Journal: Nature Communications

Article Title: PIM2 inhibition promotes MCL1 dependency in plasma cells involving integrated stress response-driven NOXA expression

doi: 10.1038/s41467-024-55572-5

Figure Lengend Snippet: a mRNA and protein levels during B cell differentiation across subsets and time points. Data are shown as floating bars (min to max, mean as line) for NBC ( n = 3) and as box plots (median ± IQR, whiskers min to max) for d4 aBC ( n = 4), CD23+ aBC ( n = 5), PB ( n = 5), and ePC ( n = 5). b Protein levels across B cell and PC subsets. Samples derived from the same experiment with two different processed gels, including one for pBAD, BAD, and BIM (same gel of Fig. 1a), and another for NOXA. The blot is representative of 2 independent experiments. c Schematic of the assay design during B cell differentiation: cells were treated at specified time points with BH3-mimetic molecules and AZD1208. Data are representative of one experiment. Cell viability was assessed by flow cytometry. Percentage of cell viability were normalized to control condition and IC 50 were calculated. d RNAseq expression of anti-apoptotic factors and PIM kinases in 33 MMCLs. Data are shown as mean ( n = 33, ‘ n ’ denotes the number of distinct MMLs). e CRISPR screening data from DepMap in 17 MMCLs: dependency scores showing stronger dependency on PIM2 and MCL1 than on BCL-XL and BCL2. No dependency was observed for PIM1 or PIM3. Data are shown as mean ( n = 17, ‘n’ denotes the number of distinct MMLs). f Protein levels of antiapoptotic and proapoptotic factors in MMCLs. Samples derived from the same experiment with different processed gels, including one for PIM2, MCL1, BCLXL, BCL2, p-BAD, BAD, BIM, NOXA and another for BAK, BAX. The blot is representative of 2 independent experiments. g Cell viability in MMCLs after treatment with BH3-mimetics and AZD1208, with calculated IC 50 values. Data are shown as mean +/− SEM (Venetoclax, n = 3; AZD5991 and AZD1208, n = 4; A1155463, n = 2). NBC, naïve B cell; d4 aBC, day-4 activated B cell; CD23+ aBC, CD23-positive activated B cell; PB, plasmablast; ePC, early plasma cell; MMCLs, multiple myeloma cell lines; PIMi, pan-PIM inhibitor AZD1208. “ n ”, denotes the number of biological replicates except for Fig. 1d and Fig. 1e as indicated. Source data are provided as a Source Data file.

Article Snippet: MCL1 activity was inhibited with either AZD5991 (HY-101533; MedChemExpress) or S86485 (HY-100741; MedChemExpress).

Techniques: Cell Differentiation, Derivative Assay, Flow Cytometry, Control, RNA sequencing, Expressing, CRISPR, Clinical Proteomics

Journal: Nature Communications

Article Title: PIM2 inhibition promotes MCL1 dependency in plasma cells involving integrated stress response-driven NOXA expression

