Journal: Cell Death Discovery

Article Title: IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform

doi: 10.1038/s41420-021-00523-y

Figure Lengend Snippet: Thirteen B-cell leukemia cell lines; 7 Ph-positive consisting of KOPN30bi (Ph1), KOPN55bi (Ph2), KOPN57bi (Ph3), KOPN66bi (Ph4), KOPN72bi (Ph5), KOPN83bi (Ph6), and KCB1 (Ph7) and 6 Ph-negative consisting of 2 TCF3-PBX1 positive, 3 ETV6-RUNX1 positive, and MLL-ENL positive, were used for analysis. A RT-PCR analysis of IKZF1 isoforms in B-cell leukemia cell lines. Primer sequences for RT-PCR analysis of IKZF1 were as follows: forward primer 5′-AAAGCGCGACGCACAAATCC-3′, reverse primer 5′-ATGGCGTTGTTGATGGCTTGGTC-3′ . B Western blot analysis of IKZF1 expression before and after LEN treatment. B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN (Cayman Chemical, Ann Arbor, MI), and IKZF1 expression was examined on western blot. Anti-IKZF1 antibody was obtained from R&D Systems (Minneapolis, MN). C RT-PCR analysis of IKZF3 isoforms in B-cell leukemia cell lines. Primer sequences for RT-PCR analysis of IKZF3 were as follows: forward primer 5′-ATGGAAGATATACAAACAAATGCGGA-3′), reverse primer 5′-AGAGAGGCCTGTGTGAGAAGGCAC-3′ . D Western blot analysis of IKZF3 expression before and after LEN treatment. B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN, and IKZF3 expression was examined on western blot. Anti-IKZF3 antibody was obtained from Proteintech (Rosemont, IL). E Comparison of decrease in IKZF1 and IKZF3 expression after LEN treatment in B-cell leukemia cell lines. In B and D , areas of IKZF1 (excluding Ik6) and IKZF3 were quantified by densitometry, and %decrease after LEN treatment was calculated as {1−[(area of LEN+)/(area of LEN−)]} × 100. The results were shown by box-whisker plots. Comparison was done by Mann–Whitney U test. Left panel: the %decrease in IKZF1 expression in Ph+ group A cell lines ( n = 4, median 90.4, range 78.4–99.9) was significantly ( p < 0.01) greater than that in non-Ph cell lines ( n = 6, median 49.5, range 32.0–65.0). Right panel: the %decrease in IKZF3 expression in Ph+ cell lines ( n = 6, median 85.0, range 58.7–89.7) was significantly ( p < 0.01) higher than that in non-Ph cell lines ( n = 6, median 50.2, range 36.3–67.5). F Time-course of IKZF1 and IKZF3 isoforms expression after LEN treatment. KOPN57bi cells were cultured with 10 µM LEN for 1, 3, 6, and 24 h, and changes in IKZF1 and IKZF3 expression was examined on western blot. G Effect of neddylation activating enzyme inhibitor MLN4924 on IKZF1 and IKZF3 isoforms expression after LEN treatment. KOPN57bi cells were cultured for 24 h in the presence or absence of 10 µM LEN with or without 30 min-pretreatment with 0.25 µM MLN4924 (ChemScene, Monmouth Junction, NJ), and changes in expression of IKZF1 and IKZF3 were examined on western blot.

Article Snippet: B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN (Cayman Chemical, Ann Arbor, MI), and IKZF1 expression was examined on western blot.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Cell Culture, Whisker Assay, MANN-WHITNEY

