Page 97 - 2021_10-Haematologica-web

CDK4 or CDK6 deletion in adult hematopoiesis
alterations with small spleens due to impaired erythroid and megakaryocytic cell development,5 and a lower red blood cell (RBC) number accompanied by increased ery- throcyte size.6 In addition CDK6 is required for normal T- cell development in the thymus5,7 and for myeloid differ- entiation.8,9 The International Mouse Phenotyping Consortium identified CDK4 knockout mice as “sub- viable” – less homozygous knockout mice than expected were born – with smaller embryo size, hypoplasia of var- ious organs and infertility, pinpointing to incomplete compensation of CDK4 functions by other proteins.10,11 Another CDK4-deficient mouse model is also smaller in size, prone to develop diabetes and sterility12,13 and, besides an increase in erythrocyte size, lacks any abnor- mality in adult hematopoiesis even in the combined absence of CDK2.14,15 Similar to other paralogues,10 CDK4 is considered to be able to compensate some, but not all of CDK6’s functions and vice versa – partly reflected in the described phenotypes of the total knockout models. These overlapping roles or additional compensatory effects cannot be studied in complete knockout mice due to adaption for the total protein loss starting in embryo- genesis.
CDK6, but not CDK4, has recently been assigned an additional function as a transcriptional regulator: as such CDK6 modulates p53 and nuclear factor κB (NFκB) responses, both important under oncogenic stress.16-19 In addition, activation of hematopoietic stem cells (HSC) depends on CDK6’s kinase-independent role; CDK6 is required to suppress the quiescence inducer Egr1.20 In B- cell lymphoid leukemia, CDK6 is part of a transcriptional complex that induces the pro-angiogenic factor VEGF-A in a kinase-independent manner.21 CDK6 acts – at least partly kinase-independent - oncogenic in JAK2V617F-driven myeloproliferative neoplasms by promoting NFκB signal- ing, proinflammatory cytokine production and inhibiting apoptosis.18 In FLT3-ITD+ myeloid leukemia, CDK6 was reported to act as a transcriptional regulator in a kinase- dependent manner and is required to induce the leuke- mogenic drivers FLT3 and PIM1.22
CDK4 and CDK6 dual inhibitors, which block the kinase activity by preventing ATP from binding to the kinase pocket, have successfully entered the clinics. Palbociclib (Ibrance® by Pfizer), ribociclib (Kisqali® by Novartis) and abemaciclib (Verzenios® by Lily) have been approved for HER2-negative, locally advanced or metastatic breast cancer23 and are currently undergoing clinical trials to test their efficacy in solid tumors and leukemia.24 Recently, CDK6 expression was identified to be a conserved and direct target of diverse NUP98-fusions driving aggressive forms of acute myeloid leukemia, which were demonstrated to be hypersensitive to CDK4/6 inhibition.25 The huge clinical success of palboci- clib is not only based on its role in interfering with the cell cycle, but also on its capacity to induce a senescence-like phenotype in breast cancer cells and to enhance cancer cell immunogenicity26 and T-cell activation.27 Side effects of CDK4/6 inhibitor treatment affect mainly the hematopoietic system and include neutropenia, lym- phopenia, anemia and thrombocytopenia. As a conse- quence the patients display an increased rate of infec- tions, especially of the upper respiratory tract.28-30
As several proto-oncogenic effects of CDK6 are kinase- independent, CDK4/6 inhibitors might target only parts of CDK6’s functions. Specific protein degraders hijacking
the cell’s proteasomal degradation machinery called PROTACs represent novel alternatives to kinase inhibitors and have been reported to specifically target CDK6.31-33 These compounds circumvent the possible upregulation of CDK6 expression in CDK4/6 inhibitor- treated patients leading to therapy resistance34,35 and tar- get the kinase-independent regulatory roles of CDK6.
We here describe the generation of Mx1-Cre Cdk6fl/fl and Mx1-Cre Cdk4fl/fl mice and their phenotype upon polyinosinic–polycytidylic acid (poly(I:C)) treatment. In contrast to total CDK4 or CKD6 knockout mice, which can develop compensatory mechanisms starting in embryogenesis and hematopoietic development, we study direct consequences of CDK4 or CDK6 loss in adult mice with a focus on hematopoiesis. Our study links the hematopoietic side effects of CDK4/6 inhibitors as ane- mia and neutropenia predominantly to CDK6 inhibition. Loss of CDK6 results in an accumulation of early HSC and common lymphoid progenitors (CLP), while CDK4- deficient mice acquire an expansion of committed myeloid progenitors.
We validated the novel Cdk4fl/fl and Cdk6fl/fl mouse mod- els as powerful tools to decipher the roles of CDK4 and CDK6 in any cell type-specific or inducible manner.
Methods
Mouse strains
Cdk4fl/+ and Cdk6fl/+ mice were obtained from the Canadian Mouse Mutant Repository. The mouse line C57BL/6N- Cdk4tm1c(NCOM)Mfgc/Tcp (referred to as Cdk4fl/fl) was gener- ated as part of the NorCOMM2 project with C57BL/6NCdk4tm1a(NCOM)Mfgc/Tcp made from NorCOMM embryonic stem (ES) cells36 at the Toronto Center for Phenogenomics, Canada. The mouse line C57BL/6N- Cdk6tm1c(EUCOMM)Wtsi/Tcp (referred to as Cdk6fl/fl) was generated with C57BL/6N-Cdk6tm1a(EUCOMM)Wtsi/Tcp made from EUCOMM ES cells36 at the Toronto Center for Phenogenomics, Canada.
Mice carrying homozygous floxed alleles were crossed with Mx1-Cre mice37 to allow inducible deletion of Cdk4 or Cdk6 in the hematopoietic system, respectively. Eight-week-old male and female mice were injected intraperitoneally (i.p.) with 200 mg poly(I:C) every 3 days (three times in total). Analysis was done 3 or 6 weeks post injection. All mice were bred and maintained under pathogen-free conditions at the Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna. All procedures were approved by the Institutional Ethics and Animal Welfare Committee and the national authority according to §§26ff. of the Animal Experiment Act, Tierversuchsgesetz 2012 - TVG 2012 (GZ BMBWF-68.205/0174-V/3b/2018).
Flow cytometry
Single-cell suspensions were prepared from bone marrow (BM), spleen (including RBC lysis) and peripheral blood (no RBC lysis for erythroid development; including RBC lysis for lineage-restricted cells) and analyzed by flow cytometry using a FACS Canto II cell analyzer (BD Biosciences, Franklin Lakes, New Jersey, USA). For stem cell analysis, cells were stained as follows: lineage panel con- taining Pacific Blue-conjugated CD3, CD11b, Gr1, Ter119 and B220, Sca1-PeCy7, cKit-PerCPCy5.5, CD34-FITC, CD150-APC, CD135-APC-Cy7 and CD48-PE. For myeloid and lymphoid pro- genitors, the following antibodies were used: lineage panel con- taining Pacific Blue-conjugated CD3, CD11b, Gr1, Ter119 and
haematologica | 2021; 106(10)
2625