The Function And Mechanism Of Cyclin-dependent Kinase 4/6 Mediated Tumor Immunity

As a global public health problem,cancer threatens human’s health and life quality.The essence of cancer is the abnormal activation of cell proliferation,that is,cell cycle disorder.Many oncogenes and tumor suppressor genes directly participate in the cell cycle regulation,and their abnormal activation makes cell cycle out of control,resulting in uncontrolled growth of cancer cells.Cyclin-dependent kinases,CDKs)are the core part of cell cycle regulation.CDKs are a series of serine/threonine protein kinases,which combine with cyclin,a regulatory subunit,to exert kinase activity.They play a crucial role in the initiation and transformation of cell cycle.Among them,CDK4/6 combines with CyclinD to form cyclinD-CDK4/6 complex,which phosphorylates Rb protein and releases E2F to promote the cell cycle from G1 phase to S phase.Studies have proved that the mutation of CKIs,an endogenous inhibitor of CDK protein,is the cause of many cancer types.For example,the mutation of endogenous CDK4/6 inhibitor pl6 occurs in many cancer types,including melanoma.CDK4 mutations were found in 3.17%of malignant solid tumor patients.While CDK6 was reported to be highly expressed in leukemia and lymphoma,and the chromosomal translocation of CDK6 was proved to be a risk factor for B lymphoid leukemia.Based on the core regulatory role of CDK4/6 in cell cycle,CDK4/6 inhibitors enter the market.Among them,palbociclib,ribociclib,abemaciclib and domestic dalpiciclib have been approved by FDA or NMPA for the treatment of ER+and HER2-metastatic breast cancer.In recent years,other physiological functions of CDK4/6 besides cell cycle regulation have become the research focus,especially its role in tumor immunity.For example,selective CDK4/6 inhibitors can down-regulate DNA methyltransferase 1(DNMT1)thus increase the level of endogenous retrovirus and trigger the anti-tumor immune responses mediated by interferon III;CDK4 destabilized PD-L1 protein by modulating the phosphorylation of speckle-type POZ protein(SPOP)to regulate cancer immune surveillance.These studies turned CDK4/6 inhibitors from small-molecule targeted drugs into immunotherapy drugs.Despite current studies on the effect of CDK4/6 inhibition on anti-tumor immunity,most of them used CDK4/6 inhibitors,which can cause off-target effect.For example,CDK4/6 inhibitors palbociclib and abemaciclib also have inhibitory activity on other CDKs such as CDK2.Therefore,the role of CDK4/6 plays in anti-tumor immunity still needs more accurate research.Through a series of experiments,we found a new mechanism of CDK4 and CDK6 regulating tumor immunity.Firstly,we analyzed the TCGA database and found that CDK4 and CDK6 were up-regulated in clinical cancer tissues.The expression of CDK4 and CDK6 were negatively correlated with CD8+T cell infiltration and the prognosis of patients.We used Crisper-Cas9 technology to generate Cdk4-/-and Cdk6-/-stable knockout cell lines to avoid off-target effect on CDK4/6 inhibitors.Through tumor transplantation experiment,we found that Cdk4 deletion can inhibit tumor growth in an immune-dependent way.Specifically,type Ⅰand type Ⅱ interferon signaling were enhanced in Cdk4-/-tumor tissues.Meanwhile,more CD8+T cells and DC cells were infiltrated,and the ratio of activated T cells was increased,which enhanced the anti-tumor immune response.The anti-tumor effect caused by Cdk6 knockout partly came from the direct inhibition of cell proliferation and partly depended on the immune system.Through in vitro experiments,we found that either Cdk4 and Cdk6 knockout or selective CDK4/6 inhibitor led to the activation of the type Ⅰ interferon pathway.Furthermore,We constructed Cdk4-/-/Sting-/-,Cdk4-/-/Mavs-/-,Cdk6-/-/Sting-/-and Cdk6-/-/Mavs-/-double knock cell lines to detect the mRNA expression of ISGs,protein expression of STAT1 and p-STAT1.Results showed that the activation of type Ⅰ interferon and tumor inhibition induced by Cdk4 and Cdk6 deletion depended on the STING protein.By detecting DNA damage markers,such as γH2AX,Rad51,p-CHK1,we found that both Cdk4 and Cdk6 knockout could cause intracellular DNA damage.Finally,we collected clinical samples of breast cancer patients for immunohistochemical staining and found that the expression of CDK4 and CDK6 in cancer tissues was significantly higher than that in adjacent tissues.The expression of ISGs in PBMC of patients treated with CDK4/6 inhibitors was up-regulated compared with control patients.The clinical sample results were consistent with previous laboratory research conclusions.In a word,through clinical and laboratory research,we found a new mechanism responsible for CDK4 and CDK6 participating in anti-tumor immunity.That is,endogenous DNA damage caused by CDK4/6 knockout or inhibition activates cGAS-STING signaling pathway and induces the transcription and expression of typeⅠ interferon.Type Ⅰ interferon promotes the activation and infiltration of immune cells in tumor microenvironment thus stimulates anti-tumor immune responses.This study suggests that CDK4/6 could be used as an immunotherapy target.Moreover,it provides a new idea for CDK4/6 inhibitors combined with immunotherapy used clinically.