CN115998885A - Inhibitor combination and application thereof in preparation of medicines for treating MYC high-expression cancers - Google Patents

Abstract

The invention discloses a pharmaceutical composition, a kit and application thereof in preparing medicines for treating MYC high-expression cancers. The pharmaceutical composition comprises a CTPS inhibitor and an ATR inhibitor. The kit of parts comprises a kit a and a kit B, wherein the kit a comprises a CTPS inhibitor and the kit B comprises an ATR inhibitor. Also disclosed is a method for selectively killing tumor cells with high expression of MYC, wherein the pharmaceutical composition or the kit is contacted with the tumor cells; and the application of CTPS1 inhibitor in preparing medicine for treating MYC high expression cancer. The invention selectively kills MYC high-expression tumor cells by jointly inhibiting CTPS and ATR activities, thereby reducing the toxic and side effects on normal cells.

Description

Inhibitor combination and application thereof in preparation of medicines for treating MYC high-expression cancers

Technical Field

The invention relates to the field of biological medicine, in particular to an application of an inhibitor combination in preparing a medicine for treating cancers, and particularly relates to an application of 3-deazauridine and one or more of BAY-1895344, VE-822 and AZD6738 in preparing medicines for treating MYC high-expression cancers.

Background

The uncontrolled expression of MYC protein is involved in the occurrence and development of 70-80% of human cancers, and can be attributed to several aspects such as gene translocation, increase of gene copy number, up-regulation of mRNA transcription and resistance of protein degradation. Studies based on mouse tumor models indicate that silencing MYC gene expression can significantly inhibit or even regress tumors. Silencing MYC gene expression, for example, in a Tet-off MYC-based liver cancer model and osteogenic sarcoma model, can induce tumor regression and differentiation; in an APC-deficiency induced intestinal tumor model, small intestine-specific knockout of MYC significantly inhibited intestinal tumorigenesis. These excellent mouse experimental results greatly motivate the enthusiasm of people to treat tumors by targeting MYC. However, MYC has long been considered "undruggable target" (a non-pharmaceutically acceptable target) due to the lack of a distinct small molecule drug binding pocket on the MYC protein surface and the long-term inhibition of serious toxic side effects caused by MYC.

Metabolic reprogramming is an important feature of tumor cells. MYC maintains the various substances required for tumor cell proliferation, growth and survival by simultaneously activating various anabolic processes, such as nucleotide synthesis, protein synthesis and lipid synthesis, which must be tightly regulated to maintain metabolic balance. An important function of MYC is to activate rRNA synthesis by synergistically modulating RNA polymerase I, II and III, thereby meeting the ribosome demand for various protein syntheses required for rapid proliferation of MYC-expressing cells. Ribosomal RNA (rRNA) accounts for more than 50% of the total ribosomal biomass, and more than 85% of the RNA in the cell is present in the form of rRNA. Thus, rRNA is the most prominent reservoir for nucleotides within cells, and increased synthesis of rRNA must be accompanied by a significant increase in nucleotide demand by cells. To meet the great demand for nucleotides, MYC activates on the one hand the expression of a variety of enzymes involved in nucleotide metabolism, while activating glycolysis and pentose phosphate pathways provides the necessary precursors for purine and pyrimidine nucleotide synthesis. The synthesized nucleotides will be mainly used for rRNA synthesis on the one hand to meet the large demand for proteins for MYC high expressing cell growth and on the other hand will be converted into deoxyribonucleotides for DNA replication. Thus limiting intracellular purine or pyrimidine nucleotide supply while maintaining MYC activity, thereby breaking the nucleotide supply-demand balance established by metabolic reprogramming of MYC would be likely to selectively inhibit or even kill MYC-expressing cells. For example, in MYC high-expression small cell lung cancer, the IMPDH which specifically inhibits guanine nucleotide synthesis rate-limiting enzyme can obviously inhibit tumor growth. MYC high expressing cells can be specifically killed by inhibiting PRPS2 activity and limiting the supply of PRPP, a common precursor for purine and pyrimidine nucleotide synthesis.

