Coculture of primary CLL cells promotes resistance to venetoclax that was overcome by sunitinib independent of IGHV mutation status. Primary CLL cells from 6 patients were cultured in suspension (unstimulated) or in coculture with bone marrow stromal cells (OP9) expressing human CD40L (CD40L) and treated with the indicated concentrations of venetoclax, sunitinib (SUN) alone or in combination or as a positive cell death control with STS and venetoclax. Drug response was determined by flow cytometric analysis of Annexin V (AV) and propidium iodide (PI)-stained CLL cells 48 hours after drug treatment. (A) Dot plots are shown for unstimulated suspension CLL cells (i) and CD40L-stimulated CLL cells (ii) for 1 representative patient. Numbers in dot blots indicate percentages of PI⁺ cells (top left quadrant) and Annexin V + PI⁺⁺ cells (top right quadrant). (B) Percentage of PI/Annexin V + PI⁺ cells for unstimulated CLL cells (i) and CD40L-stimulated CLL cells (ii) from 6 patients 48 hours after the indicated drug treatment. Cells double stained with Annexin V and PI (AV + PI, top right in dot blots) or PI⁺ cells (top left corner in dot blots) were counted as nonviable. Cells positive for only Annexin V (bottom right corner) were excluded due to possible interference from sunitinib fluorescence. On average, venetoclax induced only 8% cell kill (n = 6) in CD40-cocultured cells compared with 60% cell kill in unstimulated cells. Sunitinib (3 µM) increased the total kill to >80%. Similar results were obtained for cells cocultured with OP9 stromal cells (supplemental Figure 3). (C) DNA methylation maturation scores for 24 CLL samples separated into methylation subtypes as assessed by MassARRAY. Consensus clustering of DNA methylation levels of a panel of 6 genes was used to classify 3 clusters of CLL cases based on their degree of DNA methylation maturation (blue: low [LP-CLL]; yellow: intermediate [IP-CLL]; and red: high [HP-CLL]) programmed CLL (LP-CLL, n = 7; IP-CLL, n = 5; HP-CLL, n = 12). (D) Drug response to venetoclax (10 nM), sunitinib (1 µM) alone or in combination in 2S-stimulated CLL cells from 16 patients separated by DNA methylation subgroup as in panel C and IGHV mutation status. Gray filled symbols, mutated IGHV; white filled symbols, unmutated IGHV; solid color symbols, IGHV status data not available. IGHV sequence homology of <98% vs germline was considered as mutated. Percentage positive indicates percentage of dead cells determined by multiparametric image analysis as described in Figure 2. Data for CLL cells cocultured with OP9 control stromal cell lines are shown in supplemental Figure 2.