doi: 10.1038/s41467-024-55572-5

Figure Lengend Snippet: a ASCs were treated with increasing doses of AZD1208 for 6 h or with SSO-PIM for 24 h. Protein levels were assessed by immunoblotting. The blot is representative of 2 independent experiments. b MMCLs were treated with AZD1208 for 24 h. Protein levels were assessed by immunoblotting. Samples were from the same experiment, with different gels processed parallel. The blot is representative of 2 independent experiments. c Cells were treated with SSO-PIM for 24 h. Protein levels were assessed by immunoblotting with different processed gels, including one for PIM1, PIM2, another for PIM3 and pBAD, and another for NOXA and Caspase 3. The blot is representative of 2 independent experiments. d Correlation analysis between MCL1/PMAIP1 expression ratio with AZD1208 IC50 values. e Cells were transfected with NOXA or control siRNA, and then treated with AZD1208 for 6 h. Protein levels were assessed by immunoblotting. The blot is representative of 2 independent experiments. f ASCs were transfected with NOXA or control siRNA, and then treated with increasing doses of AZD1208 for 6 h. Caspase 3-active ASCs were analyzed by flow cytometry. Data were normalized to the control. Data are shown as mean ± SD ( n = 7). Protein levels were assessed by immunoblotting. The blot is representative of 2 independent experiments. g Cells were transfected with NOXA, BIM, BIM + NOXA (BN) siRNA, or control siRNA, and then treated with AZD1208 for 24 h. Caspase 3-active cells and cell viability were analyzed by flow cytometry. Data are shown as mean ± SD ( n = 6). Protein levels were assessed by immunoblotting. The blot is representative of 2 independent experiments. h ASCs were transfected with NOXA, BIM, BIM + NOXA (BN) or control siRNA, and then treated with AZD1208 for 6 h. Caspase 3-active ASCs were analyzed by flow cytometry. Data are shown as mean ± SD ( n = 4). Protein levels were assessed by immunoblotting, with different processed gels, including one for NOXA, pBAD, and BIM, and another for Caspase 3. The blot is representative of 2 independent experiments. P -values were calculated using Pearson’s test (two-tailed) with linear regression for ( d ), two-way ANOVA with Holm-Sidak’s adjustment for ( f ), two-way ANOVA with Tukey adjustment for ( f ) and ( g ), and two-way ANOVA with Dunnett adjustment for ( h ). P < 0.05 is indicated as * P < 0.01 as ** P < 0.001 as *** and P < 0.0001 as ****. Exact p -values are indicated in the Source Data file. “ n ”, denotes the number of biological replicates. Source data are provided as a Source Data file. ASCs, antibody-secreting cells; PIMi, pan-PIM inhibitor AZD1208; CTL, control; MMCLs, multiple myeloma cell lines; SSO, splice-switching oligonucleotide.

Article Snippet: MCL1 activity was inhibited with either AZD5991 (HY-101533; MedChemExpress) or S86485 (HY-100741; MedChemExpress).

Techniques: Western Blot, Expressing, Transfection, Control, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: PIM2 inhibition promotes MCL1 dependency in plasma cells involving integrated stress response-driven NOXA expression

doi: 10.1038/s41467-024-55572-5

Figure Lengend Snippet: a MMCLs were treated with AZD1208 for 16 h, followed by BH3-profiling. Data are shown as mean +/− SEM ( n = 3). b Cells were treated with specific SSO-PIM for 24 h, followed by BH3 profiling. Data are shown as mean +/− SEM (LP1, n = 3; U266, n = 4). c MMCLs were treated with AZD1208 for 24 h. Protein levels were assessed by immunoblotting (the same gel as in Fig. , β-actin was the same as in Fig. ). The blot is representative of 2 independent experiments. d Cells were treated with AZD1208 for 16 h, followed by MG132 addition for 3 h. Protein levels were assessed by immunoblotting. The blot is representative of 2 independent experiments. e Cells were treated with AZD1208 for 24 h with or without the pan-Caspase inhibitor QVD-OPH. Protein levels were assessed by immunoblotting. The blot is representative of 2 independent experiments. f Cells were treated with AZD1208 for 6 h. Immunoprecipitation of BAD and BCL-XL evaluated their interactions with BCL-XL, BIM, pBAD, and BAD. Samples of the IP-BCLXL were processed on different gels, one for BAD and other for BIM and BAK. The blot is representative of 1 independent experiment. g Cells were treated with AZD1208 for 6 h. Immunoprecipitation of MCL1 assessed its interaction with BAK, BIM, and NOXA. The blot is representative of 1 independent experiment. h Cells were treated with ISRIB for 2 h before addition of AZD1208 for 6 h. Immunoprecipitation of MCL1 evaluated its interaction with BAK, BIM, and NOXA. The blot is representative of 1 independent experiment. i Apoptotic Priming Pattern upon PIM Inhibition: plasma cells exhibit basal priming levels. PIM2 inhibition increases the pool of unphosphorylated BAD available for BCL-XL binding and BIM release. BIM and BAK bind to free MCL1 sites. NOXA induction further enhances MCL1 binding. Excess free pro-apoptotic molecules may cross the apoptotic threshold. Created with BioRender.com. P -values were calculated using ordinary two-way ANOVA with Sidak’s adjustment for a and b . “ n ”, denotes the number of biological replicates. Source data are provided as a Source Data file. PIMi, pan-PIM inhibitor AZD1208, MMCLs, multiple myeloma cell lines; SSO, splice-switching oligonucleotide.

