Application of CDK1 inhibitor in preparation of medicine for treating cancer with high CDK1 expression

Abstract

The invention discloses an application of a CDK1 inhibitor in preparation of a medicine for treating cancer with high CDK1 expression. The CDK1 inhibitor has a remarkable effect in the aspect of restoring the sensitivity of CRC cells to oxaliplatin, good tolerance is achieved in vivo when oxaliplatin and the CDK1 inhibitor are jointly used, and good clinical application prospects are achieved for oxaliplatin resistant patients. The invention provides a solid foundation for treating oxaliplatin-resistant patients by using the CDK1 inhibitor.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and particularly to a drug for enhancing the sensitivity of cancer cells to oxaliplatin. Background Art

[0002] Colorectal cancer (CRC) is the most common malignant tumor in the digestive tract. Chemotherapy is an indispensable strategy for treating CRC. Oxaliplatin is a third-generation platinum-based anti-tumor drug, mainly used as first-line treatment for advanced CRC and adjuvant treatment after complete resection of primary tumors. Although chemotherapy has initial efficacy, nearly half of stage II and III CRC patients have increased cancer cell resistance to oxaliplatin, further increasing the treatment difficulty. Therefore, elucidating the key molecules and potential mechanisms underlying the increased resistance to oxaliplatin may help develop new treatment strategies to address the current problems in CRC treatment.

[0003] Ferroptosis is a novel iron-dependent programmed cell death, which is different from apoptosis, necrosis, and autophagy in terms of mechanism and morphology. It is triggered by the fatal accumulation of lipid peroxides on the cell membrane. Ferroptosis is dynamically controlled by the oxidation and antioxidant systems involving multiple signaling pathways, such as solute carrier family 7 member 11 (SLC7A11) / glutathione / glutathione peroxidase 4 (GPX4), FSP1 / CoQH2, DHODH / CoQH2, and GCH1 / BH4 axes. Tumor cells can evade ferroptosis, thus unable to control disease progression and resistance, while pharmacological or genetic induction of ferroptosis can effectively restore cell chemosensitivity. MT-1G contributes to the resistance of hepatocellular carcinoma to sorafenib, and its inhibition promotes glutathione depletion and ferroptosis, enhancing the anti-tumor activity of sorafenib. Roblitinib is a fibroblast growth factor receptor 4 inhibitor that induces lipid peroxidation by regulating the β-catenin / TCF4 / SLC7A11 / FPN1 axis, thereby overcoming the intrinsic and acquired trastuzumab resistance in HER2-positive breast cancer patients.

[0004] Lipid peroxidation is a free radical-driven reaction that mainly affects polyunsaturated fatty acid-containing phospholipids (PUFAPLs). Due to the presence of the diallyl moiety in PUFAs, it is a prerequisite for inducing ferroptosis. Acyl-CoA synthetase long-chain family 4 (ACSL4) is a key enzyme mediating the synthesis of PUFA-PLs. Specifically, it catalyzes the ligation of free PUFAs and converts them into PUFA-CoA, which is then esterified and incorporated into phospholipids mediated by lysophosphatidylcholine acyltransferase 3 to form PUFA-PLs. The phosphorylation of ACSL4 at T328 by protein kinase C (PKC)-βII is crucial for the activation of ACSL4, which leads to the formation of a PKC-βII-ACSL4 positive feedback loop for lipid peroxidation, thus amplifying lipid peroxidation and inducing ferroptosis. These data indicate that ACSL4 is a sensitive monitor and a decisive factor for ferroptosis. ACSL4 is regulated by multiple transcription factors and microRNAs (miRNAs), such as YAP, miR-23a-3p, and miR-424-5p. However, we know little about the post-translational modification of ACSL4, especially the turnover of the ACSL4 protein.

[0005] Genome-wide screening based on the CRISPR / Cas9 gene editing technology is high-throughput and efficient, and it is widely used in studying disease mechanisms and drug development, providing a scientific basis for clinical treatment. If in vitro and in vivo genome-wide CRISPR / Cas9 functional screening is carried out to successfully identify the key genes that block ACSL4-induced ferroptosis leading to oxaliplatin resistance in CRC, it will provide strong support for the progress of CRC treatment. Summary of the Invention

[0006] The object of the present invention is to provide the use of a CDK1 inhibitor in the preparation of a drug for treating cancers with high expression of CDK1.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The use of CDK1 in the preparation of a drug for treating cancers.

[0009] The present invention also provides the use of a CDK1 inhibitor in the preparation of a drug for treating cancers.

[0010] The present invention also provides the use of a CDK1 inhibitor in the preparation of a drug for treating cancers with high expression of CDK1.

[0011] The present invention also provides the use of a CDK1 inhibitor in the preparation of a drug for treating colorectal cancer.

[0012] The present invention also provides the use of a CDK1 inhibitor in the preparation of a drug for treating colorectal cancer with high expression of CDK1.

[0013] The present invention also provides the use of a CDK1 inhibitor in the preparation of a drug for promoting ferroptosis of cancer cells.

[0014] The present invention also provides the use of a CDK1 inhibitor in the preparation of a drug for increasing the sensitivity of cancer cells to oxaliplatin.

