BCR-ABL uncouples canonical JAK2-STAT5 signaling in chronic myeloid

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1 Supplementary Results BCR-ABL uncouples canonical JAK2-STAT5 signaling in chronic myeloid leukemia Oliver Hantschel*, Wolfgang Warsch*, Eva Eckelhart*, Ines Kaupe, Florian Grebien, Kay-Uwe Wagner, Giulio Superti-Furga & and Veronika Sexl &

2 Supplementary Figure 1 Figure 1. JAK TKIs inhibit JAK2 activity in the low nanomolar range. (a) Kinase assays depicting the inhibitory effect of JAK TKIs TG101348, TG101209, JAK-inhibitor I and INCB on JAK2 activity. (b, c) Dose response curves for imatinib alone and combined with JAK-inhbitor I [1 µm], TG [0.5 µm] and TG [0.5 µm] were analyzed in (b) a p185 BCR-ABL+ murine cell line and in (c) the p185 BCR-ABL+ human ALL cell line Sup-B15. Each data point in the graphs represents averages ± s.d. from a representative experiment done in triplicates. (d) Percentage apoptotic K562 cells upon treatment with imatinib [0.3, 1.0 or 3.0 µm] alone or in combination with JAK2 inhibitors as indicated. (e) Immunoblot of human and murine p185 BCR-ABL+ and p210 BCR-ABL+ cell lines for the expression of JAK2, STAT5 and pstat5.

3 Supplementary Figure 2 Figure 2. PCR of BM derived from conditional Jak2 knock-out mice. (a) PCR for Jak2 flox, Jak2 wt and Jak2 Δ demonstrating the successful deletion of Jak2 flox upon Poly(i:C) treatment. (b) Anti-BCR-ABL immunoblot of Ba/F3p 185 BCR-ABL and Ba/F3 p210 BCR-ABL cells.

4 Supplementary Figure 3 Figure 3. Characterization of Jak2fl/+, Jak2fl/+ Mx1-Cre and Jak2fl/fl Mx1-Cre lymphoid cells. (a) FACS staining for the pro-b cell surface marker CD19, CD43 and B220 of Jak2 fl/fl Mx1-Cre cells. (b, c) Proliferation assay for Jak2 fl/fl, Jak2 fl/+ Mx1-Cre and Jak2 fl/fl Mx1-Cre cell lines transformed by p160 v-abl via (b) 3 [H]-thymidineincorporation assay and (c) CFSE staining over a period of 24 hours. (d) Confirmation of INF-β induced Jak2 deletion of p160 v- Abl+ and p185 BCR-ABL+ cell lines via PCR. (e) Proliferation assay for Jak2 fl/fl and Jak2 Δ/Δ cell lines transformed by p160 v-abl or p185 BCR-ABL via 3 [H]-thymidine-incorporation assay (f) FACS staining for the pro-b cell surface marker CD19, CD43 and B220 of p160 v-abl+ and p185 BCR- ABL+ Jak fl/fl and Jak2 _/_ cells.

5 Supplementary Figure 4 Figure 4. JAK2 deletion does not influence expression of surface markers. (a) FACS analyzes for the expression of the surface-markers Gr1, Mac1, ckit and Sca1 of GFP+ (p210 BCR-ABL+ ) spleen cells derived from diseased animals.

6 Supplementary Figure 5 Figure 5. Deletion of Jak2 does not alter pstat5 level and response of BCR-ABL + cells on imatinib. (a) Dose response curves of p160 v-abl+ and p185 BCR-ABL+ Jak2 fl/fl and Jak2 Δ/Δ cell lines for imatinib (n = 2/genotype). For calculation of the curves, the values for both genotypes were implicated. (b) Intracellular FACS staining for pstat5 of a p160 BCR-ABL+ Jak2 fl/fl and Jak2 Δ/Δ cell line. (c) SupB15, Ba/F3 p185 BCR-ABL, Ba/F3 p210 BCR-ABL and a murine p185 BCR-ABL+ cell line were treated for 72 hours with indicated JAK2 TKIs. FACS blots depict apoptotic stages of the cells as measured via Annexin V/probidium iodide staining. IC 50 values for BCR-ABL for the applied TKIs are depicted at the top of the panel.

