Resistance in 2S-stimulated cells is mainly mediated by Bcl-xl. (A) Dose-response heatmap of total cytotoxicity expressed as percentage-positive cells for cell death induced by sunitinib, A1210477, and navitoclax as single agents (top panels) and in combination with venetoclax (10 nM) (bottom panels) in unstimulated and 2S-stimulated cells from 6 patients. Drug response was determined by multiparametric analysis as described in Figure 2. The shade indicates the mean for all the cells in 8 micrographs for each condition according to the grayscale bar shown below. Columns are individual patient samples and rows are drug concentrations. Cell death (percentage positive) in negative control (DMSO) and for venetoclax (10 nM) as a single agent are shown as bars below the heatmaps. (B) Model illustrating potential cellular responses to venetoclax and sunitinib. (i) Apoptotic response of unstimulated CLL cells treated with venetoclax. Bcl-2 family proteins control cell death by regulating the permeabilization of the mitochondrial outer membrane (MOM) through a series of competitive binding interactions among themselves. For illustrative purposes, inhibition of BH3 proteins (mode 2) is shown although the process is predicted to be similar for activated Bax bound to and inhibited by antiapoptotic proteins (mode 1). Upon receiving an apoptotic stress signal, inactive proapoptotic BH3 proteins (red) are activated (red and white) and migrate to mitochondria where they are either bound by 1 of the antiapoptotic proteins (Bcl-2, Bcl-xl, Mcl-1, or A1) or trigger apoptosis by binding to inactive Bax (light green) and activating it at the membrane (dark green). Activated Bax oligomerizes and permeabilizes the MOM, enabling release of proapoptotic proteins including cytochrome c, endonuclease G, and Smac (orange circles) from the mitochondrial intermembrane space into the cytoplasm to activate the effector caspases that execute the cell. Venetoclax (yellow) binds Bcl-2 displacing an activator BH3 protein, allowing the downstream activation of Bax, permeabilization of MOM, and cell death. (ii) Apoptotic response of CLL cells stimulated by microenvironment. Microenvironmental survival signals increase expression of the antiapoptotic Bcl-2 family members Bcl-xl, Mcl-1, and A1 that are not targeted by venetoclax and/or the downregulation of the proapoptotic protein Bax. The excess antiapoptotic proteins bind active BH3 proteins displaced from Bcl-2 by venetoclax preventing them from activating Bax thereby inhibiting apoptosis. (iii) Sunitinib turns off microenvironmental prosurvival signals preventing increased expression of the antiapoptotic proteins other than Bcl-2 and thereby restoring sensitivity to venetoclax.