Figure 1.
Pevonedistat suppresses TNFα-mediated NFκB signaling in MPNs. (A) Cell viability curve of JAK2 V617F mutant HEL cells treated with increasing concentrations of pevonedistat and ruxolitinib. Cells were treated for 72 hours and viability was normalized to the control treatment. Data points denote mean and standard error of mean at each drug concentration; n = 6 replicates. Cell viability measured with AlamarBlue fluorescence detection. (B) Cell viability assay of HEL cells treated with pevonedistat, ruxolitinib, or in combination. Cells were treated for 72 hours at the indicated drug concentration. Cell viability measured with AlamarBlue fluorescence detection; n = 3 replicates. (C) Synergy score calculations of pevonedistat and ruxolitinib treatment from panel B. Synergy score >10: synergistic; from −10 to 10: additive; <−10: antagonistic. (D) Cell cycle assay of HEL cells treated with DMSO control, pevonedistat (80 nM), pevonedistat (2 µM), or combination, assessed across multiple time points; n = 3 replicates. (E) Annexin V apoptosis assay of HEL cells treated with DMSO control, pevonedistat (80 nM), pevonedistat (2 µM), or combination, assessed across multiple time points; n = 3 replicates. (F) Flow cytometry of IkBα in HEL cells treated with 20 ng/mLTNFα, pevonedistat, or in combination. HEL cells were treated with pevonedistat for 2 hours and TNFα for the last 30 minutes. (G) RT-qPCR of NFκB pathway-related genes in HEL cells treated with TNFα, pevonedistat, or in combination for the indicated durations. Relative NFκB pathway-related mRNA expression was determined by 2^-dCt and normalized to ACTB. Samples were run in duplicate for each experimental condition and timepoint. (H) Colony formation assays of sorted CD34+ cells from normal BM donor and MF patient samples treated with pevonedistat, TNFα, ruxolitinib, or combinations at the indicated concentrations. 1,000 CD34+ cells were plated in MethoCult containing SCF and IL-3 in triplicate per condition. Colonies were counted after 14 days in culture. The number of patient samples (n) per condition is denoted. *P < .05; **P < .01 by Mann-Whitney U test. (I) RT-qPCR of NFκB pathway-related genes in sorted CD34+ cells from normal BM donor (n = 4) and myelofibrosis patient (n = 6) samples treated with 1 µM pevonedistat, 20 ng/mL TNFα, or in combination. CD34+ cells were treated for 4 hours. Relative NFκB pathway-related mRNA expression was determined by 2^-dCt and normalized to ACTB. *P < .05; **P < .01 by Mann-Whitney U test. NS, not significant.

Pevonedistat suppresses TNFα-mediated NFκB signaling in MPNs. (A) Cell viability curve of JAK2 V617F mutant HEL cells treated with increasing concentrations of pevonedistat and ruxolitinib. Cells were treated for 72 hours and viability was normalized to the control treatment. Data points denote mean and standard error of mean at each drug concentration; n = 6 replicates. Cell viability measured with AlamarBlue fluorescence detection. (B) Cell viability assay of HEL cells treated with pevonedistat, ruxolitinib, or in combination. Cells were treated for 72 hours at the indicated drug concentration. Cell viability measured with AlamarBlue fluorescence detection; n = 3 replicates. (C) Synergy score calculations of pevonedistat and ruxolitinib treatment from panel B. Synergy score >10: synergistic; from −10 to 10: additive; <−10: antagonistic. (D) Cell cycle assay of HEL cells treated with DMSO control, pevonedistat (80 nM), pevonedistat (2 µM), or combination, assessed across multiple time points; n = 3 replicates. (E) Annexin V apoptosis assay of HEL cells treated with DMSO control, pevonedistat (80 nM), pevonedistat (2 µM), or combination, assessed across multiple time points; n = 3 replicates. (F) Flow cytometry of IkBα in HEL cells treated with 20 ng/mLTNFα, pevonedistat, or in combination. HEL cells were treated with pevonedistat for 2 hours and TNFα for the last 30 minutes. (G) RT-qPCR of NFκB pathway-related genes in HEL cells treated with TNFα, pevonedistat, or in combination for the indicated durations. Relative NFκB pathway-related mRNA expression was determined by 2^-dCt and normalized to ACTB. Samples were run in duplicate for each experimental condition and timepoint. (H) Colony formation assays of sorted CD34+ cells from normal BM donor and MF patient samples treated with pevonedistat, TNFα, ruxolitinib, or combinations at the indicated concentrations. 1,000 CD34+ cells were plated in MethoCult containing SCF and IL-3 in triplicate per condition. Colonies were counted after 14 days in culture. The number of patient samples (n) per condition is denoted. *P < .05; **P < .01 by Mann-Whitney U test. (I) RT-qPCR of NFκB pathway-related genes in sorted CD34+ cells from normal BM donor (n = 4) and myelofibrosis patient (n = 6) samples treated with 1 µM pevonedistat, 20 ng/mL TNFα, or in combination. CD34+ cells were treated for 4 hours. Relative NFκB pathway-related mRNA expression was determined by 2^-dCt and normalized to ACTB. *P < .05; **P < .01 by Mann-Whitney U test. NS, not significant.

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