7 Supplementary Figure 6 Figure 6. Evidence for BCR-ABL being the STAT5 phosphorylating kinase (a) Immunoblot for JAK1, JAK2, JAK3, TYK2, STAT5 and pstat5 in Ku812 cells after sirna mediated knock-down of the respective kinases. Knock-down levels relative to the controls are indicated. For raw gel image see Supplementary Fig. 9. (b) In vitro kinase assay depicting the inhibition of the Abelson kinase by the SU6656. (c) Immunoblot for py (4G10) in K562 cells after treatment with the TKI nilotinib, dasatinib, TG101348, JAK-inhibitor I or SU6656. (d, e) Immunoblot of Ba/F3 cells expressing either wild type p210 BCR-ABL or the BCR-ABL TKI resistant p210 BCR-ABL T315I mutant. Levels for py(4g10) are depicted after treatment with (d) 10, 100, or 1000nM dasatinib or (e) DMSO, TG101348, Bosutinib or Sunitinib [1µM each].

8 Supplementary Figure 7 Figure 7. STAT5 shows a higher sensitivity for tyrosine dephosphorylation upon dasatinib exposure compared to CRKL. (a) K562 cells were treated with various concentrations of nilotinib for 4 hours. py (4G10) levels were quantified and IC 50 values from the sigmoidal dose-response curves were calculated. (b) K562 cells were treated with various concentrations of nilotinib for 4 hours. STAT5, pstat5, CRKL and pcrkl levels were quantified and IC 50 values from the sigmoidal dose-response curves were calculated. (c) Sequences of the peptides used for in vitro kinase assays corresponding to Tyr-694 in STAT5 and Tyr-207 in CRKL in comparison to the optimal ABL phosphorylation consensus and a commonly used optimal ABL substrate peptide. (d) Table with the enzymatic parameters derived from the Michaelis-Menton graphs. (e) The recombinant ABL kinase domain was used to perform kinase assays in the presence of 100 µm ATP and increasing concentrations of an in vitro selected optimal ABL substrate peptide. Specific activity was calculated and plotted over the substrate concentration (Michaelis-Menten graph). The graph shows the mean with s.d. of two experiments done in triplicates.

9 Supplementary Figure 8 Figure 8. Raw gel images corresponding to indicated figures

10 Supplementary Figure 9 Figure 9. Raw gel images corresponding to indicated figures

11 Supplementary Figure 10 Figure 10. Raw gel images corresponding to indicated figures

12 Supplementary Table 1 Table 1. Summary of TKIs used throughout the paper

13 Supplementary Table 2 Table 2. Decrease in STAT5 phosphorylation after TKI treatment is independent of JAK2. Summary of relative decrease in MFI for pstat5 before (100 %) and after treatment with indicated JAK2 TKIs for 4 hours. Shown are the results for a p160 v-abl+ and p185 BCR-ABL+ Jak2 fl/fl and Jak2 Δ/Δ cell line.

14 References for Table 1: 33 Pardanani, A. et al. TG101209, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V617F and MPLW515L/K mutations. Leukemia 21, (2007). 34 Wernig, G. et al. Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera. Cancer Cell 13, (2008). 40 Blake, R.A. et al. SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling. Mol Cell Biol 20, (2000). 41 Rix, U. et al. Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib and dasatinib reveal novel kinase and non-kinase targets. Blood 110, (2007). 42 Remsing Rix, L.L. et al. Global target profile of the kinase inhibitor bosutinib in primary chronic myeloid leukemia cells. Leukemia 23, (2009). 46 Quintás-Cardama, A. et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood 115, (2010). 51 Gazit, A., Yaish, P., Gilon, C. & Levitzki, A. Tyrphostins I: synthesis and biological activity of protein tyrosine kinase inhibitors. J. Med. Chem. 32, (1989). 52 Bantscheff, M. et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat Biotechnol 25, (2007). 53 Carter, T.A. et al. Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases. Proc Natl Acad Sci U S A 102, (2005).