[0015] The advantages of the present invention are as follows: The CDK1 inhibitor has a significant effect on restoring the sensitivity of CRC cells to oxaliplatin. The combined use of oxaliplatin and the CDK1 inhibitor has good tolerance in vivo and has good clinical application prospects for patients resistant to oxaliplatin. The present invention provides a solid foundation for the treatment of patients resistant to oxaliplatin using a CDK1 inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and do not constitute an improper limitation of the present invention. In the drawings:

[0017] Figure 1 is a diagram showing the construction result of an oxaliplatin-resistant colorectal cancer cell line of the present invention;

[0018] Figure 2 is a diagram showing the upregulation result of CDK1 in cancers (CRC) with oxaliplatin resistance;

[0019] Figure 3-1 is one of the diagrams showing the result that knocking out CDK1 enhances the antitumor effect of oxaliplatin;

[0020] Figure 3-2 is another diagram showing the result that knocking out CDK1 enhances the antitumor effect of oxaliplatin;

[0021] Figure 3-3 is yet another diagram showing the result that knocking out CDK1 enhances the antitumor effect of oxaliplatin;

[0022] Figure 4 is a diagram of an experiment related to the relationship between CDK1 and oxaliplatin resistance;

[0023] Figure 5-1 is one of the diagrams showing the result of exploring the reason for the upregulation of CDK1;

[0024] Figure 5-2 is another diagram showing the result of exploring the reason for the upregulation of CDK1;

[0025] Figure 6-1 is one of the diagrams showing the result of CDK1 being regulated by m6A;

[0026] Figure 6-2 is another diagram showing the result of CDK1 being regulated by m6A;

[0027] Figure 6-3It is the third result diagram of CDK1 regulated by m6A;

[0028] Figure 6-4 It is the fourth result diagram of CDK1 regulated by m6A;

[0029] Figure 7-1 It is the first diagram of the interaction between CDK1 and ACSL4 protein and the promotion of its degradation;

[0030] Figure 7-2 It is the second diagram of the interaction between CDK1 and ACSL4 protein and the promotion of its degradation;

[0031] Figure 7-3 It is the third diagram of the interaction between CDK1 and ACSL4 protein and the promotion of its degradation;

[0032] Figure 7-4 It is the fourth diagram of the interaction between CDK1 and ACSL4 protein and the promotion of its degradation;

[0033] Figure 7-5 It is the fifth diagram of the interaction between CDK1 and ACSL4 protein and the promotion of its degradation;

[0034] Figure 8-1 It is the first result diagram of CDK1 reducing the stability of ACSL4 protein;

[0035] Figure 8-2 It is the second result diagram of CDK1 reducing the stability of ACSL4 protein;

[0036] Figure 8-3 It is the third result diagram of CDK1 reducing the stability of ACSL4 protein;

[0037] Figure 8-4 It is the fourth result diagram of CDK1 reducing the stability of ACSL4 protein;

[0038] Figure 9-1 It is the first result diagram of ACSL4 phosphorylated by CDK1 at S447;

[0039] Figure 9-2 It is the second result diagram of ACSL4 phosphorylated by CDK1 at S447;

[0040] Figure 9-3 It is the third result diagram of ACSL4 phosphorylated by CDK1 at S447;

[0041] Figure 10-1 It is the first result diagram of CDK1 phosphorylating ACSL4 protein at S447;

[0042] Figure 10-2 It is the second result diagram of CDK1 phosphorylating ACSL4 protein at S447;

[0043] Figure 10-3 It is the third result graph of the phosphorylation of ACSL4 protein by CDK1 at S447;

[0044] Figure 11-1 It is one of the result graphs of CDK1 inhibiting ACSL4-mediated ferroptosis;

[0045] Figure 11-2 It is the second result graph of CDK1 inhibiting ACSL4-mediated ferroptosis;

[0046] Figure 11-3 It is the third result graph of CDK1 inhibiting ACSL4-mediated ferroptosis;

[0047] Figure 11-4 It is the fourth result graph of CDK1 inhibiting ACSL4-mediated ferroptosis;

[0048] Figure 12-1 It is the fifth result graph of CDK1 inhibiting ACSL4-mediated ferroptosis;

[0049] Figure 12-2 It is the sixth result graph of CDK1 inhibiting ACSL4-mediated ferroptosis;

[0050] Figure 13-1 It is one of the result graphs of CDK1 inhibitor restoring the sensitivity of oxaliplatin in the CDX model;

[0051] Figure 13-2 It is the second result graph of CDK1 inhibitor restoring the sensitivity of oxaliplatin in the CDX model;

[0052] Figure 13-3 It is the third result graph of CDK1 inhibitor restoring the sensitivity of oxaliplatin in the CDX model;

[0053] Figure 14-1 It is one of the result graphs of CDK1 inhibitor restoring the sensitivity of oxaliplatin in vivo;

[0054] Figure 14-2 It is the second result graph of CDK1 inhibitor restoring the sensitivity of oxaliplatin in vivo;

[0055] Figure 14-3 It is the third result graph of CDK1 inhibitor restoring the sensitivity of oxaliplatin in vivo;