CTP is the lowest of the four ribonucleotides (UTP, ATP, GTP and CTP) in the cell, which makes CTP the rate-limiting molecule for nucleic acid (DNA and RNA) synthesis and other CTP-dependent biological processes. CTPs can be synthesized by the de novo synthesis pathway (de novo synthesis pathway) which provides about 70% CTP supply in cells and the salvage pathway (salvage pathway). The rate-limiting step in de novo CTP synthesis is also the last step catalyzed by CTP Synthase (CTPs) to convert UTP to CTP. The expression level and the enzyme activity of CTPS protein are obviously increased in various tumor tissues including colorectal cancer and the like. There are two subtypes of human CTPS: CTPS1 and CTPS2 have similar enzymatic activities, and no obvious difference in functions between them has been found in current research.

At present, no clinical medicine for targeting MYC protein exists, and a method for selectively killing tumor cells with high expression of MYC protein is needed.

Disclosure of Invention

In order to overcome the defect that a clinical medicine for targeting MYC protein and a method for selectively killing MYC protein high-expression tumor cells are lacking in the prior art, the invention provides an inhibitor combination and application thereof in preparing medicines for treating MYC high-expression cancers. In particular, the invention provides an inhibitor combination in the form of a pharmaceutical composition or a kit and application thereof in preparing medicines for treating cancers, in particular to a combination of a CTPS inhibitor and an ATR inhibitor such as 3-deazauridine and one or more of BAY-1895344VE-822 and AZD6738 and application thereof in preparing medicines for treating cancers with high MYC expression.

MYC meets the massive demand for proteins by activating ribosomal RNA (rRNA) synthesis, which leads to a dramatic increase in nucleotide demand, by rapidly proliferating malignant cells. CTP is the lowest of the four ribonucleotides (UTP, ATP, GTP and CTP) in the cell, is the rate-limiting molecule of the relevant biological process, and is mainly synthesized by CTPs-mediated de novo synthesis pathway. The present invention also found that inhibition of CTPS selectively induces MYC-expressing cells to produce DNA replication stress that protects genomic stability by activating ataxia telangiectasia and Rad3-related protein (ATR) and its downstream Chk 1-mediated S-phase checkpoint (checkpoint). The combined inhibition of CTPS and ATR selectively induces MYC high-expression tumor cells to generate DNA damage and apoptosis. The invention discovers that the CTPS inhibitor and the ATR inhibitor are combined for the first time, and has better effect of inhibiting MYC high-expression cells.

In order to solve the technical problems, one of the technical schemes of the invention is as follows: a pharmaceutical composition is provided, characterized in that it comprises a CTPS inhibitor and an ATR inhibitor.

Preferably, the CTPS inhibitor is 3-Deazauridine (DAU), which is represented by the following structural formula:

there are two subtypes of human CTPS: CTPS1 and CTPS2.MYC increases intracellular CTP concentration by specifically inducing CTPS1 expression; the CTPS1 is interfered independently, but not CTPS2, so that the MYC high-expression cells can be selectively induced to generate DNA replication stress, and MYC high-expression tumor cells can be selectively killed when the ATR inhibitor is combined. Since normal cells can provide CTPS for normal growth and proliferation of cells through CTPS2 and the salvage pathway. Therefore, preferably, the combined inhibition of CTPS1 and ATR can further improve the killing selectivity of MYC high expression tumor cells and reduce the toxic and side effects on normal cells. Thus, preferably, the CTPS inhibitor is a CTPS1 inhibitor, for example compound a shown by the formula:

or, preferably, the ATR inhibitor is BAY-1895344, VE-822 and/or AZD6738 as shown below.