Article Snippet: MCL1 activity was inhibited with either AZD5991 (HY-101533; MedChemExpress) or S86485 (HY-100741; MedChemExpress).

Techniques: Western Blot, Immunoprecipitation, Inhibition, Clinical Proteomics, Binding Assay

Journal: Nature Communications

Article Title: PIM2 inhibition promotes MCL1 dependency in plasma cells involving integrated stress response-driven NOXA expression

doi: 10.1038/s41467-024-55572-5

Figure Lengend Snippet: a Cells were pretreated with AZD1208 before adding increasing doses of MCL1 inhibitor. Percentage of depolarized cells were analyzed by flow cytometry. Data are shown as mean ( n = 3). b Cells were pretreated with AZD1208 before adding increasing doses of MCL1 inhibitor. Immunoprecipitation of MCL1 and BCL-XL assessed their interaction with BIM, NOXA, BAK, MCL1, and BCL-XL. The input for RPMI8226 is the same as for RPMI8226 in Fig. , because the cells for IP were from the same experiments. The blot is representative of 1 independent experiment. c Cells were pretreated with AZD1208 before adding increasing doses of MCL1 inhibitor. Immunoprecipitation of BAX-6A7 (BAX active) evaluated its interaction with BAK. The blot is representative of 1 independent experiment. d Cells were pretreated with AZD1208 before adding increasing doses of MCL1 inhibitor. Immunofluorescence of full-length of BAX (red), activated BAX (BAX 6A7; green) and mitochondria (MitoTracker dye; white). Provided images are representative of 3 images. e Cells were pretreated with AZD1208 before adding increasing doses of MCL1 inhibitor. Immunofluorescence of Cytochrome c (red) and mitochondria (MitoTracker dye; white). Provided images are representative of 3 images. f Schematic Illustration: PIM inhibition prevents reuptake of BIM and BAK by BCL-XL after MCL1 inhibition, leading to BAX activation and mitochondrial depolarization. Created with BioRender.com. g RPMI8226 cells were transfected with NOXA, BIM, BIM + NOXA siRNA or control siRNA, then treated with AZD1208 before adding increasing doses of MCL1 inhibitor. The percentage of depolarized cells was analyzed by flow cytometry. Data are shown as mean +/− SEM ( n = 6). h Cells were treated with AZD1208 overnight in the presence or absence of ISRIB before adding MCL1 inhibitor. Percentage of depolarized cells were analyzed by flow cytometry. Data are shown as mean +/− SEM ( n = 7). i Enhancement of depolarization by PIM inhibitor was assessed by normalizing depolarization levels after combined PIMi/MCL1i treatment to those with PIM inhibitor alone, with or without ISRIB. Data are shown as mean ± SEM ( n = 7). P -values were calculated using two-way ANOVA with Sidak’s adjustment for ( a ), ( h ), and ( i ), and two-way ANOVA with Tukey’s adjustment for ( g ). P < 0.05 is indicated as * P < 0.01 as ** P < 0.001 as *** P < 0.0001 as ****. Exact p -values are indicated in the Source Data file. “ n ”, denotes the number of biological replicates. Source data are provided as a Source Data file. ASCs, antibody-secreting cells; PIMi, pan-PIM inhibitor AZD1208; MCL1i, MCL1 inhibitor AZD5991; BN, BIM + NOXA.

Article Snippet: MCL1 activity was inhibited with either AZD5991 (HY-101533; MedChemExpress) or S86485 (HY-100741; MedChemExpress).