[0056] Figure 14-4 It is the fourth result graph of CDK1 inhibitor restoring the sensitivity of oxaliplatin in vivo;

[0057] Figure 15-1 It is one of the result graphs of CDK1 inhibitor restoring the sensitivity of oxaliplatin in the PDX model;

[0058] Figure 15-2 It is the second figure showing the results of CDK1 inhibitor restoring the sensitivity of oxaliplatin in the PDX model;

[0059] Figure 15-3 It is the third figure showing the results of CDK1 inhibitor restoring the sensitivity of oxaliplatin in the PDX model;

[0060] Figure 15-4 It is the fourth figure showing the results of CDK1 inhibitor restoring the sensitivity of oxaliplatin in the PDX model;

[0061] Figure 16 It is a summary figure showing the role of CDK1 in oxaliplatin resistance in colorectal cancer (CRC). Detailed implementation manners

[0062] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. Here, the illustrative embodiments and descriptions of the present invention are used to explain the present invention, but not to limit the present invention.

[0063] 1. Explore the relationship between CDK1 and CRC's resistance to oxaliplatin

[0064] Different CRC cells were incubated with oxaliplatin respectively, and the results are as Figure 1 shown in A. The IC50 values of HCT8 and HT29 cells are lower, 0.453 μM for HCT8 and 0.496 μM for HT29. As Figure 1 shown in B, HCT8 and HT29 cells were incubated in gradually increasing concentrations of oxaliplatin. Each concentration of oxaliplatin was administered for two weeks, and the total duration was about 9 months to obtain stable oxaliplatin-resistant cancer cells HCT8-OR and HT29-OR, and cultured for more than 6 months at the final oxaliplatin concentration.

[0065] The CCK-8 assay was used to detect the cell viability of HCT8, HT29, HCT8-OR, and HT29-OR cells cultured in different concentrations of oxaliplatin. The results are as Figure 1 shown in C-D. The cell viability of HCT8-OR and HT29-OR is higher than that of HCT8 and HT29. Flow cytometry was used to analyze the apoptosis rate of HCT8, HT29, HCT8-OR, and HT29-OR after treatment with 1 μM oxaliplatin for 48 hours. The results are as Figure 1 shown in E-F. The apoptosis rate of HCT8-OR and HT29-OR is lower than that of HCT8 and HT29.

[0066] qRT-PCR was used to analyze the mRNA levels of CDK1, CDK2, CDK4, CDK6, and CDK9 in oxaliplatin-sensitive (n = 39) and oxaliplatin-resistant (n = 30) CRC tissues. The results are as Figure 1As shown in G-K.

[0067] As Figure 2 shown in A, a genome-wide CRISPR / Cas9 loss-of-function screen was performed using the GeCKO v2 human library containing 123,411 sgRNAs targeting 19,050 human genes, as well as in vitro and in vivo screens. Oxaliplatin significantly reduced the sgRNAs associated with cancer cell survival. As Figure 2 shown in B, some genes related to oxaliplatin resistance in the HCT8-OR were also found in the screening model, such as PLK1, HuR, and CHK2. CDK1 ranked highly in both in vitro and in vivo screens and was more druggable, and subsequent research was carried out based on this.

[0068] In oxaliplatin-resistant CRC tissues, as Figure 2 C, D, Figure 1 shown in G-K, the mRNA and protein levels of CDK1 were significantly upregulated, while the levels of other CDK family members such as CDK2, CDK4, CDK6, and CDK9 were not upregulated. The expression of CDK1 in paraffin-embedded tissues was verified by immunohistochemistry (IHC) staining, and the results were as Figure 2 shown in E and F. 80.95% of oxaliplatin-resistant patients had high CDK1 expression. As Figure 2 shown in G, the overall survival time of CRC patients with high CDK1 expression was shorter than that of CRC patients with low CDK1 expression. These data suggest that the upregulation of CDK1 in CRC may be the cause of oxaliplatin resistance.

[0069] 2. Knockdown of CDK1 reduces the resistance of CRC cells to oxaliplatin

[0070] The CDK1 mRNA levels in the control group and HCT8-OR / HT29-OR cells were detected by qRT-PCR, and the results were as Figure 3-1 shown in A. The immunoblotting (IB) results of CDK1 protein expression in the control group and HCT8-OR / HT29-OR cells were as Figure 3-1 shown in B. The statistical results of CDK1 protein expression levels in the control group and HCT8-OR / HT29-OR cells were as Figure 4 shown in A. It can be seen that the CDK1 mRNA and protein levels in HCT8-OR and HT29-OR cells were higher than those in the control parental cells.

[0071] After knocking out CDK1 in HCT8-OR / HT29-OR cells using CRISPR / Cas9 gene editing, the immunoblotting of CDK1 protein expression in HCT8 / HT29-OR cells with knocked-out CDK1 was as Figure 3-1As shown in C, the CCK-8 assay was used to detect the survival rates of HCT8-OR and HT29-OR cells with / without CDK1 knockout after oxaliplatin treatment, and the results are as Figure 3-1 shown in D. CDK1 knockout significantly reduced the survival rates of HCT8-OR and HT29-OR cells after oxaliplatin treatment. As Figure 3-1 shown in E-F, in the presence of oxaliplatin, after knocking out CDK1, the number of clones formed by resistant cells almost decreased to the level of parental cells. After treatment with oxaliplatin, Figure 3-1 as shown in G, more apoptotic cells were observed in HCT8-OR and HT29-OR cells with CDK1 knockout than in control cells.