Inhibiting CTPS1 can selectively induce MYC high-expression cells to generate DNA damage, the cells participate in DNA damage repair by activating CHK1/2, ATM, PARP1/2, WEE1 and DNA-PK channels, and inhibiting CTPS1 to induce DNA damage and simultaneously inhibiting key signal channels involved in DNA damage repair can lead to serious DNA damage and apoptosis. Thus, in some preferred embodiments, the pharmaceutical composition further comprises one or more inhibitors targeting CHK1/2, ATM, PARP1/2, WEE1, or DNA-PK.

Preferably, the pharmaceutical composition further comprises one or more other CTPS inhibitors other than 3-deazauridine, other ATR inhibitors other than BAY-1895344, VE-822 and AZD6738.

In order to solve the technical problems, the second technical scheme of the invention is as follows: a kit of parts is provided comprising a kit a and a kit B, wherein the kit a comprises a CTPS inhibitor and the kit B comprises an ATR inhibitor.

Preferably, the CTPS inhibitor is a CTPS1 inhibitor and the ATR inhibitor is one or more of BAY-1895344, VE-822 and AZD6738.

More preferably, the CTPS inhibitor is 3-deazauridine.

Even more preferably, the kit a is administered before the kit B, the kit a is administered after the kit B, or the kit a is administered simultaneously with the kit B.

In some preferred embodiments, the kit of parts further comprises a kit C; the kit C comprises one or more inhibitors targeting CHK1/2, ATM, PARP1/2, WEE1, DNA-PK, and/or one or more other CTPS inhibitors other than 3-deazauridine, other ATR inhibitors other than BAY-1895344, VE-822 and AZD6738.

Preferably, the administration sequence of the kit A, the kit B and the kit C is as follows: medicine box A, medicine box B and medicine box C; or a medicine box A, a medicine box C and a medicine box B; or a medicine box B, a medicine box A and a medicine box C; or a medicine box B, a medicine box C and a medicine box A; or a medicine box C, a medicine box A and a medicine box B; or a medicine box C, a medicine box B and a medicine box A; or simultaneously.

In order to solve the technical problems, the third technical scheme of the invention is as follows: a method for selectively killing MYC-expressing tumor cells for non-therapeutic purposes is provided, wherein the method is carried out by contacting the tumor cells with the pharmaceutical composition according to any one of the technical schemes of the invention and the kit according to any one of the second technical schemes of the invention. The non-therapeutic approach may be used, for example, to kill MYC-expressing tumor cells in the laboratory to study the prepared cell model, or to study the synergy between drugs to develop more compounds or combinations that target MYC-expressing tumor cells.

Preferably, the tumor cell is a hematological tumor or a solid tumor.

More preferably, the hematological neoplasm comprises multiple myeloma or lymphoma; the solid tumor is breast cancer, liver cancer, melanoma, papilloma, osteogenic sarcoma, neuroblastoma, pancreatic cancer, prostate cancer, renal cancer or intestinal cancer such as carcinoma of large intestine or colon cancer.

In order to solve the technical problems, the fourth technical scheme of the invention is as follows: provides a pharmaceutical composition according to any one of the technical schemes of the invention, and application of the kit according to any one of the two technical schemes of the invention in preparing medicines for treating MYC high-expression cancers.

In some preferred embodiments, the MYC-expressing cancer is a hematological tumor or a solid tumor.

Preferably, the hematological neoplasm comprises multiple myeloma or lymphoma; the solid tumor is breast cancer, liver cancer, melanoma, papilloma, osteogenic sarcoma, neuroblastoma, pancreatic cancer, prostate cancer, renal cancer or intestinal cancer such as carcinoma of large intestine or colon cancer.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

because of the non-drug property of MYC protein, it is very difficult to develop anti-tumor drugs by directly targeting MYC. The invention bypasses MYC itself, selectively induces MYC high expression cells to generate replication stress by inhibiting CTPS, and selectively kills MYC high expression tumor cells when inhibiting ATR activity in a combined way. In a preferred technical scheme, the selective interference CTPS1 activity combined with an ATR inhibitor such as BAY-1895344, VE-822 or AZD6738 can further improve the selectivity of MYC high-expression cells and reduce the toxic and side effects on normal cells.

Drawings

FIG. 1 is a graph showing the results of compounds selectively inhibiting cell viability of MYC-expressing cells.

FIG. 2 is the effect of DAU on MYC high expressing cell cycle.

FIG. 3 shows the Chk-1Ser345 and ATR Thr1989 phosphorylation modification of DAU to induce MYC-high expressing cells in a concentration-dependent manner.

FIG. 4 is a graph showing the selective induction of DNA damage in MYC-highly expressing cells ARPE-19-MYC by the combined inhibition of CTPS and ATR.

FIG. 5 is a graph showing the selective induction of DNA damage in MYC-highly expressing cells RKO, HCT116 and SW480 by combined inhibition of CTPS and ATR.

FIG. 6 is a graph of the combined inhibition of CTPS and ATR-induced DNA damage as a function of MYC activity.

Fig. 7 is a graph showing that CTPS and ATR are combined inhibited to induce apoptosis in MYC-expressing cells.

Fig. 8 is a graph of the combined inhibition of CTPS and ATR-induced apoptosis dependent MYC activity.

Fig. 9 is a graph of inhibition of tumor growth in vivo by combination of CTPS and ATR inhibition.

Fig. 10 is a graph showing that combined inhibition of CTPS and ATR induced MYC-expressing apoptosis was not affected by P53 and HRAS status.

FIG. 11 shows that CTPS1 knockdown in combination with ATR inhibitor induces apoptosis of MYC high expressing cells.

FIG. 12 shows that knockdown CTPS1 in combination with ATR inhibitor induces apoptosis of MYC high expressing cells.

FIG. 13 is a graph depicting the correlation of MYC with CTPS1 and CTPS2 mRNA expression examined by methods of over-expressing and knocking down MYC.

FIG. 14 is a graph showing the effect of inhibiting CTPS1 and CTPS2, alone or in combination, on the viability of MYC-highly expressing tumor cells.

Fig. 15 is a schematic diagram of the present invention.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 inhibition of CTPS Selective inhibition of cell viability of MYC high expressing cells

To study the role of CTPS in MYC-driven tumor cell growth, a stable cell line ARPE-19-Tet-On-MYC-CMV-Puro capable of inducing expression of MYC was first constructed in normal human retinal epithelial cell ARPE-19. The stably transformed cell line will hereinafter be abbreviated as ARPE-19-MYC, whereby cells to which DOX was added to activate MYC expression were designated as ARPE-19-MYC (DOX+), and cells to which DOX was not added to activate were designated as ARPE-19-MYC (DOX+). Two ARPE-19-MYC models were then validated with two compounds, purvalanol A and VX-680, reported previously, that selectively induced apoptosis in MYC-highly expressing cells, which were targeted to inhibit CDK1 and aurora-B kinase, respectively. The results show that both compounds can selectively inhibit the cell viability (A and B of FIG. 1) and cell death (C of FIG. 1) of ARPE-19-MYC (DOX+). ARPE-19-MYC (DOX+) and ARPE-19-MYC (DOX-) cells were then treated with the CTPS inhibitor DAU, respectively, and the effect of inhibiting CTPS on cell viability and the correlation with MYC expression were examined. The results show that DAU has a stronger inhibitory effect on ARPE-19-MYC (DOX+) cell viability than ARPE-19-MYC (DOX-) (D and E of FIG. 1). In addition, shRNA was used to knock down MYC expression in a variety of MYC-highly expressing tumor cells RKO, HCT116 and SW480, and then the sensitivity of cells expressing control and MYC shRNA to DAU was compared. The results show that knocking down MYC significantly attenuated the inhibitory effect of DAU on RKO (F and G of fig. 1), SW480 (H and I of fig. 1) and HCT116 (J and K of fig. 1) cell viability. These experimental results show that inhibiting CTPS can selectively inhibit cell viability of MYC-expressing cells.

Example 2 DAU Selective Induction of MYC high expressing cell replication stress

To reveal the reason for inhibiting CTPS selectively inhibiting MYC high expressing cell viability, the effect of DAU on cell cycle was examined. The present inventors found that DAU was able to significantly block ARPE-19-MYC (DOX+) cells in S phase, while having only a weak effect on ARPE-19-MYC (DOX-) cells (FIGS. 2A and B). In addition, RKO, HCT116 and SW480 also showed a significant increase in S phase cell proportion after DAU treatment, with a significant decrease in G1 and G2 phase cells (C, D and E of FIG. 2), whereas in MYC knockdown RKO, HCT116 and SW480 cells, DAU had no significant effect on cell cycle (F, G and H of FIG. 2). Experimental results show that inhibiting CTPS selectively prevents completion of S-phase DNA replication of MYC high-expression cells.

Based on the above data that DAU prevents completion of MYC-expressing cell S-phase DNA replication, inhibition of CTPS is presumed to be likely to selectively lead to MYC-expressing cell DNA replication stress. The present invention evaluates DNA replication stress by detecting phosphorylation modifications of Chk-1Ser34 and ATR Thr 1989. The experimental results show that DAU induced the phosphorylation modification of ARPE-19-MYC (DOX+) cells Chk-1Ser345 and ATR Thr1989 in a concentration-dependent manner, whereas ARPE-19-MYC (DOX-) cells had no significant change in the phosphorylation modification of Chk-1Ser345 and ATR Thr1989 before and after DAU treatment (FIG. 3A). In addition, cells expressing MYC in RKO, HCT116, SW480 and Raji also underwent significant modification of Chk-1Ser345 and ATR Thr1989 phosphorylation after DAU treatment (FIG. 3B), and more importantly, DAU induced modification of Chk-1Ser345 and ATR Thr1989 phosphorylation was significantly reduced after MYC expression knockdown (FIGS. 3C and D). These results indicate that inhibition of CTPS induces MYC-high expressing cell DNA replication stress in a MYC-dependent manner.

Example 3 combined inhibition of CTPS and ATR induces DNA damage in MYC-dependent manner

DNA replication stress protects genomic stability by activating ATR-Chk1 signaling pathways, and sustained, unreliable DNA replication stress will lead to DNA damage and apoptosis. Next, it was examined whether combined inhibition of CTPS and ATR could selectively induce DNA damage in MYC-expressing cells. DNA damage was assessed by detecting the phosphorylation modification of H2AX Ser 139. Immunofluorescent staining results showed that more than 60% of ARPE-19-MYC (DOX+) cells showed positive H2AX Ser139 phosphorylation modification after 16 hours of treatment with DAU and ATR inhibitor BAY-1895344, whereas less than 10% of ARPE-19-MYC (DOX-) cells showed positive H2AX Ser139 phosphorylation modification (FIGS. 4A and B). Furthermore, immunoblotting (WB) results also showed that the combination of DAU and BAY-1895344 or both the other two ATR inhibitors AZD6738 and VE-822 significantly increased the H2AX Ser139 phosphorylation modification in ARPE-19-MYC cells, while having no significant effect on ARPE-19-Puro cells (C of fig. 4). In addition, the comet assay results also show that the combination of DAU and ATR inhibitor BAY-1895344 significantly increases the DNA content of the comet tail of ARPE-19-MYC (dox+) cells without significantly affecting ARPE-19-MYC (DOX-) cells (D and E of fig. 4). In addition, RKO, HCT116 and SW480 cells also showed significant increases in H2AX Ser139 phosphorylation modification after combined DAU and ATR inhibitor treatment (A, B and C of FIG. 5). Comet assay results also showed that the DNA content of the comet tails of RKO, HCT116 and SW480 cells was significantly increased in combination with DAU and ATR inhibitor BAY-1895344 (fig. 5D and E). The H2AX Ser139 phosphorylation modification induced by the combination of DAU and ATR inhibitors was significantly reduced after shRNA knockdown MYC expression (a and B of fig. 6). Comet assay results also show that knockdown MYC significantly reversed the DNA content increase in comet tail induced by the combination of DAU and ATR inhibitors (C and D of fig. 6).

