TP53 is a tumor suppressor gene that encodes for the transcription factor p53. It plays important functions throughout human life, including regulation of genomic stability, proliferation, apoptosis, cell cycling, autophagy, metabolism, and stem cell homeostasis. [1], [2], [3], [4] Moreover, TP53 is also the most frequently mutated gene, which is found mutating in 50 % of human solid tumors, and results in the adverse prognosis to conventional therapies across multiple cancer types. TP53 rarely mutates in hematological malignancies, but in many hematological malignancies including AML (acute myeloid leukemia), p53 activity is also suppressed due to multiple antagonistic mechanisms. [5], [6] Therefore, activating p53 has always been a focus of anticancer research.

CK1α encoding gene is located in the region commonly deleted in MDS (myelodysplastic syndromes) and AML–del (5q32). Gene expression profiling of the cells with CK1α silence or inhibition can reactivate p53. [7], [8] Therefore, targeting CK1α thus appears to be a promising approach for the treatment of MDS and AML. Nevertheless, the only CK1α ablation provokes DNA damage and induces the upregulation of the driver oncogenes MYC and MDM2 in various hematological malignancies. [9], [6] CDK7 and CDK9 are members of the cyclin dependent kinases (CDKs) family, which are the essential drivers of cell-cycle regulatory machinery. [10], [11] CDK7 and CDK9 overexpression has been observed in AML and other hematologic malignancies and in solid tumors, making them attractive targets for cancer therapeutics. [12], [13], [14], [15], [16], [17] CDK7 and CDK9 have been implicated in the regulation of super-enhancer (SE) mediated signaling path. Inhibiting or downregulating CDK7/9 could effectively reduce the protein levels of MCL-1, BCL-2, MYC and MDM2, which leads to potent growth inhibition of tumor cells. [18], [12], [19], [20], [21], [22], [23], [24] Therefore, inhibiting CDK7/9 may offset the upregulation of MYC and MDM2 caused by CK1α degradation. Moreover, inhibiting MDM2 also restores p53. [25], [26] Yinon Ben-Neriah’s team reported a series of pyrazole-pyrimidine scaffold molecules, which can target both CK1α and CDK7/9. The study showed that blocking CK1α together with CDK7 and CDK9 synergistically can stabilize p53 and downregulate of MYC, MCL-1 and MDM2. In addition, the selected compounds induced apoptosis in AML leukemia progenitor cells and led to a growth suppression in the patient-derived xenograft mouse models. [27], [28].

Proteolysis targeting chimera (PROTAC) is an emerging strategy to use small molecules to degrade the targeting protein via hijacking the ubiquitin–proteasome degradation system. PROTAC is a bifunctional molecule comprising an appropriate linker binds to a ligand for the target protein and a ligand for E3 ligase recruitment. [29] After the formation of a ternary complex, the target protein can be ubiquitinated and degraded by the proteasome. [30], [31] PROTACs have the advantage of higher selectivity, better activity, and the ability to reduce drug resistance in comparison to inhibitors, and have the potential to target proteins considered “undruggable”. Thus, PROTACs have emerged as a new modality of drug design tools and potential therapeutic strategies to treat cancers. [32], [33], [34], [35] An increasing number of selective CDK9 degraders have been developed, including THAL-SNS-032, [36] B03 [37] and CP-07 [38]. These all showed potent anticancer effect including AML. [39] However, reports on PROTAC degradation of CK1 and CDK7 proteins are rare.

In this study, we designed and synthesized a series of PROTACs based on the structure of compound A86, and evaluated their effects of reactivating p53 through co-degradation of CK1α, CDK7 and CDK9 and their application in the treatment of AML cancer cells. The selected PROTAC 13i exhibited remarkable CK1α, CDK7 and CDK9 degradation activity and considerably promoted the p53 expression. The PROTAC molecules effectively suppressed cell proliferation and led to cell apoptosis in MV4-11 cells by reducing the levels of MCL-1, MYC，BCL-2 and MDM2. Furthermore, the PROTACs can also downregulate the levels of TNF-α, IL-1β and IL-6 in peripheral blood mononuclear cells (PBMCs). Our findings indicate that PROTAC 13i is a potent CK1α, CDK7 and CDK9 PROTAC and can activate p53 with the potential to treat AML cancer. Moreover, hopefully, the designed strategy in this study becomes a potential therapy for the treatment of AML and other types of tumors.