Figure 7.
Figure 7. CDK6 kinase activity is not required to regulate genes involved in apoptosis and HSC quiescence in JAK2V617F mutant LSKs. (A) Methylcellulose replating assay of whole BM cells (VavCre; JAK2V617F; Cdk6−/−, VavCre; JAK2V617F; Cdk6+/+, VavCre; Jak2+/+; Cdk6−/−, and VavCre; Jak2+/+; Cdk6+/+), 2/7 VavCre; JAK2V617F; Cdk6−/− could not be used for third replating experiments due to too small cell number. Error bars indicate + standard error of the mean (n ≥ 6 per genotype). Two-way ANOVA with Bonferroni posttest was used, and significance is indicated. *P < .05. EPO, erythropoietin; SCF, stem cell factor. (B) Representative histograms showing annexin V positivity of LSK cells in BM of indicated genotype. (C) Heat map depicts expression of validated signature genes involved in apoptosis, NF-κB signaling, and stem cell quiescence in LSKs. Regularized log-transformation of count data was used as input for the heat map. Color code: red, upregulation; blue, downregulation. Mean of 3 replicates is depicted. For full data set, see supplemental Figure 13A. (D) Overview of gene set enrichment analysis in indicated contrasts. The Hallmark Gene Set Collection was used as reference. False discovery rate <0.25 was considered significant. no: gene sets only enriched in VavCre; JAK2V617F; Cdk6−/− vs VavCre; JAK2V617F; Cdk6+/+; Yes, gene sets enriched in both VavCre; JAK2V617F; Cdk6−/− vs VavCre; JAK2V617F; Cdk6+/+ and VavCre; JAK2V617F; Cdk6+/+ palbociclib vs untreated. (E) Primary mononuclear cells were exposed to increasing concentrations of palbociclib or dimethyl sulfoxide (DMSO) for 48 hours. Apoptosis was evaluated by labeling primary CD34+CD38−CD45dim stem/progenitor cells with annexin V and 4′,6-diamidino-2-phenylindole (DAPI) followed by flow cytometry analysis. (F) Primary patient samples (1.2 × 105) were embedded in methocult with or without palbociclib. Colonies were picked at days 13 to 15, and allele-specific polymerase chain reaction was performed. Allelic burden is presented as % JAK2V617F+/− colonies vs total colony number DMSO control or palbociclib treated. DN, down; TGF, transforming growth factor; TNFA, tumor necrosis factor-α.

CDK6 kinase activity is not required to regulate genes involved in apoptosis and HSC quiescence in JAK2V617Fmutant LSKs. (A) Methylcellulose replating assay of whole BM cells (VavCre; JAK2V617F; Cdk6−/−, VavCre; JAK2V617F; Cdk6+/+, VavCre; Jak2+/+; Cdk6−/−, and VavCre; Jak2+/+; Cdk6+/+), 2/7 VavCre; JAK2V617F; Cdk6−/− could not be used for third replating experiments due to too small cell number. Error bars indicate + standard error of the mean (n ≥ 6 per genotype). Two-way ANOVA with Bonferroni posttest was used, and significance is indicated. *P < .05. EPO, erythropoietin; SCF, stem cell factor. (B) Representative histograms showing annexin V positivity of LSK cells in BM of indicated genotype. (C) Heat map depicts expression of validated signature genes involved in apoptosis, NF-κB signaling, and stem cell quiescence in LSKs. Regularized log-transformation of count data was used as input for the heat map. Color code: red, upregulation; blue, downregulation. Mean of 3 replicates is depicted. For full data set, see supplemental Figure 13A. (D) Overview of gene set enrichment analysis in indicated contrasts. The Hallmark Gene Set Collection was used as reference. False discovery rate <0.25 was considered significant. no: gene sets only enriched in VavCre; JAK2V617F; Cdk6−/− vs VavCre; JAK2V617F; Cdk6+/+; Yes, gene sets enriched in both VavCre; JAK2V617F; Cdk6−/− vs VavCre; JAK2V617F; Cdk6+/+ and VavCre; JAK2V617F; Cdk6+/+ palbociclib vs untreated. (E) Primary mononuclear cells were exposed to increasing concentrations of palbociclib or dimethyl sulfoxide (DMSO) for 48 hours. Apoptosis was evaluated by labeling primary CD34+CD38−CD45dim stem/progenitor cells with annexin V and 4′,6-diamidino-2-phenylindole (DAPI) followed by flow cytometry analysis. (F) Primary patient samples (1.2 × 105) were embedded in methocult with or without palbociclib. Colonies were picked at days 13 to 15, and allele-specific polymerase chain reaction was performed. Allelic burden is presented as % JAK2V617F+/− colonies vs total colony number DMSO control or palbociclib treated. DN, down; TGF, transforming growth factor; TNFA, tumor necrosis factor-α.

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