Journal: Cell Death Discovery

Article Title: IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform

doi: 10.1038/s41420-021-00523-y

Figure Lengend Snippet: A Analysis of thymidine uptakes after LEN treatment. Seven Ph+ and 12 non-Ph cell lines were cultured for 72 h in the presence or absence of 10 µM LEN, and % inhibitions of thymidine uptakes by LEN were compared by Mann–Whitney U test. Left panel: Comparison of % inhibitions between Ph+ cell lines ( n = 7) including group A (closed triangles, n = 4) and group B (closed circles, n = 3) and non-Ph cell lines (open circles, n = 12). The % inhibition in group A Ph+ cell lines ( n = 4, median 52.5%, range 46.7–58.3%) was significantly ( p < 0.01) greater than in non-Ph cell lines ( n = 12, median 30.4%, range 15.1–47.9%). Right panel: Comparison of % inhibition between Ph+ group A ( n = 4) and non-Ph ( n = 12) cell lines. The results were shown as the box-and-whisker plots. B Time-course of % inhibition of thymidine uptakes in Ph+ and non-Ph cell lines after LEN treatment. Ph3 (KOPN57bi) and non-Ph (KOPN63) cells were cultured in the presence or absence of 10 µM LEN, and thymidine uptake was measured at days 1, 2, 3, 4, and 5. P values were calculated by unpaired t -test. A significant difference in % inhibition was observed in Ph3 starting day 3 and thereafter. C Analysis of cell cycle progression after LEN treatment. KOPN57bi cells were cultured for 72 h in the presence or absence of 10 µM LEN and cell cycle progressions were analyzed by PI staining. Left panel: The representative flow cytogram showing a decrease in the S (M3) and G2/M (M4) phases in the presence of LEN. The X and Y axes indicate DNA content and cell numbers, respectively. Right panel: Experiments were performed 4 times. A significant decrease in the S (from 21.5 ± 3.0 % to 14.9 ± 2.2%, p < 0.002) and the G2/M (from 16.0 ± 5.4% to 11.2 ± 2.2%, p < 0.05) phases and a significant increase in the subdiploid region (from 2.8 ± 1.3% to 7.5 ± 4.4%, p < 0.05) were documented after LEN treatment. The G0/G1 phase was increased from 59.9 ± 8.4% to 66.1 ± 6.7%, after LEN treatment, but this difference was not significant. D Western blot analysis of changes in expression of CDKs, CDKIs, and cyclins after LEN treatment. KOPN57bi and non-Ph (KOCL58) cells were cultured for 72 h in the presence or absence of 10 µM LEN and expression of cell cycle regulators was examined on western blot. The bands were quantified by densitometry and shown as the relative to the control after compensation by α-tubulin expression. Primary antibodies were from Cell Signaling Technology (Beverly, MA) for cyclin B1, CDK6, and p27, from BD Bioscience (Franklin Lakes, NJ) for cyclin D3, cyclin E, CDK4, and p21, from Santa Cruz Biotechnology (Dallas, TX) for cyclin A, from BioLegend (San Diego, CA) for CDK2, and from SIGMA-Aldrich for α-tubulin. Expression of cyclins D3, E, and CDK2 was downregulated in the presence of LEN.

Article Snippet: B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN (Cayman Chemical, Ann Arbor, MI), and IKZF1 expression was examined on western blot.

Techniques: Cell Culture, MANN-WHITNEY, Inhibition, Whisker Assay, Staining, Western Blot, Expressing

Journal: Cell Death Discovery

Article Title: IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform

doi: 10.1038/s41420-021-00523-y

Figure Lengend Snippet: KOPN57bi cells were cultured for 72 h in the assays A-G or for 14 days in the colony formation assay I in the presence or absence of either 5 μM LEN or 0.5 μM IM alone (Selleck Chemicals; Houston, TX), or both. The results were compared by unpaired t -test. A Thymidine uptake assays. The LEN-induced inhibition of thymidine uptake was significantly ( p < 0.01) enhanced to 87.4 ± 1.2% in the presence of IM when compared with LEN alone (34.5 ± 2.5%). B AlamarBlue cytotoxic assays. Leukemia cells were cultured in the presence or absence of reagents and followed by 5h-incubation with alamarBlue (Bio-Rad Laboratories, Hercules, CA, RRID:SCR_008426). The LEN-induced decrease in the cell viability was significantly ( p < 0.01) enhanced to 38.7 ± 2.6% in the presence of IM when compared with LEN alone (75.7 ± 8.8%). C Changes in CH50 of IM in the absence or presence of LEN in alamarBlue cytotoxic assays. alamarBlue cytotoxic assays were performed at different concentrations of IM in the presence or absence of 5 μM LEN. The IC50 of IM was shifted to 0.088 μM in the presence of LEN from 1.05 μM in the absence of LEN. The X and Y axes indicate log concentrations of IM and cell viability, respectively. D Demonstration of synergism. alamarBlue cytotoxic assays were performed at 6 concentrations (0.07, 0.2, 0.67, 2.0, 6.0, and 18 μM) of LEN and/or 3 concentrations (0.125, 0.25, and 0.5 μM) of IM. For The combination index (CI) was determined by median-effect analysis using the CalcuSyn software (Biosoft, Ferguson, MO). The CI was calculated in each culture condition and defined as synergistic ≤0.8, additive 0.8<, and antagonistic 1.2<, according to the report by Chou TC et al. . The X and Y axes indicate log concentrations of LEN and CI, respectively. E Flow cytometric analysis of apoptosis by PI staining. The marked increase in the subdiploid apoptotic fraction (42.3%) by LEN and IM treatment was revealed, although this population was only 14.3% or 18.1% by either LEN or IM treatment, respectively. The X and Y axes indicate DNA content and cell numbers, respectively. F Effect of pretreatment with pan-caspase inhibitor Z-VAD FMK. Cells were cultured with or without 30 min-pretreatment with pan-caspase inhibitor Z-VAD FMK (20 μM), and flow cytometric analysis of PI staining was performed. The subdiploid population was decreased to 12.2% accompanied with an increase in the G0/G1 population (74.4%) by pretreatment. The X and Y axes indicate DNA content and cell numbers, respectively. G Flow cytometric analysis of apoptosis by Annexin-V/PI staining. The marked increase in the late apoptotic population from 9.5 to 53.0% (in the right/upper quadrant) and the decrease in the viable population from 84.8 to 28.0% (in the left/lower quadrant) were demonstrated by LEN and IM treatment. The X and Y axes indicate log fluorescence intensities of Annexin-V and PI, respectively. H Flow cytometric analysis of apoptosis by cleaved caspase 3 antibody. Cells were cultured with or without 30 min-pretreatment with pan-caspase inhibitor Z-VAD FMK (20 μM), and were stained using active caspase-3 apoptosis kit (BD Biosciences, San Jose, CA, USA) after permeabilization and fixation. The X and Y axes indicate log fluorescence intensities and cell numbers, respectively. I Colony formations assays. KOPN57bi cells (5.0 × 10 4 per dish) were suspended in a semi-solid α-MEM medium containing 1% methyl cellulose, 20% FCS, 10% fetal bovine albumin, and 100 µM 2-mercaptoethanol, and incubated in duplicate in 35 mm dishes at 37 °C for 14 days with or without LEN and/or IM. The number of colonies (>50 cells) was counted on light microscopy. LEN alone suppressed colony formation significantly stronger than did IM alone, and cotreatment with LEN and IM further suppressed the colony formation up to 4.2% of the control.

Article Snippet: B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN (Cayman Chemical, Ann Arbor, MI), and IKZF1 expression was examined on western blot.

Techniques: Cell Culture, Colony Assay, Inhibition, Incubation, Software, Staining, Fluorescence, Light Microscopy

Journal: Cell Death Discovery

Article Title: IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform

doi: 10.1038/s41420-021-00523-y

Figure Lengend Snippet: A Flow cytometric analysis of apoptosis after POM plus IM treatment. KOPN57bi cells were cultured for 72 h in the presence or absence of either POM (2.5 μM) or IM (0.5 μM) or both, and apoptosis assay was performed by flow cytometry using PI/Annexin-V double staining. The X and Y axes indicate log fluorescence intensities of Annexin-V and PI, respectively. B AlamarBlue cytotoxic assays after POM plus IM. KOPN57bi cells were cultured for 72 h in the presence or absence of either 5 μM LEN or 2.5 μM POM (Tokyo Chemical Industry, Tokyo, JAPAN) alone or LEN plus IM or POM plus IM, and alamarBlue cytotoxic assays were performed in each culture condition. P values were calculated by unpaired t -test. C Flow cytometric analysis of apoptosis after LEN plus DA. KOPN57bi cells were cultured for 72 h in the presence or absence of either 5 μM LEN or 100 nM DA (Selleck Chemicals) alone or both, and apoptosis assays were performed by flow cytometry after PI/Annexin-V double staining. The X and Y axes indicate log fluorescence intensities of Annexin-V and PI, respectively. D Flow cytometric analysis of apoptosis after LEN plus PO in the IM resistant Ph+ cell line. The IM-resistant SU/SR cells harboring T315I mutation were cultured for 72 h in the presence or absence of either 0.5 μM IM or 5 nM PO (ChemScene) with or without 5 μM LEN, and flow cytometric analysis of apoptosis was performed after PI/Annexin-V double staining. The X and Y axes indicate log fluorescence intensities of Annexin-V and PI, respectively.

Article Snippet: B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN (Cayman Chemical, Ann Arbor, MI), and IKZF1 expression was examined on western blot.