[0072] HCT8 and HT29 cells overexpressing CDK1 were constructed, and their CDK1 expression levels were as Figure 4 shown in B. The colony formation and apoptosis were measured by the CCK8 assay, and the results are as Figure 3-1 shown in I- Figure 3-2 L. Overexpression of CDK1 in HCT8 and HT29 cells enhanced the resistance of these cells to oxaliplatin.

[0073] We subcutaneously injected HCT8-OR / HT29-OR cells and HCT8-OR / HT29-OR cells with CDK1 knockout into NOD / SCID mice respectively, and then intraperitoneally injected oxaliplatin. The results are as Figure 3-2 shown in M- Figure 3-3 R. The tumors of mice carrying HCT8-OR and HT29-OR did not respond to oxaliplatin; however, in mice carrying HCT8-OR and HT29-OR with CDK1 knocked out, the tumor mass and volume were significantly reduced after oxaliplatin treatment, and IHC staining of Ki-67 (a proliferation marker) also confirmed this. In addition, HCT8 cells and HCT8 cells overexpressing CDK1 were subcutaneously injected into mice respectively, and then intraperitoneally injected with oxaliplatin. The tumor volumes of each experimental group were measured, as Figure 4 shown in C-E. Oxaliplatin significantly inhibited the growth of HCT8-derived tumors, while overexpression of CDK1 blocked the inhibitory effect of oxaliplatin on tumors, and the tumors derived from HCT8 cells overexpressing CDK1 were larger.

[0074] 3. N6-methyladenosine (m 6 A) hypomethylation upregulates CDK1 expression

[0075] To explore the mechanism of CDK1 upregulation, HCT8-OR and HT29-OR cells were treated with 5-azacytidine (5-Aza-dC, a DNA methyltransferase inhibitor) and trichostatin A (TSA, a histone deacetylase inhibitor). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used for detection, and the results are asFigure 5-1 As shown in A-B, the CDK1 mRNA did not change, indicating that DNA methylation and histone acetylation are not involved in the regulation of CDK1, as Figure 5-1 shown in C-D, the knockout of some transcription factors (such as TP53 and HIF-1α) had a slight effect on the expression of CDK1. As Figure 5-1 shown in E, there was no significant difference in the CDK1 copy number between oxaliplatin-resistant CRC tissues and oxaliplatin-sensitive CRC tissues.

[0076] m 6 A plays an important role in RNA metabolism. Now, it is explored whether CDK1 is regulated by m 6 A. As shown in Figure 6A-B, compared with parental cells, the total m 6 A level was significantly decreased in HCT8-OR and HT29-OR cells. The levels of some known m 6 A writers and erasers were tested, and the results Figure 6-2 shown in C, in HCT8-OR and HT29-OR cells, only the m 6 A writer METTL3 was significantly downregulated (red to blue in the figure represents a decrease in level). As Figure 6-2 shown in D-E, Figure 5-1 F-G, the enhanced expression of METTL3 decreased the CDK1 mRNA and protein levels. At the same time, as Figure 6-2 shown in F-G, Figure 5-1 H, the m 6 A enrichment in CDK1mRNA increased. As Figure 5-2 shown in I, a luciferase reporter gene was constructed by inserting the full-length CDK1 mRNA behind the luciferase gene of the pmirGLO vector, and then transfected into HCT8-OR and HT29-OR cells. The results Figure 6-2 shown in H, the luciferase protease activity of CDK1 mRNA was significantly decreased after METTL3 overexpression. Using the sequence-based m 6 A modification site predictor SRAMP, the results Figure 5-2 shown in J, we found six putative m 6 A motifs in CDK1 mRNA, and performed stepwise mutations by replacing adenosine with thymine. The results of the luciferase reporter gene assay Figure 6-2 shown in I indicated that the mutations at positions 499–503 (ms2) and 1669–1673 (ms6) on CDK1 mRNA increased the luciferase activity, indicating that these two m 6The A motif is essential for METTL3-mediated CDK1 regulation. The effects of overexpressing METTL3 on CDK1 mRNA levels in CDK1 knockout cells re-introduced with wild-type or mutant CDK1 were analyzed by qRT-PCR, and the results are shown in Figure 6-2 J and K. Consistently, METTL3 significantly decreased the expression of CDK1 in HCT8-OR and HT29-OR CDK1 knockout cells re-introduced with wild-type CDK1.

[0077] For METTL3-overexpressing HCT8-OR and HT29-OR cells treated with 5 μg / mL actinomycin D, the CDK1 mRNA levels were analyzed by qRT-PCR, and the results are shown in Figure 6-3 L - M. After METTL3 overexpression, the half-life of CDK1 mRNA was significantly shortened. To study the m 6 A reader involved in this process, the CDK1 mRNA levels of METTL3-overexpressing HCT8-OR and HT29-OR cells were analyzed by qRT-PCR, and the results are shown in Figure 6-3 N. Only knocking out YTHDF2 could improve the decrease in CDK1 mRNA levels caused by METTL3 overexpression. The enrichment of YTHDF2 on CDK1 mRNA was determined by RIP, and the results are shown in Figure 6-3 O - P. Compared with parental cells, less enrichment of YTHDF2 on CDK1 mRNA was observed in HCT8-OR and HT29-OR. The YTHDF2 mRNA levels in parental cells, HCT8-OR and HT29-OR were analyzed by qRT-PCR, and the YTHDF2 protein levels in parental cells, HCT8-OR and HT29-OR were detected by IB, and the results are shown in Figure 5-2 K - M. There was no difference in YTHDF2 expression among parental cells, HCT8-OR and HT29-OR.

[0078] The luciferase reporter assay was used to detect the effect of YTHDF2 knockout on the m 6 A activity of CDK1 mRNA, and the results are shown in Figure 6-3 Q. The knockout of YTHDF2 increased the luciferase activity of the wild-type CDK1 reporter gene, but did not increase the activity of the m 6 A mutant reporter gene (mutations in ms2 and ms6). The wild-type CDK1 luciferase reporter gene and m 6 A mutant CDK1 (ms2 and ms6 mutations) luciferase reporter gene were respectively introduced into HCT8-OR and HT29-OR cells overexpressing YTHDF2, as shown in Figure 6-4As shown in R-S, overexpression of YTHDF2 decreased the expression of CDK1 in HCT8-OR and HT29-OR cells transfected with wild-type CDK1 luciferase reporter gene, but had less effect on the expression of CDK1 in HCT8-OR and HT29-OR cells transfected with m 6 A mutant CDK1 (ms2 and ms6 mutations) luciferase reporter gene.

[0079] 4. CDK1 binds to and degrades ACSL4

[0080] CDK1 is a typical driver of the G2 / M phase transition. The cell cycle progression of oxaliplatin-resistant and oxaliplatin-sensitive cells was tested, and the results showed that: compared with parental cells, the cell cycle of HCT8-OR and HT29-OR cells did not accelerate; on the contrary, as Figure 5-2 shown in N, a slight G2 / M phase arrest was observed in HT29-OR cells, indicating that CDK1 drives cell resistance to oxaliplatin in a cell cycle-independent manner.

[0081] CDK1-binding proteins were enriched and analyzed by mass spectrometry. The results were as Figure 7-1 shown in A. Some known CDK1-binding proteins, such as CCNB1 and HNRNPUL1, were identified. It was also found that ACSL4 is a key factor in ferroptosis related to oxaliplatin resistance and can be immunoprecipitated by CDK1. Furthermore, it was inferred that CDK1 causes cancer cell resistance to oxaliplatin through ACSL4. As Figure 8-1 shown in A, Figure 7-1 B, endogenous co-immunoprecipitation (co-IP) showed that CDK1 and ACSL4 bind to each other in HCT8-OR and HT29-OR cells. As Figure 7-1 shown in C, co-localization of CDK1 and ACSL4 was observed in immunofluorescence staining.

[0082] As Figure 8-1 shown in B, the Ni-NTA pull-down assay showed that CDK1 directly interacts with ACSL4 in vitro. In addition, we transfected labeled exogenous CDK1 and ACSL4 proteins into CRC cells. As Figure 8-1 shown in C, Figure 7-2 D, it was found that there was binding between them. As Figure 8-1 shown in D, Figure 7-2 E-G, compared with control cells, the expression of ACSL4 protein was significantly increased in HCT8-OR and HT29-OR CDK1 cells with CDK1 knocked out, but the mRNA expression did not increase. Figure 8-1 shown in E-F, Figure 7-2 H, after CDK1 knockout, the half-life of ACSL4 protein was significantly prolonged. Figure 8-2 shown in G, Figure 7-2As shown in I-7-3K, re-introducing CDK1 eliminated the above effect. The reduction of ACSL4 caused by CDK1 overexpression was alleviated by MG132, a proteasome inhibitor, rather than chloroquine, an autophagy inhibitor, indicating that the ubiquitin-proteasome system is responsible for CDK1-mediated ACSL4.

[0083] Figure 8-2 H, Figure 7-3 As shown in L, CDK1 knockout significantly reduced the endogenous ubiquitination level of ACSL4, while with the increasing intensity of re-introducing CDK1, the endogenous ubiquitination level of ACSL4 increased in a gradient form at the same expression level, indicating that re-introducing CDK1 increased its level in a dose-dependent manner. By analyzing the UbiBrowser database, we found 15 known lysine ubiquitination sites on the ACSL4 protein. Through stepwise mutation by replacing lysine with arginine and then performing ubiquitination analysis. Figure 8-2 As shown in I~8-3J, the results showed that only K388R, K498R, and K690R could block the increase in the ubiquitination level of ACSL4 caused by CDK1. Figure 8-3 As shown in K, the branched-chain ubiquitin chain of K48, rather than K63, mediates the degradation of ACSL4 by CDK1. Figure 8-3 L, Figure 7-3 As shown in M-N, overexpression of CDK1 in cells reduced the protein level of ACSL4, but after mutating K388, K498, and K690 on ASCL4, overexpression of CDK1 in cells did not reduce the protein level of ACSL4.

[0084] CDK1 is not an E3 ubiquitin ligase. To explore the type of ubiquitin ligase involved in the regulation of ACSL4 by CDK1, the intersection of CDK1 enrichment mass spectrometry and the predicted ACSL4 E3 ligase by UbiBrowser is as Figure 7-3 As shown in O, the results showed that five E3 ligases might bind to both CDK1 and ACSL4 simultaneously. Then we silenced them one by one, Figure 8-3 As shown in M, Figure 7P-R, it was found that only knocking out UBR5 could counteract the reduction in the ACSL4 level caused by re-introducing CDK1 in HCT8-OR and HT29-OR cells. Figure 8-3 N, Figure 7-4 The endogenous Co-IP results in S showed that both CDK1 and ACSL4 were immunoprecipitated by UBR5. Knocking out CDK1 reduced the binding of UBR5 to ACSL4 in HCT8-OR and HT29-OR cells, and re-introducing CDK1 increased the binding of UBR5 to ACSL4 in HCT8-OR and HT29-OR cells. Figure 8-3 O, Figure 7-5As shown in T-V, overexpression of wild-type UBR5 and overexpression of the ubiquitin ligase-deficient mutant (C2768A) significantly reduced the expression of ACSL4 protein; however, in the absence of CDK1, this effect was eliminated. Figure 8-4 P, Figure 7-5 As shown in W, the K48-linked ubiquitination level of ACSL4 increased due to UBR5 overexpression, did not increase due to overexpression of UBR5-C2768A, and knockout of CDK1 or mutation of K388 / K498 / K690 eliminated the above effects. In vitro ubiquitination assays showed that Figure 7-5 As shown in X, UBR5 can directly ubiquitinate ACSL4, rather than UBR5-C2768A.

[0085] 5. CDK1 directly phosphorylates ACSL4 at S447

[0086] CDK1 is a Ser / Thr protein kinase. To investigate whether ACSL4 is phosphorylated by CDK1. As Figure 9-1 A and Figure 10-1 shown in A, compared with control cells, the Ser / Thr phosphorylation level of ACSL4 was significantly reduced in HCT8-OR and HT29-OR cells with CDK1 knockdown, and the total protein of ACSL4 increased. However, after reintroduction of CDK1, the Ser / Thr phosphorylation level of ACSL4 returned to normal.

[0087] We used the Scansite online tool and found that CDK1 might phosphorylate ACSL4 at S57 and S447. Exogenous IP assays were performed in CDK1-overexpressing cells, and then mass spectrometry analysis was performed on the phosphorylation sites. Figure 9-2 The results in B showed that S447 was identified, but S57 was not. As Figure 9-2 shown in the C sequence alignment analysis, ACSL4 S447 was highly conserved among species. The results of in vitro phosphorylation assays Figure 9-2 in D showed that CDK1 phosphorylated ACSL4, but after serine at position 477 was mutated to alanine (a phosphorylation-deficient mutant) or treated with the CDK1 inhibitor RO-3306, CDK1 could not phosphorylate ACSL4.

[0088] We constructed an antibody specifically targeting p-ACSL4 S447 and verified its effectiveness. The results, as Figure 10-1 shown in B, showed that CDK1 promoted p-ACSL4 S447. Figure 9-2 E, Figure 10-1 As shown in C-D, after knockout of CDK1, the level of p-ACSL4S447 decreased, and after re-expression of CDK1, the level of p-ACSL2S447 was restored. Figure 9-2 F,Figure 10-2 As shown in E - G, overexpression of CDK1 or UBR5 decreased the level of ACSL4 in cells overexpressing ACSL4 - WT or ACSL4 - S447D (a phosphomimetic mutant), but the level of ACSL4 was not decreased in cells overexpressing the phosphodefective mutant ACSL4 - S427A.

[0089] As Figure 9-3 G, Figure 10-2 H shown, ACSL4 - S447A blocked the interaction between UBR5 and ACSL4 in HCT8 - OR and HT29 - OR cells. As Figure 9-3 H shown, in cells overexpressing ACSL4 - WT or ACSL4 - S447D, UBR5 or CDK1 increased the level of K48 - linked ubiquitination of ACSL4. As Figure 10-3 I shown, a similar phenomenon was not observed in cells overexpressing ACSL4 - S447A. Figure 9-3 I, Figure 9-3 J, Figure 10-3 J, Figure 10-3 J shown, the expression of CDK1 was negatively correlated with the level of ACSL4 in clinical CRC tissues, but positively correlated with the level of p - ACSL4 S447. Figure 9-3 J shown, compared with the overexpression of ACSL4 - S447 in HCT8 - OR and HT29 - OR cells re - expressing CDK1 after oxaliplatin treatment, the overexpression of ACSL4 - S447A significantly reduced the cell viability. It can be seen that in oxaliplatin - resistant CRC cells, phosphorylation of ACSL4 S447 is necessary for CDK1 to degrade ACSL4 through UBR5.

[0090] 6. CDK1 confers oxaliplatin resistance by blocking ACSL4 - mediated ferroptosis

[0091] As Figure 11-1 shown in A - 11 - 2D, HCT8 - OR and HT29 - OR cells were more resistant to the ferroptosis inducers Erastin or RSL3 than their parental cells. As Figure 11-3 shown in I - J, Figure 11-2 E - H, Figure 12-1 shown in A - D, CDK1 knockout or RO - 3306 treatment significantly increased the sensitivity of HCT8 - OR and HT29 - OR cells to Erastin or RSL3, accompanied by high levels of lipid peroxidation; re - expression of CDK1, knockout of ACSL4 or treatment with rosiglitazone (ROSI, an ACSL4 inhibitor) eliminated these effects. Figure 12-1 shown in A - D, Figure 11-2As shown in E-H, in Erastin- or RSL3-treated cells, the increased lipid peroxidation and cell death caused by CDK1 knockout can be effectively blocked by adding ferritin-1 (an iron poisoning inhibitor), deferoxamine (an iron chelator), or N-acetylcysteine (a ROS scavenger), but not by adding Z-VAD-FMK (a pan-caspase inhibitor) or Necrostatin-1 (a necroptosis inhibitor). Figure 11-2 E-H, Figure 12-1 A-D, Figure 11-3 As shown in K-L, 12-1E-F, overexpression of CDK1 reduced cell death and lipid peroxidation induced by Erastin or RSL3 in HCT8 and HT29 cells, while knockout of UBR5 or overexpression of ACSL4-WT significantly increased cell death and lipid peroxidation, especially more significantly after overexpression of ACSL4-S447A. Figure 12-2 G-I, Figure 11-3 M~ Figure 11-4 O, the above results were further verified by detecting the ferroptosis markers 4-hydroxy-2-nonenal (4-HNE) and PTGS2. Figure 12-2 J-K, Figure 11-4 As shown in P-Q, overexpression of CDK1 blocked the increase in PUFAPL levels induced by Erastin or RSL3, such as phosphatidylethanolamine (PE) 18:0_20:4 and PE 18:0_22:4; however, knockout of UBR5 or overexpression of ACSL4-WT, especially overexpression of ACSL4-S447A, could eliminate the blocking effect of CDK1 overexpression on the increase in PUFAPL levels induced by Erastin or RSL3. These data indicate that inhibiting ACSL4-driven lipid peroxidation and ferroptosis is crucial for CDK1 to promote CRC resistance to oxaliplatin.

[0092] 7. CDK1 inhibitors overcome oxaliplatin resistance in cell / patient-derived xenograft (CDX / PDX) models

[0093] Regarding the effect of CDK1 inhibitors on oxaliplatin resistance, preclinical evaluations were conducted using animal models. As Figure 13-1 shown in A, for the CDX model, NOD / SCID mice were subcutaneously injected with HCT8-OR or HT29-OR cells, and then oxaliplatin, RO-3306, and Liproxstatin-1 were given at different times. The results were as Figure 13-1 shown in B-C, all mice survived at the end of the treatment, and no mice showed signs of severe weight loss (>15%), infection, or wounds, indicating that the combination of the above drugs had no obvious toxicity in vivo. Figure 14-1A-C, Figure 13-1 As shown in D-F, HCT8-OR / HT29-OR-derived tumors were insensitive to oxaliplatin. When used in combination with RO-3306, tumor mass and volume were greatly reduced, and individual tumors were hardly detectable. However, these anti-tumor effects disappeared after adding the ferroptosis inhibitor Liproxstatin-1. Figure 14-1 D~ Figure 14-2 I, Figure 13-1 G~ Figure 13-3 As shown in R, compared with the other three groups, higher levels of lipid peroxidation, ACSL4, 4-HNE, PTGS2, and Fe 2+ levels, as well as lower levels of p-ACSL4 S447 and Ki-67, were observed in the oxaliplatin + RO-3306 group.

[0094] Figure 15-1 As shown in A, we established a PDX model with better clinical relevance by using two clinically oxaliplatin-resistant CRC tissues, and then treated them with oxaliplatin, RO-3306, and Liproxstatin-1. The results were as Figure 15-1 shown in B and C. None of the mice showed signs of severe weight loss (>15%), infection, or wounds. Figure 14-3 J-L, Figure 15-2 As shown in D-F, there was no significant statistical difference in tumor size between the control group and the oxaliplatin group, while the tumors in the oxaliplatin + RO-3306 group were significantly reduced, and the addition of Liproxstatin-1 significantly restored tumor growth. Compared with the control group or oxaliplatin alone, the combination of oxaliplatin and RO-3306 increased lipid peroxidation, ACSL4, 4-HNE, PTGS2, and Fe 2+ levels, while reducing the expression of p-ACSL4 S447 and Ki-67; Figure 14-3 M~ Figure 14-4 R, Figure 15-2 G~ Figure 15-4 As shown in R, these effects disappeared after adding Liproxstatin-1.