Techniques: Flow Cytometry, Immunoprecipitation, Immunofluorescence, Inhibition, Activation Assay, Transfection, Control

Journal: Scientific Reports

Article Title: Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma

doi: 10.1038/s41598-022-04916-6

Figure Lengend Snippet: Expression of BCL2 family genes in the LL-100 panel. ( a ) Heat map of RNA-seq gene expression data of BCL2 family members. Framed in red are expression levels of MCL1 and BCL2 in ALCL and PEL. ( b ) Results of qRT-PCR showing expression of BCL2 family members. ( c ) Western blot analysis showing the protein expression of MCL1, BCL2 and BCLXL in ALCL and PEL cell lines. DLBCL and MM cell lines were included as controls. Note that ALCL and PEL cell lines are consistently MCL1 pos /BCL2 neg .

Article Snippet: Life cell imaging data showing the effect of the MCL1 inhibitor AZD-5991 (300 nM) and the BCL2/BCLXL inhibitor ABT-263 (300 nM) on growth (48 h) and apoptosis (12 h) of ( a ) L-82, SU-DHL-1 and ( b ) SR-786, KARPAS-299. ( c ) shows effects of AZD-5991 and BEZ-235 on growth and apoptosis of cell lines L-82 and SU-DHL-1.

Techniques: Expressing, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma

doi: 10.1038/s41598-022-04916-6

Figure Lengend Snippet: SiRNA-mediated knockdown of BCL2 family members in ALCL cell lines. Life cell imaging shows effects of knockdown of BCL2 family members on growth (cell counts) and apoptosis (caspase 3/7 activity) 48 h after onset of treatment with siRNA oligos. ( a ) Knockdown of MCL1 and BCLXL; ( b ) knockdown of MCL1 and BCL2A1. Note synergistic effects of MCL1 and BCLXL knockdown in cell line L-82 and additive effects of MCL1 and BCL2A1 knockdown in cell line SU-DHL-1. The horizontal line shows the expected additive effects of the combination of siRNA oligos.

Article Snippet: Life cell imaging data showing the effect of the MCL1 inhibitor AZD-5991 (300 nM) and the BCL2/BCLXL inhibitor ABT-263 (300 nM) on growth (48 h) and apoptosis (12 h) of ( a ) L-82, SU-DHL-1 and ( b ) SR-786, KARPAS-299. ( c ) shows effects of AZD-5991 and BEZ-235 on growth and apoptosis of cell lines L-82 and SU-DHL-1.

Techniques: Knockdown, Imaging, Activity Assay

Journal: Scientific Reports

Article Title: Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma

doi: 10.1038/s41598-022-04916-6

Figure Lengend Snippet: Effect of AZD-5991 and ABT-263 on growth and apoptosis of ALCL cell lines. Life cell imaging data showing the effect of the MCL1 inhibitor AZD-5991 (300 nM) and the BCL2/BCLXL inhibitor ABT-263 (300 nM) on growth (48 h) and apoptosis (12 h) of ( a ) L-82, SU-DHL-1 and ( b ) SR-786, KARPAS-299. ( c ) shows effects of AZD-5991 and BEZ-235 on growth and apoptosis of cell lines L-82 and SU-DHL-1. Apoptosis was assessed by caspase 3/7 activity. The horizontal line shows the expected additive effects of the combination of both inhibitors. Note that the only cell line that does not respond to ABT-263 is the BCLXL low cell line KARPAS-299.

Article Snippet: Life cell imaging data showing the effect of the MCL1 inhibitor AZD-5991 (300 nM) and the BCL2/BCLXL inhibitor ABT-263 (300 nM) on growth (48 h) and apoptosis (12 h) of ( a ) L-82, SU-DHL-1 and ( b ) SR-786, KARPAS-299. ( c ) shows effects of AZD-5991 and BEZ-235 on growth and apoptosis of cell lines L-82 and SU-DHL-1.

Techniques: Imaging, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: BH3 Mimetic Sensitivity of Colorectal Cancer Cell Lines in Correlation with Molecular Features Identifies Predictors of Response

doi: 10.3390/ijms22083811

Figure Lengend Snippet: Overview of the targets of the inhibitors and combinations.

Article Snippet: A-1155463 and AZD5991 were purchased from Chemietek, Indianapolis, IN, US.

Techniques: