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Supplementary Figure 1. Combination treatment with olaparib/AZD1775 is effective in select SCLC CDX models. Mice bearing CDX3 (a), CDX4 (b), CDX8 (c), or CDX8p (d) tumors were treated with vehicles (black) AZD1775 (red), olaparib (green), or the combination (blue) for 21 days. Each line depicts an individual relative tumor volume from all mice. e) CDX4 tumors were treated with vehicles (black) AZD1775 (red), topotecan (green), or the combination (blue). Topotecan was administered on days 1-3 and AZD1775 was administered on days 1-21. Supplementary Figure 2. CDX8p is more chemoresistant than CDX8. a) The clinical timeline for the patient that gave rise to CDX8/8p. Mice bearing CDX8 (b) or CDX8p (c) tumors were treated with vehicles (black) or cisplatin/etoposide (red). Individual relative tumor volumes are depicted. Supplementary Figure 3. Olaparib/AZD1775 is less effective after treatment with cisplatin/etoposide. Mice bearing CDX3 tumors were treated with cisplatin/etoposide on days 1-3 and allowed to recover for 3 weeks, at which time they were randomised and treated with vehicles (black) AZD1775 (red), olaparib (green), or the combination (blue) for 21days. The treatment periods are marked by orange lines. Individual relative tumor volumes are depicted for each mouse. Cohort sizes > 7. Supplementary Figure 4. AZD1775 and olaparib monotherapy activity in representative SCLC CDX models. CDX3 (a, b), CDX4 (c, d), CDX8 (e, f), or CDX8p (g, h) ex vivo cultures were treated with increasing concentrations of either AZD1775 (a, c, e, g) or olaparib (b, d, f, h) for 1 week and relative cell viability was assessed. One representative experiment of at least three is shown. Supplementary Figure 5. Representative immunohistochemical images for phospho-histone H3 (a), cleaved caspase 3 (b), phospho-CHK1 (c), and �H2AX (d) from CDX3 tumours treated with vehicle, AZD1775, olaparib, or the combination. Supplementary Figure 6. Lack of a BRCAness signature in olaparib-sensitive models. a) Heatmap of average log2 RPKM values are depicted for CDX3, CDX4, CDX8, and CDX8p following supervised clustering of models based on olaparib sensitivity. b) The number of synonymous (black) and nonsynonymous (white) mutations as determined by whole exome sequencing are graphed for CDX3, CD8, and CDX8p. The lack of a matched germline sample precluded somatic mutation calling in CDX4. c) Filtered RNAseq reads for PALB2 were loaded into the Broad Integrative Genomics Viewer and the E178* was highlighted in green for each read. Supplementary Figure 7. Published mechanisms of olaparib resistance. a) Lysates from two representative CDX tumors were immunoblotted for indicated proteins. b) PARP1 RNA expression was assessed in tumor lysates from CDX3, CDX4, DX8, and CDX8p. Three representative tumors from each model were examined. c) Lysates from two representative CDX tumors were immunoblotted for indicated proteins. Supplementary Figure 8. Treatment with AZD1775, olaparib, or the combination does not alter p21 expression. Duplicate tumor lysates taken 24 hours after indicated treatments were immunoblotted for indicated proteins. Supplementary Figure 9. Published mechanisms of AZD1775 resistance. a) SETD2 and PKMYT1 RNA expression was assessed in tumor lysates from CDX3, CDX4, CDX8, and CDX8p. Three representative tumors from each model were examined, and mean values are plotted against the cohort average maximal in vivo response to AZD1775. b) RRM2 levels were measured by immunoblotting lysates from CDX tumors harvested 2 hours after a single dose of vehicle (white), AZD1775 (red), olaparib (green), or the combination (blue). Samples were quantified and normalised to vinculin in each sample. Cohort sizes > 4 tumors per condition. Supplementary Table 1. Mass Specrometer and UPLC system parameters Supplementary Table 2. Optimization parameters for mass spectrometry analysis |