[0095] From the above experiments, it can be seen that oxaliplatin resistance is a difficult problem in the clinical treatment of CRC. We used in vitro and in vivo genome-wide CRISPR / Cas9 functional screening to determine that CDK1 is a key factor in oxaliplatin resistance. CDK1 was significantly overexpressed in oxaliplatin-resistant CRC and was closely related to poor prognosis. CDK1 blocked ferroptosis by inhibiting ACSL4, leading to oxaliplatin resistance. As Figure 16As shown, CDK1 directly interacts with and degrades ACSL4 by recruiting the E3 ubiquitin ligase UBR5, a process that depends on the phosphorylation of ACSL4 at S447. In vitro and in vivo models showed that genetic and pharmacological blockade of CDK1 restored the sensitivity of tumor cells to oxaliplatin. The present invention reveals a close relationship between CDK1 and oxaliplatin resistance, which provides a preclinical basis for using CDK1 as a therapeutic target for oxaliplatin-resistant CRC patients.

[0096] CDK1 is a founding member of the cyclin-dependent protein kinase (CDK) family and is overexpressed in a variety of human cancers, closely associated with malignant phenotypes and poor prognosis. Evidence suggests that upregulation of CDK1 is due to copy number changes. However, no statistical difference in CDK1 copy number was observed between oxaliplatin-resistant and oxaliplatin-sensitive CRC tissues, suggesting the existence of other mechanisms regulating CDK1. 6 A is the most common modification of eukaryotic RNA. 6 A methylase (writer) and demethylase, reversibly and dynamically controlled. 6 A writer METTL3 is m 6 The only catalytic subunit of the METTL3 modification system is widely believed to be crucial for the development and progression of cancer. 6 The total level of A was reduced, indicating that METTL3 may inhibit oxaliplatin resistance in CRC. The present invention also found that CDK1 is a new target of METTL3, and CDK1 mRNA is expressed by METTL3. 6 A modification. m 6 The fate of A-modified RNA is determined by m 6 A binding proteins, especially the YT521-B homology (YTH) family, including YTHDF1 / 2 / 3 and YTHDC1 / 2. YTHDF1, YTHDF3 and YTHDC2 are closely related to RNA translation efficiency. YTHDC1 controls m 6 YTHDF2 plays an important role in the regulation of RNA stability through A-mediated alternative splicing. Overexpression of METTL3 reduced the stability of CDK1 mRNA, while knockout of YTHDF2 but not other m 6 A receptor blocks this effect, indicating that YTHDF2 recognizes the m 6 A modified CDK1 mRNA, CDK1 mRNA decay, leading to CDK1 downregulation. The present invention discloses m 6 A strong association between A and oxaliplatin resistance in CRC.

[0097] ACSL4 is a novel phosphorylation substrate of CDK1, and CDK1 binds to and degrades ACSL4 through direct phosphorylation. ACSL4 is phosphorylated by CDK1 at S447 and undergoes K48-linked polyubiquitination mediated by UBR5 at K388 / K498 / K690. The degradation of ACSL4 requires the phosphorylation group generated by CDK1 to recognize UBR5. Overexpression of ACSL4, especially the phospho-deficient mutant ACSL4-S447A, significantly abolishes the resistance to oxaliplatin caused by overexpression of CDK1. The in vivo effect of CDK1 inhibition on restoring oxaliplatin sensitivity is antagonized by ferroptosis inhibitors, indicating that CDK1 confers oxaliplatin resistance by inhibiting ACSL4-mediated ferroptosis.

[0098] Preclinical CDX and PDX models have shown that the selective CDK1 inhibitor RO-3306 has a significant effect on restoring the sensitivity of CRC cells to oxaliplatin. CDK1 inhibitors have target selectivity, which is reflected in that their killing effect on tumor cells is four times that on normal cells. No obvious side effects were observed after in vivo administration of the CDK1 inhibitor. Even in the case of multi-drug use, no signs of severe weight loss, infection, or wounds were found in any treated mice, which means that the combination of oxaliplatin and the CDK1 inhibitor has good tolerance in vivo and thus has good clinical application prospects for patients with oxaliplatin resistance. The present invention provides a solid basis for using CDK1 inhibitors to treat patients with oxaliplatin resistance.

[0099] In summary, it can be seen that CDK1 is a target in oxaliplatin resistance, and the combination of CDK1 inhibitors and oxaliplatin is a promising strategy for treating patients with oxaliplatin-resistant CRC.

[0100] The technical solutions provided by the embodiments of the present invention have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present invention. The descriptions of the above embodiments are only applicable to help understand the principles of the embodiments of the present invention; at the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. Use of CDK1 in the preparation of drugs for treating cancer.

2. Use of a CDK1 inhibitor in the preparation of drugs for treating cancer.

3. Use of a CDK1 inhibitor in the preparation of drugs for treating cancers with high CDK1 expression.

4. Use of a CDK1 inhibitor in the preparation of drugs for treating colorectal cancer.

5. Use of a CDK1 inhibitor in the preparation of drugs for treating colorectal cancer with high CDK1 expression.

6. Use of a CDK1 inhibitor in the preparation of drugs for promoting ferroptosis of cancer cells.

7. Use of a CDK1 inhibitor in the preparation of drugs for increasing the sensitivity of cancer cells to oxaliplatin.

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