Screening And Functional Study Of Small Molecule Inhibitors Targeting Cyclin-dependent Kinase 9(CDK9) In Clear Cell Renal Cell Carcinoma

Renal cell carcinoma(RCC)is a malignancy with high incidence,which accounts for around 4%of all cancers worldwide.RCC is a heterogeneous cancer which is classified into three major types,which includes clear cell Renal Cell Carcinoma(ccRCC),papillary Renal Cell Carcinoma(pRCC)and chromophobe Renal Cell Carcinoma(chRCC).ccRCC is the most frequently occurred renal cell carcinoma and accounts for 75%of all renal cell carcinoma.Compared with other subtypes of renal cell carcinoma,ccRCC is characterized by high metastasis,which remains as a major challenge,such as limitation of currently available therapeutic strategies and the associated side-effects in clinic.Cyclin-dependent kinase 9(CDK9)is a serine/threonine kinase which plays a key role in gene transcriptional elongation.CDK9 controls gene transcriptional elongation through phosphorylating carboxy-terminal domain at serine 2 of RNA polymerase Ⅱ(RNA Pol Ⅱ p-SER2).Numerous reports suggested that CDK9 over-expression is associated with poor prognoiss of various types of cancers,which prompts the development of CDK9 inhibitors,aiming to treat cancers.In this dissertation,CDK9 was found to be over-expressed in ccRCC and associated with poor prognosis in clinic patients.Knocking down of CDK9 attenuates ccRCC cell growth and migration.Compared to breast cancer,liver cancer and ovarian cancer cell lines,which express low levels of CDK9,iCDK9,a CDK9 inhibitor,is more potent in killing ccRCC cells.The IC50 in ccRCC is much lower than that in breast cancer,liver cancer and ovarian cancer cells.However,iCDK9 displays no specificity between normal kidney cells and ccRCC cells,which limits its development as a drug in clinical trials.We therefore designed a series of compounds that were based on iCDK9 structure,and eventually obtained a compound named ICDK9-7-1 which inhibits the growth of ccRCC specifically.More importantly,iCDK9 and ICDK9-7-1 have the same efficacy to inhibit tumor growth in mouse xenograft models.However,ICDK9-7-1 shows no toxicity while iCDK9 treatment significantly decreased mouse body weight.Specifically,HE staining revealed that,compared with control and ICDK9-7-1 panels,iCDK9-treated mice exhibited severe damage in many organs including lung,liver,spleen and kidney,among others.It is demonstrated that bromodomain containing 4(BRD4)recruits positive transcription elongation factor b(P-TEFb),a protein complex composed of CDK9 and Cyclin T1,to maintain the transcriptional elongation of oncogenic and anti-apoptotic genes and therefore tumor growth.Interestingly,the high expression of BRD4 is associated with poor prognosis in ccRCC based on TCGA data,and studies showed that BRD4 indeed plays an important role in ccRCC.We further demonstrated that inhibition of BRD4 and CDK9 in combination in ccRCC is more effective than inhibition of BRD4 and CDK9 alone in terms of down-regulating oncogenic protein,anti-apoptotic proteins and immune-checkpoint proteins.In summary,the current study revealed that CDK9 plays a critical role in ccRCC and a CDK9 inhibitor named ICDK9-7-1 was identified,which is effective in inhibiting tumor growth in mouse xenograft models but has low toxicity.Meanwhile,the combination of ICDK9-7-1 with BRD4 inhibitor,JQ1,further improve the inhibitory effects,which will be a potential avenue in battling ccRCC in clinic.