Example 4 combined inhibition of CTPS and ATR induces apoptosis in MYC-dependent manner

The effect of combined inhibition of CTPS and ATR on apoptosis was next examined by Annexin V/PI staining. The results indicate that neither ARPE-19-MYC (DOX+) nor ARPE-19-MYC (DOX-) induced significant apoptosis when either DAU or BAY-1895344 were administered alone, whereas about 60% of ARPE-19-MYC (DOX+) had significant apoptosis after the combined DAU and BAY-1895344 treatments, while ARPE-19-MYC (DOX-) cells were not significantly altered (A of FIG. 7). The other two ATR inhibitors AZD6738 and VE-822 were also able to significantly induce apoptosis in ARPE-19-MYC (dox+) cells after treatment in combination with DAU (B of fig. 7), and apoptosis induced by the combination DAU and ATR inhibitors could be reversed by the pan Caspase inhibitor Q-VD (C of fig. 7), indicating that Caspase activation is the main cause of apoptosis induced by the combination DAU and ATR inhibitors. Tumor cells with uncontrolled MYC expression may have been adapted to high MYC expression due to various mutations and thus are insensitive to combined inhibition of CTPS and ATR-induced apoptosis. The apoptosis-inducing effect of the combined inhibition of CTPS and ATR was then examined in tumor cells with deregulated MYC expression. In HCT116, RKO, SW480 and Raji cells, neither DAU nor BAY-1895344 alone induced significant apoptosis, whereas the combined DAU and BAY-1895344 treatment for 48 hours significantly increased the proportion of apoptotic cells (D, F and G of FIG. 7). In addition, the proportion of apoptotic cells was also significantly increased after DAU treatment of ATR shRNA-expressing HCT116 and RKO cells compared to control shRNA-expressing cells (a and B of fig. 8). More importantly, apoptosis induced by the combination of DAU and BAY-1895344 was significantly reversed after MYC knockdown (C, D and E of FIG. 8). These results indicate that combined inhibition of CTPS and ATR can selectively induce DNA damage and apoptosis in MYC-expressing cells.

Example 5 inhibition of tumor growth in vivo in combination with DAU and BAY-1895344

To examine the in vivo therapeutic effects of the combination of DAU and BAY-1895344, 3D cell culture models of RKO, HCT116 and ARPE-19-MYC (DOX+) were first constructed. It was found that although both DAU and BAY-1895344 administered alone reduced the volume of the tumor spheres to some extent, when combined, the volume of the tumor spheres was only about 5% -10% of the control group (a of fig. 9). A nude mouse transplant tumor model was then constructed with HCT116 cells, then DAU and BAY-1895344 were given either alone or in combination, and tumor growth curves were recorded (FIG. 9B), tumor tissues were isolated and weighed after the end of the experiment (FIG. 9C and D), and the results indicated that the tumor inhibition effect of the combination DAU and BAY-1895344 was significantly better than that of the treatment with both drugs alone.

Since MYC high-expression cells are often accompanied by RAS mutation and P53 deletion, tumor fineness is synergistically promotedCell survival and proliferation, followed by investigation of over-expressed sustained activation HRAS (HRAS ^G12V ) Or whether the P53 knockdown cells remain susceptible to treatment with the combined DAU and BAY-1895344. The results show that HRAS is overexpressed ^G12V Or ARPE-19-MYC (DOX+) cells knocked down by P53 still underwent significant apoptosis after the combined treatment of DAU and BAY-1895344 (A, B and C of FIG. 10).

Example 6 Combined inhibition of CTPS1, but not CTPS2, and ATR-induced apoptosis of MYC-highly expressing cells

CTPS activity is catalyzed by both CTPS1 and CTPS2 subtypes, and in order to elucidate the role of CTPS1 and CTPS2 in MYC-driven tumor cell growth, cell lines for knocking out CTPS1 and CTPS2 separately and CTPS1 and CTPS2 simultaneously are constructed in RKO cells using CRISPR technology, respectively. When ATR inhibitor BAY-1895344 treatment was administered, the proportion of apoptosis of CTPS1 alone to CTPS1 and CTPS2 simultaneously knocked out increased significantly, without significant impact on CTPS2 knocked out cells (a of fig. 11). WB results showed that the single knockout of CTPS1 resulted in severe DNA replication stress to a degree not significantly different from the simultaneous knockout of CTPS1 and CTPS2, whereas the knockout of CTPS2 had no significant effect on DNA replication stress (B of fig. 11). To rule out the off-target effects of CRISPR, flag-CTPS1 and Flag-CTPS2 were overexpressed in CTPS1 knocked-out cells, respectively, and the results showed that overexpression of CTPS1 or CTPS2 was able to completely reverse BAY-1895344 induced apoptosis (C and D of fig. 11). The same phenomenon was also seen with shRNA in HCT116 and RKO cells when CTPS1 and CTPS2 were knocked down, respectively, i.e., when ATR inhibitor BAY-1895344 treatment was administered, both CTPS1 knockdown alone and CTPS1 and CTPS2 knockdown cells were significantly apoptotic and to a similar extent, while CTPS2 knockdown was not significantly affected (a and B of fig. 12). Meanwhile, knocking down CTPS1 alone resulted in DNA replication stress to a similar extent as that of simultaneously knocking down CTPS1 and CTPS2, whereas knocking down CTPS2 had no significant effect on DNA replication stress (C and D of fig. 12). These results indicate that CTPS1 plays a key role in MYC-driven tumors.

In order to reveal the molecular mechanism of the key role of CTPS1 in MYC-driven tumors, the correlation of MYC with CTPS1 and CTPS2 mRNA expression was examined by the method of over-expression and knock-down of MYC, and RT-PCR results showed that CTPS1 significantly increased or decreased mRNA levels when MYC was over-expressed and knocked down, while CTPS2 mRNA expression was only slightly changed under the same conditions (A, B and C of fig. 13). WB results also showed that CTPS1 protein levels increased or decreased significantly when MYC was overexpressed or knocked down, while CTPS2 protein levels were only slightly altered under the same conditions (D, E and F of fig. 13). These results indicate that MYC is likely to provide CTP for rapid cell proliferation by selectively inducing CTPS1 rather than CTPS2 gene expression.

Example 7 inhibition of CTPS1 Selective inhibition of cell viability of MYC high expressing cells

Next, we examined the effect of gene intervention CTPS1 expression on MYC high expressing tumor cell viability. CCK8 experimental results show that the knocking-out CTPS1 can remarkably inhibit the activity of RKO cells, and the degree of the knocking-out CTPS1 is equivalent to that of the CTPS1 and CTPS2 which are knocked out simultaneously, while the CTPS2 which is knocked out singly has no obvious effect on the activity of the cells (A of figure 14). In addition, the same phenomenon is seen when CTPS1 and CTPS2 are knocked down in HCT116 and RKO cells respectively and simultaneously by shRNA, i.e., both the single and simultaneous knockdown of CTPS1 and CTPS2 can effectively inhibit cell viability, while the knockdown of CTPS2 has no obvious effect (B and C of fig. 14). In the cells expressing Myc shRNA, the knock-down CTPS1 had no obvious effect on cell viability (D of fig. 14), indicating that inhibiting CTPS1 selectively inhibited cell viability of Myc-expressing tumor cells.

Thus, a technical principle diagram (figure 15) of the invention is constructed, wherein MYC upregulates CTPS1 through metabolic reprogramming to provide CTP for rapid proliferation of tumor cells for ribosome synthesis and DNA replication, and the CTPS1 is inhibited from selectively inducing MYC high-expression tumor cells to generate DNA replication stress, and CTPS1 and ATR are jointly inhibited from selectively inducing MYC high-expression tumor cells to generate DNA damage and apoptosis.

Claims (10)

1. A pharmaceutical composition comprising a CTPS inhibitor and an ATR inhibitor.

2. The pharmaceutical composition of claim 1, wherein the CTPS inhibitor is 3-deazauridine.

3. The pharmaceutical composition of claim 1, wherein the CTPS inhibitor is a CTPS1 inhibitor, such as compound a of the formula:

4. the pharmaceutical composition of claim 1, wherein the ATR inhibitor is BAY-1895344, VE-822 and/or AZD6738.

5. The pharmaceutical composition of any one of claims 1-4, further comprising one or more inhibitors targeting CHK1/2, ATM, PARP1/2, WEE1, DNA-PK;

preferably, the pharmaceutical composition further comprises one or more other CTPS inhibitors other than 3-deazauridine, other ATR inhibitors other than BAY-1895344, VE-822 and AZD6738.

6. A kit of parts comprising a kit a and a kit B, wherein the kit a comprises a CTPS inhibitor and the kit B comprises an ATR inhibitor;

preferably, the CTPS inhibitor is a CTPS1 inhibitor and the ATR inhibitor is one or more of BAY-1895344, VE-822 and AZD 6738;

more preferably, the CTPS inhibitor is 3-deazauridine;

even more preferably, the kit a is administered before the kit B, the kit a is administered after the kit B, or the kit a is administered simultaneously with the kit B.

7. The kit of claim 6, wherein the kit of parts further comprises a kit C; the kit C comprises one or more inhibitors targeting CHK1/2, ATM, PARP1/2, WEE1, DNA-PK, and/or the kit C comprises one or more other CTPS inhibitors other than 3-deazauridine, other ATR inhibitors other than BAY-1895344, VE-822 and AZD 6738;

preferably, the administration sequence of the kit A, the kit B and the kit C is as follows: medicine box A, medicine box B and medicine box C; or a medicine box A, a medicine box C and a medicine box B; or a medicine box B, a medicine box A and a medicine box C; or a medicine box B, a medicine box C and a medicine box A; or a medicine box C, a medicine box A and a medicine box B; or a medicine box C, a medicine box B and a medicine box A; or simultaneously.

8. A method for selectively killing MYC-expressing tumor cells for non-therapeutic purposes, comprising contacting the tumor cells with the pharmaceutical composition of any one of claims 1-5, the kit of parts of any one of claims 6-7;

preferably, the tumor cell is a hematological tumor or a solid tumor;

more preferably, the hematological neoplasm comprises multiple myeloma or lymphoma; the solid tumor is breast cancer, liver cancer, melanoma, papilloma, osteogenic sarcoma, neuroblastoma, pancreatic cancer, prostate cancer, renal cancer or intestinal cancer such as carcinoma of large intestine or colon cancer.

9. Use of the pharmaceutical composition according to any one of claims 1 to 5, the kit of parts according to any one of claims 6 to 7 for the manufacture of a medicament for the treatment of MYC-expressing cancers;

preferably, the MYC-expressing cancer is a hematological tumor or a solid tumor;

more preferably, the hematological neoplasm comprises multiple myeloma or lymphoma; the solid tumor is breast cancer, liver cancer, melanoma, papilloma, osteogenic sarcoma, neuroblastoma, pancreatic cancer, prostate cancer, renal cancer or intestinal cancer such as carcinoma of large intestine or colon cancer.

Application of CTPS1 inhibitor in preparing medicine for treating MYC high expression cancer;

preferably, the MYC-expressing cancer is a hematological tumor or a solid tumor;

more preferably, the hematological neoplasm comprises multiple myeloma or lymphoma; the solid tumor is breast cancer, liver cancer, melanoma, papilloma, osteogenic sarcoma, neuroblastoma, pancreatic cancer, prostate cancer, renal cancer or intestinal cancer such as carcinoma of large intestine or colon cancer.

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