Fig. 3
From: Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo
ADAM10 is essential for PDX acute leukemia in vivo. A Publicly available data from the BloodSpot databank were analyzed for ADAM10 mRNA expression profile in leukemia samples with the indicated BCP-ALL and AML subtypes compared to healthy donor BM controls (dataset 202603_at; Leukemia MILE study GSE13159). Box indicates median, 25th and 75th percentile; whiskers indicate min/max. **** p < 0.0001, *** p < 0.001 by multiple t-test compared to healthy BM group. ns: not significant, CN: cytogenetically normal. B Flow cytometry analysis of ADAM10 surface expression in ALL (n = 14) and AML (n = 10) PDX samples compared to healthy donor BM (n = 10) samples. Isotype control of representative PDX and BM samples are included for comparison. C Correlation of ADAM10 expression in AML blasts with overall survival in 172 AML patients (dataset for 202603_at; Human AML cells GSE13159). D-F In vivo competitive ADAM10 validation in AML and ALL PDX samples. D ADAM10 surface protein expression in control (CTRL and Isotype) and KO ALL and AML PDX samples transduced with the indicated sgRNAs and re-isolated following 8 weeks of in vivo growth. Histograms of cells transduced with two independent ADAM10-sgRNAs are shown. E Quantification of in vivo competitive validation assay. Percentage of the ADAM10 KO population in the injection mix (Input) and in the corresponding re-isolated PDX from BM (black) or spleen (grey) at advanced leukemia (Output) for ALL-199 (n = 7; 4 animals with 4 BM and 3 spleen measurements), ALL-265 (n = 12; 9 animals with 9 BM and 3 spleen measurements), AML-356 (n = 9; 5 animals with 4 BM and 5 spleen measurements), AML-388 (n = 6, 3 animals with 3 BM and 3 spleen measurements), AML-393 (n = 9; 6 animals with 6 BM and 3 spleen measurements), AML-602 (n = 6; 3 animals with 3 BM and 3 spleen measurements) and AML-661 (n = 14; 7 animals with 7 BM and 7 spleen measurements). **** p < 0.0001, *** p < 0.001, ** p < 0.01 by paired t-test. F Dropout of ADAM10 KO cells is more prominent in spleen compared to BM in some samples. Percentages of ADAM10 KO cells in the BM and spleen derived from E. **** p < 0.0001 by paired t-test. G Quantification of in vitro competitive validation assay for ADAM10. Percentage of the ADAM10 KO population before (Input) and after the in vitro cultivation period (Output) (all PDX samples n = 3, 3 individual sgRNAs). *p < 0.05 by paired t-test, nd (not determined), ns (not significant). H In vivo competitive validation assays for ADAM10 in ALL-199 and ALL-265 after the indicated in vivo growth times. Percentage of the KO populations in the injection mixture and in PDX cells re-isolated from the BM is depicted. Grey dotted line is the interpolation of the data using Pade (1,1) approximant, robust fit