected as well as targeted genes, sequences, MRN complex mutation frequency, and strains utilized.
Volume 4, Issue 9