Techniques: Cell Culture, Apoptosis Assay, Flow Cytometry, Double Staining, Fluorescence, Mutagenesis

Journal: Cell Death Discovery

Article Title: IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform

doi: 10.1038/s41420-021-00523-y

Figure Lengend Snippet: KOPN57bi cells were cultured for 24 h in the presence or absence of LEN (5 μM) or IM (0.5 μM) alone or both, and harvested. The isolated RNAs were reverse-transcribed and labeled using the GeneChip HT 3′ IVT Expression kit (Affymetrix, Santa Clara, CA) as instructed by the manufacturer. The labeled probes were hybridized in triplicate to GeneChip Human Genome-U133 Plus 2.0 Arrays (Affymetrix) as described previously . The arrays were analyzed using GeneChip Operating Software 1.2 (Affymetrix) and GeneSpring GX 14.9 software (Agilent Technologies, Santa Clara, CA). Genes with a p value lower than 0.01 by one-way ANOVA were selected. After performing fold-change analysis, genes that exhibited two times higher or lower expression were selected in each of the culture conditions. By using selected gene lists, single experiment pathway analysis was performed. A Upregulated genes. Among upregulated genes showing 2-fold higher by LEN plus IM treatment than LEN alone treatment, fold changes in RNAs of 3 genes MDM2, BTG2, and IKZF3 were presented in each culture condition. B Downregulated genes. Among downregulated genes showing 2-fold lower by LEN plus IM treatment than by LEN or IM alone treatment, fold changes in RNAs of 4 genes HELLS, CDC6, MCM4, and MCM8 were presented in each culture condition.

Article Snippet: B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN (Cayman Chemical, Ann Arbor, MI), and IKZF1 expression was examined on western blot.

Techniques: Cell Culture, Isolation, Labeling, Expressing, Software

Journal: Cell Death Discovery

Article Title: IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform

doi: 10.1038/s41420-021-00523-y

Figure Lengend Snippet: A Flow cytometric analysis of DEX effect on LEN plus IM-induced apoptosis. KOPN57bi cells were cultured for 48 h in the presence or absence of LEN (5 μM) plus IM (0.5 μM) with or without the addition of 2.5 nM DEX (SIGMA-Aldrich), and flow cytometric analysis was performed after Annexin-V/PI staining. B Comparative analysis of apoptosis-inducing effects between 2-drugs and 3-drugs. KOPN57bi cells were cultured for 48 h in the presence or absence of LEN (5 μM) plus IM (0.5 μM), IM plus DEX (2.5 nM), or LEN plus IM plus DEX, and flow cytometric analysis was performed after Annexin-V/PI staining. The X and Y axes shown in A and B indicate log fluorescence intensities of Annexin-V and PI, respectively. C Effect of DEX on LEN plus IM-induced cytotoxicity. KOPN57bi cells were cultured for 48 h in the presence or absence of LEN (5 μM) plus IM (0.5 μM) with or without the addition of DEX (2.5 nM) and alamarBlue cytotoxic assays were performed. D Dose-dependent effect of DEX on LEN plus IM-induced cytotoxicity. KOPN57bi cells were cultured for 48 h in the presence or absence of LEN (5 μM) plus IM (0.5 μM) with different concentrations (0, 1.25, 2.5, and 5 nM) of DEX, and alamarBlue cytotoxic assays were performed. E Comparative analysis of LEN and POM in 2-drugs and 3-drugs-induced cytotoxicity. KOPN57bi cells were cultured for 48 h in the presence or absence of LEN (5 μM), POM (2.5 μM), or IM (0.5 μM) with or without the addition of DEX (2.5 nM), and alamarBlue cytotoxic assays were performed. F Synergistic effect of LEN, PO, and DEX on cytotoxicity in SU/SR cells harboring T315 I mutation. The Ph+ SU/SR cells with T315I mutation were cultured for 48 h in the presence or absence of PO (5 nM) alone or LEN (5 μM) plus PO with or without the addition of DEX (2.5 nM), and alamarBlue cytotoxic assays were performed.

Article Snippet: B-cell leukemia cell lines were cultured for 24 h in the presence or absence of 10 µM LEN (Cayman Chemical, Ann Arbor, MI), and IKZF1 expression was examined on western blot.

Techniques: Cell Culture, Staining, Fluorescence, Mutagenesis