Transitory dasatinib-resistant states in KITmut t(8;21) acute myeloid leukemia cells correlate with altered KIT expression  Markus D. Herrmann, Jochen K. Lennerz, Lars Bullinger, Stephan Bartholomae, Karlheinz Holzmann, Mike-Andrew Westhoff, Selim Corbacioglu, Klaus-Michael Debatin  Experimental Hematology  Volume 42, Issue 2, Pages 90-100 (February 2014) DOI: 10.1016/j.exphem.2013.10.006 Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 Acute inhibition of c-KIT by dasatinib and knockdown of KIT in treatment-naive cells results in induction of apoptosis. (A) Apoptosis induction in treatment-naive Kasumi-1 cells upon acute treatment with dasatinib for 24, 48, 72, and 96 hours as assayed DNA fragmentation. (B) Representative Western blot demonstrates knockdown of KIT protein levels and resulting interruption of downstream signaling as well as induction of Caspase3 cleavage in treatment-naive Kasumi-1 cells upon either electroporation alone, electroporation and treatment with control RNAi, or electroporation and treatment with KIT-specific RNAi after 6, 24, and 48 hours. (C) Apoptosis induction upon KIT knockdown in treatment-naive Kasumi-1 cells as quantified by DNA fragmentation. Experimental Hematology 2014 42, 90-100DOI: (10.1016/j.exphem.2013.10.006) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 Dasatinib treatment schedule and analytical protocol. One experimental set representative out of four independent sets of experiments (I–IV). In each set, we analyzed clones generated under long-term dasatinib exposure (R) and post–dasatinib exposure (PR) conditions. Experimental Hematology 2014 42, 90-100DOI: (10.1016/j.exphem.2013.10.006) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 3 Long-term dasatinib-exposure induced persistence characterized by KIT and downstream signaling alterations. (A, B) Determination of drug sensitivity by H3-thymidine incorporation in four independent clones (I-IV) after 12 weeks of consecutive treatment with maximally 12, 24, and 48 nmol/L dasatinib (R12, R24, R48). Three subsequently molecularly identical clones (I–III) were combined in (A) (n = 9 measurements per concentration and exposure) whereas clone IV is shown in (B) (n = 3 measurements). (C, D) Representative Western blots demonstrate alterations in KIT protein levels and downstream signaling: (C) representative clone II and (D) clone IV. Note that R48 in (C) showed overexpression of KIT, whereas R48 in (D) shows loss of KIT expression. (E) Representative Western blot demonstrates KIT knockdown and subsequent induction of Caspase3 cleavage in untreated controls (K) in comparison with each of the three independent dasatinib-resistant clones (I–III) that overexpressed KIT following the 12-week treatment protocol. (F) Rate of apoptosis induction upon KIT knockdown in K compared with resistant clones (I–III) that overexpress KIT as quantified by DNA fragmentation. Experimental Hematology 2014 42, 90-100DOI: (10.1016/j.exphem.2013.10.006) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 4 Dasatinib-resistant states were reversed upon discontinuation of inhibitor treatment. (A, B) Determination of drug sensitivity by H3-thymidine incorporation in the same four independent clones (I–IV) 2 weeks after dasatinib cessation demonstrate normalization of IC50 when compared with untreated controls (K). (C) Representative Western blots after dasatinib cessation demonstrate reversal of KIT-signaling alterations in clones I–III. (D) KIT-expression as assessed by RT-PCR (expressed as an average of each clone I–III to GAPDH); statistical comparison of each condition (R/PR) to untreated control (K) by t test. (E) Western blot after dasatinib cessation demonstrates reversal of KIT-signaling alterations in clone IV. (F) KIT-expression as assessed by RT-PCR (expressed as three technical replicates of clone IV to GAPDH); statistical comparison of each condition (R/PR) to untreated control (K) by t test. *p < 0.05. n.s., not significant. Experimental Hematology 2014 42, 90-100DOI: (10.1016/j.exphem.2013.10.006) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 Gene expression signatures reflect the transient nature of dasatinib-resistant states. (A) Scheme of dasatinib treatment protocol. Color of cells denotes dasatinib exposure at each time point: green = not exposed to dasatinib at time point; red = exposed to dasatinib exposed at time point. (B) Heat map displaying all differentially expressed genes in K, R48, and PR48 cells (clone IV) of three technical replicates as measured by GeneChip array analysis (selection of genes based on p < 0.05 and >2-fold change). Red cells indicate high expression, black cells indicate intermediate expression, and green cells low expression of a gene in the respective group with respect to the overall gene level in all samples. (C) Top-ranking gene-ontology (GO) groups determined by GO-Miner analysis and selected based on p < 0.01 in R48 conditions relative to K (clone IV). (D) Connectivity map (CMAP) result showing the performance of all of the top 20 instances with the queried signature [42]. (E) Top-ranking gene-ontology (GO) groups determined by GO-Miner analysis and selected based on p < 0.01 in PR48 conditions relative to K (clone IV). Experimental Hematology 2014 42, 90-100DOI: (10.1016/j.exphem.2013.10.006) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 Gene expression signatures reflect the transient nature of dasatinib-resistant states. (A) Scheme of dasatinib treatment protocol. Color of cells denotes dasatinib exposure at each time point: green = not exposed to dasatinib at time point; red = exposed to dasatinib exposed at time point. (B) Heat map displaying all differentially expressed genes in K, R48, and PR48 cells (clone IV) of three technical replicates as measured by GeneChip array analysis (selection of genes based on p < 0.05 and >2-fold change). Red cells indicate high expression, black cells indicate intermediate expression, and green cells low expression of a gene in the respective group with respect to the overall gene level in all samples. (C) Top-ranking gene-ontology (GO) groups determined by GO-Miner analysis and selected based on p < 0.01 in R48 conditions relative to K (clone IV). (D) Connectivity map (CMAP) result showing the performance of all of the top 20 instances with the queried signature [42]. (E) Top-ranking gene-ontology (GO) groups determined by GO-Miner analysis and selected based on p < 0.01 in PR48 conditions relative to K (clone IV). Experimental Hematology 2014 42, 90-100DOI: (10.1016/j.exphem.2013.10.006) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 6 Remaining differently expressed genes characterize dasatinib-sensitive profiles of primary t(8;21) AML samples. The overlap of the differentially expressed genes associated with Kasumi-1 dasatinib resistance (PR vs. K and K vs. PR) and the genes well-measured in 38 primary t(8;21) positive AML samples (n = 89) were visualized by hierarchical clustering. Gene expression values are color coded (red = higher expression than mean; green = lower expression than mean), and the presence of KIT exon 8 and exon 17 mutations is highlighted as indicated (as light and dark green squares, respectively). Experimental Hematology 2014 42, 90-100DOI: (10.1016/j.exphem.2013.10.006) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions