Figure 2.
A subgroup of CLL does not undergo spontaneous apoptosis in absence of stroma support. (A) PBMCs were isolated from samples from 23 patients with CLL and plated in 384-well plates in BM. After 24 hours, cell viability was measured using Cell Titer-Glo. Blue line represents 75% cell viability. Percent cell viability is calculated relative to day 0 (100%). (B) Cell death was measured in 7 PBMC samples whose viability >75% (CMI) and in 5 PMBC samples whose viability <75% (CMD). PBMCs from both groups were seeded in 6-well plates in presence of BM. After 48 hours, cell death was measured by annexin V/Hoechst staining. Percent cell death is calculated as 100% live cells (viable cells [negatively stained, lower left quadrants]). Results represent the mean of 4 samples per group. Error bars represent SEM, P values were calculated using Welch t test, and P value < .05 indicate statistical significance. (C) PBMCs from both groups (CMD and CMI) were grown in CM and treated with increasing doses of venetoclax (0 nM, 1 nM, and 5 nM) for 48 hours. Cell viability was measured using Cell Titer-Glo (percentage relative to dimethyl sulfoxide). (D) IC50 values of venetoclax segregate based on CM dependence (CMD, n = 9 and CMI, n = 7). Results represent the mean per group. Error bars represent SEM, P values were calculated using Welch t test, and P value < .05 indicate statistical significance. (E) Upper, Western blot analysis of whole cell extracts using the MCL1 antibody from both CLL groups. Actin was used as loading control. Lower, densitometry analysis was performed for images shown in upper panel. P values were calculated between the 2 groups using Welch t test, P value = .0004 for the comparison of CMD vs CMI samples. (F) Upper, heatmap of BH3 profile performed on freshly isolated CLL PBMCs, 5 samples from patients were derived from each group (CMD, patient# 1, 2, 3, 4, and 7 and CMI, patient# 15, 20, 21, 22 and 23 as defined in panel A of Figure 2) using BIM and BAD peptides. Percent cytochrome C release (indicative of mitochondrial outer membrane permeabilization) is calculated for each BH3 peptide treatment relative to maximum value of negative control (dimethyl sulfoxide). Lower, graphs showing difference in cytochrome C release in response to BIM and PUMA peptides between both groups. Means are depicted as horizontal bars ± standard error bars, P values were calculated using Welch t test, and P value < .05 indicate statistical significance. (G) Left, PBMCs were cultured in BM for 24 hours and their cell viability was measured using Cell Titer-Glo. Percent Cell viability is calculated relative to day 0 (100%). IGVH status of the 23 samples were compared with their viability. Right, the table showing the odds ratio is ∼17:1 for CMI status for IGHV-U vs IgHV-M with a Fisher exact test P value = .01. IGHV-M, Immunoglobulin heavy-chain variable region-Mutated; IGHV-U, Immunoglobulin heavy-chain variable region-Unmutated.

A subgroup of CLL does not undergo spontaneous apoptosis in absence of stroma support. (A) PBMCs were isolated from samples from 23 patients with CLL and plated in 384-well plates in BM. After 24 hours, cell viability was measured using Cell Titer-Glo. Blue line represents 75% cell viability. Percent cell viability is calculated relative to day 0 (100%). (B) Cell death was measured in 7 PBMC samples whose viability >75% (CMI) and in 5 PMBC samples whose viability <75% (CMD). PBMCs from both groups were seeded in 6-well plates in presence of BM. After 48 hours, cell death was measured by annexin V/Hoechst staining. Percent cell death is calculated as 100% live cells (viable cells [negatively stained, lower left quadrants]). Results represent the mean of 4 samples per group. Error bars represent SEM, P values were calculated using Welch t test, and P value < .05 indicate statistical significance. (C) PBMCs from both groups (CMD and CMI) were grown in CM and treated with increasing doses of venetoclax (0 nM, 1 nM, and 5 nM) for 48 hours. Cell viability was measured using Cell Titer-Glo (percentage relative to dimethyl sulfoxide). (D) IC50 values of venetoclax segregate based on CM dependence (CMD, n = 9 and CMI, n = 7). Results represent the mean per group. Error bars represent SEM, P values were calculated using Welch t test, and P value < .05 indicate statistical significance. (E) Upper, Western blot analysis of whole cell extracts using the MCL1 antibody from both CLL groups. Actin was used as loading control. Lower, densitometry analysis was performed for images shown in upper panel. P values were calculated between the 2 groups using Welch t test, P value = .0004 for the comparison of CMD vs CMI samples. (F) Upper, heatmap of BH3 profile performed on freshly isolated CLL PBMCs, 5 samples from patients were derived from each group (CMD, patient# 1, 2, 3, 4, and 7 and CMI, patient# 15, 20, 21, 22 and 23 as defined in panel A of Figure 2) using BIM and BAD peptides. Percent cytochrome C release (indicative of mitochondrial outer membrane permeabilization) is calculated for each BH3 peptide treatment relative to maximum value of negative control (dimethyl sulfoxide). Lower, graphs showing difference in cytochrome C release in response to BIM and PUMA peptides between both groups. Means are depicted as horizontal bars ± standard error bars, P values were calculated using Welch t test, and P value < .05 indicate statistical significance. (G) Left, PBMCs were cultured in BM for 24 hours and their cell viability was measured using Cell Titer-Glo. Percent Cell viability is calculated relative to day 0 (100%). IGVH status of the 23 samples were compared with their viability. Right, the table showing the odds ratio is ∼17:1 for CMI status for IGHV-U vs IgHV-M with a Fisher exact test P value = .01. IGHV-M, Immunoglobulin heavy-chain variable region-Mutated; IGHV-U, Immunoglobulin heavy-chain variable region-Unmutated.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal