Journal: Lymphatics

Article Title: BCL2 Protein Progressively Declines during Robust CLL Clonal Expansion: Potential Impact on Venetoclax Clinical Efficacy and Insights on Mechanism

doi: 10.3390/lymphatics2020005

Figure Lengend Snippet: Distinct modulation of BCL2 and MCL1 protein levels during ODN + IL15-induced CLL cell cycling. ( A ) U-CLL950 cells were evaluated for intracellular BCL2 and MCL1 protein both at d0 and following 5 d of ODN + IL15 stimulation. PE-fluorescent intensity representing specific mAb (open red histograms) or respective Ig isotype controls (grey filled) was measured on viability-gated cells by flow cytometry. Inserted values represent the ratio median fluorescence intensity (RMFI) of specific versus control mAb. ( B ) Comparison of BCL2 levels in U-CLL1013 cells following d6 culture with medium alone or ODN + IL15 (non-activating and activating conditions), respectively [ , , ]. ( C ) Two-color flow cytometry with CFSE-labeled M-CLL1328 CLL cells was used to monitor BCL2 and MCL1 protein levels as a function of division status. Top 2 histograms display CFSE fluorescence within viability-gated cells from d6 cultures with IL15 alone or activating ODN + IL15. While the former remains undivided, the latter shows over 5 cycles of division, as noted earlier with this clonal population . Bottom 6 histograms represent BCL2-PE (left column) and MCL1-PE (right column) fluorescence (shaded = Ig control) within gated division subsets of ODN + IL15-stimulated cells (values = RMFI). ( D , E ) Summary plots of RMFI values per division for all ODN + IL15-stimulated CLL populations tested (( D ) = BCL2; ( E ) = MCL1). (Of note: Maximal BCL2 protein level appeared to be an intrinsic attribute of the individual CLL clone since RMFI values were quite similar when 5/6 CLL populations were assessed for BCL2 in two separate experiments. For the one exception (M-CLL1328), where a 3-fold difference in RMFI values was noted between experiments, a mean value was used. ( F , G ) Plots showing mean RMFI (±SD) for ( F ) BCL2 and ( G ) MCL1 expression per each division subset of the CLL panel represented in ( D , E ). Paired, 2-tailed t -tests were used for assessing the statistical significance of differences between levels in the undivided subset as opposed to each division subset, as well as BCL2 expression changes within cells at successive divisions (1 vs. 2 div; 2 vs. 3; and 3 vs. 4 div); all differences were found to be statistically significant. In ( F – I ) below, 2 CLL populations (M-CLL600 and M-CLL1300), whose individual BCL2/MCL1 values are plotted in D-E, were not used for statistical analyses of CLL pools in ( F – I ): M-CLL600 due to insufficient cell numbers for assessment at ≥3 divisions; M-CLL1300 due to unreliability of assessing cells with ≥4 divisions (>50% CD19-negative; ). ( H ) Comparative BCL2 levels in pooled M-CLL (n = 6) and pooled U-CLL (n = 6), gated by division subsets; no statistically significant difference was noted by unpaired t -test (2-sided or 1-sided). ( I ) Comparative MCL1 levels in pooled M-CLL (n = 4) and U-CLL (n = 6). While mean MCL1 expression in all U-CLL division subsets exceeded that of M-CLL, the difference was only of borderline statistical significance at 2 divisions ( p = 0.09 by unpaired, 1-sided t -test). In a separate statistical analysis in which M-600 and M-1300 were included in pools for comparing MCL1 levels in U-CLL versus M-CLL cohorts, the difference between U-CLL and M-CLL at 2 divisions became statistically significant ( p = 0.04) by unpaired, 1-sided t -test.

Article Snippet: To monitor cell divisions, CLL clonal populations were pre-labeled with CFSE [ ] and cultured for 5–7 days in an enriched medium optimized for B cell viability [ , ], at 10 5 cells/200 μL in 96-well culture plates, with synergistic activating stimuli [ , , ]: CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA, USA; final concentration of 0.2 mM) and recombinant human IL15 (R&D Systems, Minneapolis, MN, USA; final concentration of 15 ng/mL).

Techniques: Flow Cytometry, Fluorescence, Control, Comparison, Labeling, Expressing

Journal: Lymphatics

Article Title: BCL2 Protein Progressively Declines during Robust CLL Clonal Expansion: Potential Impact on Venetoclax Clinical Efficacy and Insights on Mechanism

doi: 10.3390/lymphatics2020005

Figure Lengend Snippet: BCL2 inhibitor and survivin inhibitor preferentially target non-cycling and cycling CLL cells, respectively. ( A ) M-CLL1031 experiments comparing quiescent (IL15 only) and cycling (ODN + IL15) d5–6 cultures for sensitivity to venetoclax (50 nM) and YM155 (333 nM): inhibitors added during the last 24–36 h of d5(6) cultures. Following harvest, CFSE-labeled cells were gated into viable and dead subsets by SSC and V450-Pacific Blue dye exclusion, and both subsets were assessed for division (open histograms = viable; shaded histograms = dead). ( B,C ) Pooled viability analysis of CLL cultures stimulated with either IL15 or ODN + IL15 and subsequently pulsed with varying doses of either ( B ) venetoclax (n = 3 CLL exp: U-CLL966, M-CLL275 and M-CLL1031) or ( C ) YM155 (n = 3 CLL exp: U-CLL321, M-CLL849, and M-CLL1031). Data expressed as % of control with vehicle (mean ± SD). In these pooled CLL experiments, inhibitors were added during the last 36–48 h of d5–6 cultures. (Note: in a separate experiment with U-CLL1013 cells cultured for 39 h with medium or IL15 alone, both populations were equally sensitive to venetoclax; data not shown). * indicates statistically significant difference in viability when compared to control cultures pulsed with vehicle (in each exp normalized to 100%). pooled analysis of gated undivided and divided blasts from ODN + IL15-stimulated cultures for ( D , E ) viability and ( F , G ) absolute yield of viable blasts following pulse with venetoclax ( D , F ) or YM155 ( E , G ). * indicates p < 0.05 and > 0.001 while ** indicates p < 0.001 when values in inhibitor-pulsed cultures are compared with parallel cultures with vehicle alone. ( H , I ) ODN + IL15-induced blasts with differing division histories were compared for ( H ) overall viability and ( I ) absolute yield of viable cells per culture following exposure to venetoclax or YM155. Data expressed as % of values in parallel cultures pulsed with vehicle alone (mean ± SD). Experiments with venetoclax (n = 5) involved the following CLL: U-284, U-675, U-966, U-1013, and M-1031. Experiments with YM155 (n = 5) involved U-321, M-849, U-1013, M-1031, and U-1692.

Article Snippet: To monitor cell divisions, CLL clonal populations were pre-labeled with CFSE [ ] and cultured for 5–7 days in an enriched medium optimized for B cell viability [ , ], at 10 5 cells/200 μL in 96-well culture plates, with synergistic activating stimuli [ , , ]: CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA, USA; final concentration of 0.2 mM) and recombinant human IL15 (R&D Systems, Minneapolis, MN, USA; final concentration of 15 ng/mL).

Techniques: Labeling, Control, Cell Culture

Journal: Lymphatics

Article Title: BCL2 Protein Progressively Declines during Robust CLL Clonal Expansion: Potential Impact on Venetoclax Clinical Efficacy and Insights on Mechanism

doi: 10.3390/lymphatics2020005

Figure Lengend Snippet: Phase microscopy of ODN + IL15-stimulated cultures exposed to YM155 survivin inhibitor or vehicle alone. U-CLL1692 cells were stimulated with ODN + IL15; pulsed on d4 with vehicle alone (DMSO) ( A ) or YM155 (333 nM) ( B ); and examined by phase microscopy 24 h later. Note the evidence of shrunken cells with membrane blebs (apoptosis) in YM155-treated cultures. Also evident are cells with intracellular structures reminiscent of statically aligned chromosomes during the M phase of the cell cycle (black stars).

Article Snippet: To monitor cell divisions, CLL clonal populations were pre-labeled with CFSE [ ] and cultured for 5–7 days in an enriched medium optimized for B cell viability [ , ], at 10 5 cells/200 μL in 96-well culture plates, with synergistic activating stimuli [ , , ]: CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA, USA; final concentration of 0.2 mM) and recombinant human IL15 (R&D Systems, Minneapolis, MN, USA; final concentration of 15 ng/mL).

Techniques: Microscopy, Membrane

Journal: Lymphatics

Article Title: BCL2 Protein Progressively Declines during Robust CLL Clonal Expansion: Potential Impact on Venetoclax Clinical Efficacy and Insights on Mechanism

doi: 10.3390/lymphatics2020005

Figure Lengend Snippet: Deletion of Chr13 loci encoding miR15/miR16 is linked to greater BCL2 protein during clonal expansion. ( A,B ) BCL2 expression (RMFI) versus division frequency within ODN + IL15-stimulated CLL populations segregated into ( A ) del(13q)−/− and ( B ) del(13q)+ CLL cohorts ( C ) Box plot analysis of pooled experiments comparing BCL2 within undivided and divided blasts of del(13q)+ and del(13q)−/− groups. Note: BCL2 expression in cells with 3 divisions was used as representative of dividing cells because (a) CLL populations vary in the extent of cycling and (b) all stimulated CLL (excepting M-CLL600 and U-CLL1300) had reliable numbers of cells representing 3 divisions for analysis. Statistical comparisons of BCL2 levels within undivided versus divided cells of the same cohort involved a 2-sided, paired t -test. When differing cohorts were compared, a 2-sided, unpaired t-test was employed to determine statistical significance. ( p values indicated in parenthesis represent the use of a 1-sided t -test), ( D ) Values for % decline in BCL2 obtained by comparing RMFI in cells with 3 divisions versus RMFI in respective undivided CLL cells. ( E,F ) MCL1 protein expression versus division frequency in ( E ) del(13q)−/− and ( F ) del(13q)+ CLL. ( G ) Box plot analysis of maximal MCL1 within viable cells of del(13q)+ versus del(13q)−/− CLL cohorts stimulated with ODN + IL15. ( H ) Comparison of del(13q)+ and del(13q)−/− CLL cohorts for cell division at which maximal MCL1 was noted.

Article Snippet: To monitor cell divisions, CLL clonal populations were pre-labeled with CFSE [ ] and cultured for 5–7 days in an enriched medium optimized for B cell viability [ , ], at 10 5 cells/200 μL in 96-well culture plates, with synergistic activating stimuli [ , , ]: CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA, USA; final concentration of 0.2 mM) and recombinant human IL15 (R&D Systems, Minneapolis, MN, USA; final concentration of 15 ng/mL).

Techniques: Expressing, Comparison

Journal: Lymphatics

Article Title: BCL2 Protein Progressively Declines during Robust CLL Clonal Expansion: Potential Impact on Venetoclax Clinical Efficacy and Insights on Mechanism

doi: 10.3390/lymphatics2020005

Figure Lengend Snippet: p53 protein rises during ODN + IL15-induced B-CLL cycling, while TP53 mRNA declines. ( A ) p53-protein PE fluorescence (solid lines) versus control IgG (dashed) in viability-gated U-CLL430 cells from d5 cultures with IL-15 alone or ODN + IL15. Values represent RMFI and Δ MFI (difference between geometric MFI with specific versus control mAb). ( B ) p53 protein within CFSE-labeled M-CLL922 cells from 5 d cultures with ODN + IL15. Two-color dot plot (right) reveals p53 protein (red) within viability-gated cells of gated division subsets (control stain = grey). ( C ) Kinetic analysis of p53 protein expression within CFSE-labeled U-CLL430 cells cultured for d3 to d5 days with IL15 alone, ODN alone, or ODN + IL15. Rectangle designates the undivided subset. In these dot plots, staining with p53-PE (red) overlaid with Ig Ctrl-PE (grey) staining in cells of varying divisions. ( D ) Summarized display of data in ( C ) as p53 levels (RMFI) per division over d3 to d6. (Of note, excepting d3, all daily p53 staining analyses included a defrosted sample of the CL-01 B cell line, used for standardization of p53 staining; all values for adjustment were < 2-fold.) ( E ) Levels of p53α (canonical isoform) were measured by densitometric analysis of both p53 and β-actin blots followed by calculation of p53α/actin ratio for each assessed lysate (M-CLL1031 exp with 31 μg lysate/lane where * indicates cultures supplemented with caspase-2 inhibitor; U-CLL675 exp with 20 μg/lane; U-CLL625 exp with 15 μg/lane, excepting ODN ** only, with 6 μg/lane). The bands above p53α likely represent its ubiquitinated forms , while any bands below p53 isoforms from differential splicing . ( F ) Box plot summarizing data (RATIO of p53/actin) from blotting experiments comparing lysates from IL15-only cultures to those from parallel ODN + IL15 cultures. Box plots are overlaid with values from individual lysates. Statistical comparisons of IL15 only versus IL15 + ODN involved the non-parametric Mann–Whitney Rank Sum test. ( G,H ) q-PCR assessments of TP53 mRNA in CLL cells; ( G ) ΔCt values computed from the difference between threshold cycle for test TP53 cDNA and threshold cycle for reference β-actin ( ACTB ); ( H ) TP53 mRNA fold change calculated using within 2 −ΔΔCt method , to facilitate comparison of TP53 mRNA levels in cells with versus without IL15 exposure. Statistical analysis by paired, 2-sided t- test.

Article Snippet: To monitor cell divisions, CLL clonal populations were pre-labeled with CFSE [ ] and cultured for 5–7 days in an enriched medium optimized for B cell viability [ , ], at 10 5 cells/200 μL in 96-well culture plates, with synergistic activating stimuli [ , , ]: CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA, USA; final concentration of 0.2 mM) and recombinant human IL15 (R&D Systems, Minneapolis, MN, USA; final concentration of 15 ng/mL).

Techniques: Fluorescence, Control, Labeling, Staining, Expressing, Cell Culture, MANN-WHITNEY, Comparison

Journal: Lymphatics

Article Title: BCL2 Protein Progressively Declines during Robust CLL Clonal Expansion: Potential Impact on Venetoclax Clinical Efficacy and Insights on Mechanism

doi: 10.3390/lymphatics2020005

Figure Lengend Snippet: IL-15-triggered STAT5/PI-3K pathway reduces BCL2 mRNA within ODN (TLR9)-primed CLL cells. These experiments sought evidence for altered BCL2 mRNA levels after IL15 exposure to ODN-stimulated CLL cells. ODN priming was important because we earlier reported that 20 h ODN exposure elicited the NF-kB-dependent upregulation of IL15 receptors (CD122 and IL15Rα) above negligible baseline levels on resting CLL cells . Any evidence that short-term IL15 exposure resulted in dampened BCL2 mRNA might be explained by an IL15-fostered rise in BCL2 repressive miR (e.g., miR15a/miR16-1) . Our rationale for suspecting a critical role for IL15 cytokine in mediating BCL2 repression was based upon the following. First, IL15 signaling was necessary for a significant rise in p53 TF protein within CLL cells receiving ODN signals ( - ), as well as for significant CLL clonal expansion within ODN-stimulated cultures [ , , ]. Second, the IL15 boost in p53 TF (a direct transactivator of miR15a/miR16-1 synthesis) was detected in undivided CLL cells by at least day 3, even prior to IL15-driven divisions characterized by sustained/further elevated p53 TF protein ( , ). For these experiments, quiescent CLL cells were primed with ODN for 20 h and subsequently pulsed with IL-15 (or medium alone) for 4 h or 20–28 h intervals before total RNA was harvested and BCL2 and MCL1 mRNA quantified by specific q-PCR. ( A,B ) ΔCt values for ( A ) BCL2 mRNA and ( B ) MCL1 mRNA within IL15-pulsed or un-pulsed cultures at differing intervals after the IL15 pulse. A paired, 2-sided t-test was used to compare ΔCt values from primed cultures with/without IL15. ( C ) More intuitive fold-change values, calculated with the 2 −ΔΔCt method , better reveal the altered levels of BCL2 and MCL1 mRNA at 20–28 h after the IL15 pulse. Bars represent the mean ± SD of the diverse experiments, with overlaid symbols representing values from individual CLL. Statistical significance was determined by the non-parametric Mann–Whitney rank sum test. ( C ) Experiments with STAT5/PI-3K inhibitors examined whether IL15 activation of STAT5/PI-3K pathways is critical for IL15-triggered BCL2 mRNA repression, as earlier noted for IL15-facilitated growth of ODN-primed CLL cells . Specific inhibitors of PI-3K (LY294002), STAT5 (pimozide or STAT5 INH II), or vehicle alone were added to ODN-primed cultures 30 min prior to a 20 h pulse with IL-15. By q-PCR, the yield of BCL2 mRNA in IL15 pulsed cultures was compared to the yield in ODN-primed cultures that had been exposed to DMSO but not IL15. Consequently, IL15-induced fold change in BCL2 mRNA was determined. (Note: in cultures pulsed with DMSO alone, IL15-elicited fold change in BCL2 mRNA was 0.57 ± 0.04 (mean ± SD) for the diverse experiments). When pooling inhibitor results from these experiments, data from each CLL experiment was normalized by comparing IL15-induced fold change “with inhibitor” to IL15-induced fold change “with DMSO alone”. From the latter determinations, plotted values for % of IL15-elicited fold change without inhibitor” could be calculated. Bar blots display median/range values for the above determination noted with the diverse CLL evaluated, with overlaid symbols representing values for individual CLL evaluated. The dotted horizontal line represents the fact that, with this approach, all values for “IL15-induced fold change without inhibitor” are effectively normalized to 100%. Any rise in this relative percentage indicates that the inhibitor blocked the IL15-induced BCL2 mRNA decline. p values for statistical significance were determined using a non-parametric Mann–Whitney rank sum test.

Article Snippet: To monitor cell divisions, CLL clonal populations were pre-labeled with CFSE [ ] and cultured for 5–7 days in an enriched medium optimized for B cell viability [ , ], at 10 5 cells/200 μL in 96-well culture plates, with synergistic activating stimuli [ , , ]: CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA, USA; final concentration of 0.2 mM) and recombinant human IL15 (R&D Systems, Minneapolis, MN, USA; final concentration of 15 ng/mL).

Techniques: MANN-WHITNEY, Activation Assay

Journal: Lymphatics

Article Title: BCL2 Protein Progressively Declines during Robust CLL Clonal Expansion: Potential Impact on Venetoclax Clinical Efficacy and Insights on Mechanism

doi: 10.3390/lymphatics2020005

Figure Lengend Snippet: B-CLL populations with del(13q) manifest heightened p53 protein during cycling. ( A ) del(13q)−/− CLL or ( B ) del(13q)+ CLL cohorts were assessed for intracellular p53 protein during ODN + IL15-triggered clonal expansion. Histograms indicate p53-PE (RMFI) within blasts of varying division history. ( C ) Box plots comparing p53 levels within undivided and divided cells within del(13q)−/− and del(13q)+ CLL cohorts. ( D–G ) Plots of p53 expression versus division frequency in ODN + IL15-stimulated CLL populations, segregated on the basis of FISH-determined chromosomal anomalies . ( H ) Box plot analysis of p53 levels (as in ( C )) with the exclusion of Tri12+ CLL from analysis. Statistical analyses involved a paired, 2-sided t -test (comparisons of undivided versus divided cells of the same cohort) and an unpaired, 2-sided t -test (inter-cohort comparisons). When non-parametric p53 data distribution was detected in the del13−/− cohort ( C ), statistical evaluations involving the latter were performed with the Mann–Whitney rank sum test.

Article Snippet: To monitor cell divisions, CLL clonal populations were pre-labeled with CFSE [ ] and cultured for 5–7 days in an enriched medium optimized for B cell viability [ , ], at 10 5 cells/200 μL in 96-well culture plates, with synergistic activating stimuli [ , , ]: CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA, USA; final concentration of 0.2 mM) and recombinant human IL15 (R&D Systems, Minneapolis, MN, USA; final concentration of 15 ng/mL).

Techniques: Expressing, MANN-WHITNEY