SUPPLEMENTARY FIGURE LEGENDS Fig. S1. Microenvironment –dependent effect of BMS-387032 on the survival of CLL cells (n=6), 3 patients were ZAP-70 positive, 3 patients had negative ZAP-70 expression. Cells were cultured in media only (native control), on HS-5 bone marrow stroma feeder layer cells, CD40Ligand expressing feeder-layer cells of incubated with anti-IgM immunobeads stimulating the B-cell receptor. Induction of apoptosis by BMS-387032 in CLL patient cells and healthy donor B-cells was assessed by Annexin V-FITC/7-AAD flow cytometry after 48 h for the quantification of double-negative viable cells. Curve-fitting and calculation of EC50 values were calculated as described in Methods. Fig. S2. Differential quantitative analysis of kinobeads-captured proteins from primary CLL cells and Ramos cells. Lysates of CLL and Ramos cells were subjected to precipitation with either kinobeads or BMS387032-beads, the captured proteins were digested with trypsin, labeled with isobaric tags, mixed, purified by HPLC and analyzed quantitatively with tandem mass spectrometry. 1200 proteins (orange symbols) were differentially quantified including 190 protein kinases (blue symbols). The x-axis represents the total abundance as calculated by the sum of all reporter ion intensities. The 20 kinases most prominently expressed in primary CLL cells vs. Ramos cells are YES, EPHA4, HCK, SRC, PKCb, FES, TAOK3, FGR, LRRK2, ALK2, AAK1, ITK, p38g, EPHB6, PIK3C2b, LYN, TAOK1, BRD3, JAK3, and SYK. 1 The 20 kinases most prominently expressed in Ramos cells vs. primary CLL cells are NEK2, CDK6, MYT1, CDC2, HRI, MELK, ACTR2B, QIK, SgK223, BIKE, TGFBR1, IKKe, ACK, PIP5K2A, LIMK2, ERK3, WEE1 ,ARG, CAMK1D, and PAK4. Fig. S3. Examples for the determination of relative changes in proteins bound to kinobeads. Data are shown for CDK2, CDK7, CDK5, CDK9, and PCTAIRE2, as well as for additional proteins known to form protein complexes with CDK kinases. The proteins were identified and quantified in kinobeads competition-binding experiments using BMS-387032 versus vehicle treated lysate. Symbols represent quantitative values for individual peptides at six different drug concentrations. Trend lines indicate results of the two-sided linear regression analysis. Fig. S4. Kinase-binding profiles of live cells treated with BMS-387032 and geldanamycin. Aliquots of freshly purified CLL cells were incubated with (A) BMS-387032, or (B) geldanamycin at various concentrations (10 µM, 1 µM, 0.1 µM, and vehicle) for 6 hours. Subsequently cells were harvested and cell lysates were prepared from each aliquot followed by addition of kinobeads. Captured proteins from each aliquot were analyzed by quantitative mass spectrometry. The bars represent relative quantities for individual kinases, defined as the free drug concentration at which half-maximal competition of kinobead binding is observed. (A) Cell aliquots treated with vehicle and with 0.1 µM, 1 µM, and 10 µM BMS-387032, respectively. The bars represent quantities for individual kinases detected relative to the quantities detected in vehicle treated cells. 2 (B) Cell aliquots treated with vehicle and with 0.1 µM, 1 µM, and 10 µM geldanamycin, respectively. The bars represent quantities for individual kinases detected in cell samples treated by 10 µM (blue), 1 µM (orange), and 0.1 µM (green) geldanamycin, respectively, relative to the quantities detected in vehicle treated cells. The B-lymphocyte kinase BLK is most strongly affected by treatment of cells with geldanamycin. Fig. S5. Heat map representation of the most selectively enriched proteins in CDK9 immunoprecipitations from Ramos and CLL cells. The color code indicates relative abundance in Ramos vs. CLL cells. 3