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B. Maurer et al.
B220, CD127-FITC, Sca1-PeCy7, cKit-PerCPCy5.5, CD16/32-PE and CD34-APC. For lineage-restricted cells, the following antibod- ies were used: Gr1-APC-eFlour780, CD3-Pacific Blue, CD19-APC, Ter119-PE and CD11b-PerCPCy5.5. Erythroid development was evaluated using Ter119-APC-eFluor780, CD71-Biotin and Streptavidin-PE-Cy7 antibodies (Ter119medCD71high (proerythrob- lasts; R1), Ter119highCD71high (basophilic erythroblasts; R2), Ter119highCD71med (late basophilic and polychromatophilic ery- throblasts; R3) and Ter119highCD71low (orthochromatophilic ery- throblasts; R4). All antibodies were purchased from BD Biosciences, eBioscience (ThermoFisherScientific, Waltham, Massachusetts, USA) or BioLegend (San Diego, California, USA). Cell cycle analysis was performed in a two-step protocol by stain- ing with antibodies directed against HSC surface markers (lineage panel containing APC-Cy7-conjugated CD3, CD11b, Gr1, Ter119 and B220, Sca1-PeCy7, cKit-PerCPCy5.5, CD150-APC and CD48- PE) followed by intracellular staining with Ki-67 FITC and 4,6 diamidino-2-phenylindole (DAPI). For intracellular staining, cells were fixed in Cytofix/Cytoperm (BD Biosciences), washed and stained with Ki-67 FITC (BD Bioscience), and then co-stained with 0.1 mg/100 ml DAPI in phosphate-buffered saline (PBS). Flow cytometry plots were evaluated by FACSDiva software version 6.1.2 or FlowJo v10.6.1 (both BD).
Statistical analysis
Statistical analysis was carried out using a one-way analysis of variance (ANOVA) with subsequent Bonferroni’s or Tukey’s mul- tiple comparison test, Mann-Whitney U test or Kruskal-Wallis test with subsequent Dunn’s test. Data are presented as mean values + standard error of the mean (SEM) and were analyzed by GraphPad Prism. Statistical significance is as follows: *P<0.05, **P<0.01; ***P<0.001; ****P<0.0001.
Results
Generation and validation of Cdk4fl/fl Mx1-Cre and Cdk6fl/fl Mx1-Cre mice
In order to explore distinct roles of CDK4 and CDK6 in adult hematopoiesis, we generated Mx1-Cre Cdk6fl/fl and Mx1-Cre Cdk4fl/fl mice (Online Supplementary Figure S1A; Figure 1A). Heterozygous Cdk4fl/+ or Cdk6fl/+ mice on C57BL/6N background were obtained from the Canadian Mouse Mutant Repository and crossed to Mx1-Cre mice, which activate CRE recombinase upon poly(I:C) injection in hematopoietic cells.37 Eight-week-old Cdk4fl/fl Mx1-Cre, Cdk6fl/fl Mx1-Cre and control littermates (Cdk4fl/fl or Cdk6fl/fl) were injected three times with poly(I:C) and ana- lyzed 3 or 6 weeks thereafter (Figure 1B). Polymerase chain reaction (PCR) analysis and western blot confirmed excellent deletion efficacy 3 weeks post poly(I:C) injec- tion of Cdk4 or Cdk6 in spleen (Figure 1C and D) and BM (Online Supplementary Figure S1B) and 6 weeks after poly(I:C) injection (Online Supplementary Figure S1D) of Mx1-Cre expressing mice (hence called Cdk4D/D or Cdk6D/D). By trend, CDK4 expression is elevated upon Cdk6 deletion and vice versa (Figure 1E; Online Supplementary Figure S1C).
CDK6 but not CDK4 influences erythroid development
Patients treated with CDK4/6 inhibitors frequently develop anemia.28,38 In order to understand the contribu- tion of CDK4 or CDK6 in this process, we studied ery- throid development in Cdk4D/D and Cdk6D/D mice and their littermate controls. Cdk4D/D and Cdk6D/D mice displayed
significantly reduced numbers of RBC in peripheral blood. The reduction was more pronounced in mice lack- ing Cdk6 (Figure 2A). Hematocrit and hemoglobin levels were only reduced in Cdk6D/D mice but not in Cdk4D/D ani- mals (Online Supplementary Figure S2A and B). In complete Cdk6-/- mice, CDK6 is involved in the transition of mature RBC from the BM to the periphery.6 This is recapitulated in Cdk6D/D mice; they harbor elevated numbers of Ter119+ erythroid cells in the BM (Figure 2B) and show an altered maturation profile with a significant accumulation of cells in developmental stage R2 (basophilic erythroblasts) and R4 (orthochromatophilic erythroblasts). Erythroid devel- opment is unaffected in Cdk4D/D mice (Figure 2C and D). The same effects were observed by trend when analyzing mice 6 weeks post Cdk4 or Cdk6 deletion (Online Supplementary Figure S2E). No changes were observed in the spleen size (Online Supplementary Figure S2C) or splenic numbers of erythroid cells (Online Supplementary Figure S2D) in Cdk6D/D and Cdk4D/D mice. These data led us to conclude that anemia observed upon CDK4/6 inhibitor treatment can be predominantly attributed to CDK6 blockade.
Cdk6D/D mice accumulate hematopoietic stem/progenitor cells in the bone marrow
CDK6 is required to exit quiescence in human HSC.39 In mice, CDK6 is crucial for HSC activation under stress while being dispensable under homeostatic conditions. Cdk6-/- mice display unaltered BM cellularity and LSK (Lin-Sca-1+c-Kit+) cell numbers.20 Based on CD150 and CD48 expression, LSK cells can be further subdivided in CD150+CD48- Fraction A cells (containing the most dor- mant and long-term HSC), the more cycling CD150+CD48+ Fraction B cells and CD150-CD48+ Fraction C cells (myeloid- or lymphoid-prone).40 Cdk6-/- mice have comparable numbers of Fraction A, B or C stem cells with a consistent increase of the most dor- mant HSC characterized by CD135-CD34- within Fraction A cells.18,20 Upon deletion of CDK4 or CDK6 in adult BM we found that Cdk4D/D mice reacted with a slight increase in BM cellularity while a subtle decrease was inflicted in Cdk6D/D animals (Online Supplementary Figure S3A). Three weeks post Cdk4 or Cdk6 deletion, the percentage of LSK cells was significantly increased in Cdk6D/D BM but stayed unaltered in Cdk4D/D BM (Figure 3A and B). The change in LSK numbers in Cdk6D/D BM is attributed to the significant accumulation of long-term Fraction A stem cells, short-term Fraction B stem cells and multipotent progenitor (MPP) Fraction C cells (Online Supplementary Figure S3B to D). Reminiscent of the findings in total Cdk6-/- mice, Cdk6D/D Fraction A stem cells were enriched with the most dormant HSC (CD150-CD48+CD135-CD34- LSK), which was unaffect- ed in Cdk4D/D BM (Figure 3C). Comparable results upon 6 weeks of poly(I:C) injection confirmed these findings (Online Supplementary Figure S3F).
In complete Cdk6-/- mice no significant changes in the cell cycle profile of Fractions A, B or C were detected under homeostatic conditions indicating a compensatory effect of CDK4.20 In Cdk4D/D, the proportion of Fraction A or B cells in each cell cycle phase was also unaltered com- pared to littermate control BM. In Cdk6D/D mice a CDK4- mediated compensation could not rescue the frequency of fraction B cells in the S/G2/M phase which was signifi- cantly decreased (Figure 3D to E; Online Supplementary
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haematologica | 2021; 106(10)