KR20220034454A - A pharmaceutical composition for preventing, treating or alleviation of cancer comprising FYN kinase inhibitor and additional kinase inhibitor - Google Patents

Abstract

The present invention found a combination of targeted therapeutic agents (FYN-IGF1R/EGFR/ABL2) that can synergize with each other, and the efficacy of the combination thereof is superior to that of a single targeted therapeutic agent. One aspect of the present invention provides a pharmaceutical composition for preventing, treating or alleviating cancer comprising: a first agent that inhibits the activity of FYN kinase; and a second agent comprising at least one selected from an agent for inhibiting the activity of kinase, an agent for inhibiting the activity of EGFR kinase, and an agent for inhibiting the activity of ABL kinase.

Description

TECHNICAL FIELD [0001] A pharmaceutical composition for preventing, treating or alleviation of cancer comprising FYN kinase inhibitor and additional kinase inhibitor TECHNICAL FIELD

The present specification relates to a pharmaceutical composition having an excellent effect on cancer, particularly breast cancer, by controlling the activity of two or more genes.

There is an urgent need for targeted therapies for triple negative breast cancer (TNBC). Triple-negative breast cancer lacks expression of estrogen receptor, progesterone receptor and HER2, limiting treatment options targeting these marker molecules. Triple-negative breast cancer has a poor prognosis compared to other subtypes of breast cancer, and standard targeted therapies involving kinase inhibitors are lacking. PARP inhibitors such as Olaparib are used to treat triple-negative breast cancer, but the mechanism of action of this drug is necessarily accompanied by BRCA1/2 mutation, which occurs in only 30% of triple-negative breast cancers. Therefore, there is an urgent need for a targeted therapy that can affect a wide range of triple-negative breast cancer.

Tyrosine kinases or tyrosine kinases have been used as major drug targets in cancer therapy for their pharmacological and major roles in cell proliferation and survival.

Tyrosine kinases are involved in most characteristic signs of cancer, including cell survival and proliferation, angiogenesis and cell invasion. Therefore, tyrosine kinase inhibitors (TKIs) have been of major interest in cancer treatment. In fact, most targeted cancer therapies are TKIs, including the first FDA-approved targeted therapy, the Bcr-Abl inhibitor Imatinib.

Despite tremendous advances in targeted cancer therapies, the efficacy of TKIs often does not achieve significant levels and is largely compromised by drug resistance. Indeed, the median time to progression-free survival (PFS) for non-small cell lung cancer (NSCLC) patients with EGFR mutations treated with the EGFR inhibitor gefitinib is only 12 months.

One of the reasons for such resistance resistance is gene interaction that enables changes in signal transduction as a compensatory mechanism upon drug treatment. For example, MET or PIK3CA amplification was used to compensate for the suppression of EGFR by gefitinib (an EGFR inhibitor) or erlotinib (an EGFR inhibitor) in ~10% of patients with NSCLC. Activation was configured.

Therefore, combination therapy that inhibits several signaling molecules that can compensate for each other's activity may be a major strategy for overcoming drug resistance or resistance of kinase inhibitor therapy. However, such targeted therapy combination therapy is currently less practiced, principally because of the lack of highly scalable methods to screen for effective combination therapy until recently.

Korean Patent No. 10-1931435 Korean Patent Publication No. 10-2018-0064403

Han K. et. al., Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nat. Biotechnol. 2017 May; 35(5): 463-474. Engelman J.A. et. al., MET Amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 2007 May; 316(5827):1039-1043.

In order to solve the above problems, the present inventors have reached the present invention by confirming a synergistic anticancer effect on cancer, preferably breast cancer, more preferably triple-negative breast cancer, when inhibiting the activity of two or more specific genes at the same time. The inventors of the present invention have invented a combination of tyrosine kinase inhibitors that have a synergistic effect on triple-negative breast cancer removal by utilizing CombiGEM-CRISPR technology. The inventors of the present invention intend to provide the combination of FYN as one of the main therapeutic targets to enhance the cytotoxic effect with other tyrosine kinases such as IGF1R, EGFR and ABL2 with the combination of such inhibitors. The inventors of the present invention confirmed that the combination of NVP-ADW742 (IGF1R inhibitor) and PP2 or saracatinib (FYN inhibitor) synergistically reduced triple negative breast cancer proliferation in vitro and in vivo. Accordingly, the inventors of the present invention aim to provide a novel and remarkably superior combination therapy targeting FYN and other tyrosine kinases simultaneously for eliminating triple negative breast cancer.

Accordingly, one aspect of the present invention is a first agent that inhibits the activity of FYN kinase; and a second agent comprising at least one selected from an agent for inhibiting the activity of IGR-1R kinase, an agent for inhibiting the activity of EGFR kinase, and an agent for inhibiting the activity of ABL kinase; Prevention, treatment or alleviation of cancer, including To provide a pharmaceutical composition for

In addition, one aspect of the present invention is to provide a method for preparing a composition for preventing, treating or alleviating the cancer.

In addition, one aspect of the present invention is to provide a method of treating a patient in need of the composition according to the present invention using the composition.

The object of the present invention is not limited to the object mentioned above. The object of the present invention will become clearer from the following description, and will be realized by means and combinations thereof described in the claims.

One aspect of the present invention is a first agent that inhibits the activity of FYN kinase; and

For the prevention, treatment or alleviation of cancer including; a second agent comprising at least one selected from an agent for inhibiting the activity of IGR-1R kinase, an agent for inhibiting the activity of EGFR kinase, and an agent for inhibiting the activity of ABL kinase A pharmaceutical composition is provided.

In one aspect of the present invention, the second agent provides a pharmaceutical composition for preventing, treating or alleviating cancer, which is an agent that inhibits the activity of IGR-1R kinase.

In one aspect of the present invention, the second agent provides a pharmaceutical composition for preventing, treating or alleviating cancer, which is an agent that inhibits the activity of EGFR kinase.

In one aspect of the present invention, the second agent provides a pharmaceutical composition for preventing, treating or alleviating cancer, which is an agent that inhibits the activity of ABL kinase.

In one aspect of the present invention, the first agent or the second agent are each independently at least one selected from a small molecule, a protein, a peptide, an RNA molecule, an endonuclease complex, and a DNA construct, prevention, treatment of cancer Or it provides a pharmaceutical composition for alleviation.

In one aspect of the present invention, the first agent and the second agent provide a pharmaceutical composition for preventing, treating or alleviating cancer, which is a kinase inhibitor that inhibits kinase activity with small molecules, respectively.

In one aspect of the present invention, the first agent is a small molecule FYN kinase inhibitor, and the agent that inhibits the activity of the IGR-1R kinase is a small molecule IGR-1R kinase inhibitor, and inhibits the activity of the EGFR kinase The agent is a small molecule EGFR kinase inhibitor, and the agent inhibiting the activity of the ABL kinase is a small molecule ABL kinase inhibitor, and provides a pharmaceutical composition for preventing, treating or alleviating cancer.

In one aspect of the present invention, the FYN kinase inhibitor is 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4- d ]pyrimidine (PP2) or N- ( 5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methyl-1-piperazinyl)ethoxy]-5-[(tetrahydro- 2H -pyran-4 -yl)oxy]-4-quinazolinamine (Saracatinib), wherein the IGF-IR kinase inhibitor is 5-(3-phenylmethoxyphenyl)-7-[3-(pyrrolidin-1-ylmethyl)cyclo Butyl]pyrrolo[2,3-d]pyrimidin-4-amine (NVP-ADW742), wherein the EGFR kinase inhibitor is N- (3-chloro-4-fluoro-phenyl)-7-methoxy-6 -(3-morpholin-4-ylpropoxy)quinazolin-4-amine (gefitinib), wherein the ABL kinase inhibitor is 4-[(4-methylpiperazin- 1 -yl)methyl]-N- [4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]-phenyl]-benzamide (imatinib), provides a pharmaceutical composition for the prevention, treatment or alleviation of cancer.

In one aspect of the present invention, the composition is applied to a patient group having any one or more of FYN overexpression gene, IGF-1R overexpression gene, EGFR overexpression gene and ABL overexpression gene, preventing, treating or alleviating cancer pharmaceutical composition to provide.

In one aspect of the present invention, the DNA construct includes an endonuclease gene encoding a CRISPR-related (Cas) protein, and provides a pharmaceutical composition for preventing, treating or alleviating cancer.

In one aspect of the present invention, the cancer is breast cancer, it provides a pharmaceutical composition for preventing, treating or alleviating cancer.

In one aspect of the present invention, the cancer is triple negative breast cancer, it provides a pharmaceutical composition for the prevention, treatment or alleviation of cancer.

In one aspect of the present invention, the first agent and the second agent are administered in a weight ratio of 1 to 0.1 to 10, and provides a pharmaceutical composition for preventing, treating or alleviating cancer.

In one aspect of the present invention, the first agent is administered in an amount of 10 to 50 mg/kg, and the second agent is administered in an amount of 10 to 50 mg/kg, a pharmaceutical composition for preventing, treating or alleviating cancer to provide.

The composition according to one aspect of the present invention can provide a breakthrough toward effective targeted therapy while minimizing the risk of drug resistance or resistance.

The composition according to one aspect of the present invention can inhibit cancer growth more effectively than would be expected as a simple sum of the respective therapeutic effects of two tyrosine kinase inhibitor treatments.

In the composition according to one aspect of the present invention, the combination of synergistic tyrosine kinase inhibitors can achieve therapeutic efficacy with a much smaller dose compared to the effect that can be achieved when each is used as a single dose, so that the administration dose of the drug can be lowered there is.

The composition according to one aspect of the present invention has the effect of reducing side effects by reducing the dose for each drug, thereby extending the treatment period.

The effects of the present invention are not limited to the above-mentioned effects. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

1 summarizes the expression of each sgRNA pair in a combinatorial library, on the left is a histogram of the frequency of each sgRNA combination, and on the right is a histogram of the number of sgRNAs targeting gene pairs.
Figure 2 shows the accuracy of sgRNA-barcode pairs in each library.
3 is a result confirming that the growth phenotype score Z is correlated between two different permutations of the same pair of sgRNA.
4 is a scatter plot of the growth phenotype score Z of two independent biological replicates.
In Figure 5, the left is a table of r values between the growth phenotype scores of each sgRNA pair targeting the same gene pair, and the right is a scatter plot of the growth phenotype scores Z of different sgRNA pairs targeting the same gene pair.
6A is a scatter plot of predicted growth phenotype Z scores and observed growth phenotype Z scores for each gene combination, FIG. 6B is a scatter plot of growth phenotype Z scores and normalized GI scores for each gene combination, and FIG. Scatter plots of gene-level GI scores and RIGER p values calculated with the GI score of each sgRNA pair targeting the pair. Green dots indicate gene combinations in which the same gene was targeted by two sgRNAs. Red dots indicate candidate synthetic lethal gene pairs listed in Table 1.
7 is the predicted and observed growth phenotype score Z of all sgRNA pairs.
8 is the standard deviation of the GI scores of 200 neighboring gene pairs by predicted growth phenotype scores.
9 is a T7 endonuclease assay of MDA-MB-231 cells infected with sgRNA expressed with a lentivirus targeting the indicated genes.
10 is a summary of synergistic killing by sgRNAs targeting the indicated gene pairs. Raw data comparing changes in cell viability by a single sgRNA and combinations thereof are in FIG. 2 .
11 is the change in cell viability of the indicated sgRNA(s). The dotted line represents the change in cell viability predicted by the Bliss independent model.
In FIG. 12 , the left is a scatterplot of growth phenotype score Z and changes in cell viability by the indicated sgRNA combinations, and the right is a scatterplot of GI scores and synergistic killing by the indicated sgRNA combinations.
13 is a network analysis of 30 candidate synthetic lethal gene pairs. The size of each node represents the number of linkages in the gene.
14 is a dose response curve of TKIs displayed in the presence and absence of PP2.
15 is a change in cell viability upon treatment of the HCC38 cell line with the indicated TKIs.
16 is apoptosis and cell proliferation in MDA-MB-231 cells treated with NVP-ADW742 (4uM) and PP2 (10uM).
17 is an MTT assay using MDA-MB-231 cells treated with the indicated TKI combinations.
18 is the tumor volume of xenografts treated with the indicated TKI combinations.
19 is the tumor weight at day 16 after the first TKI treatment of xenografts treated with the indicated TKI combinations.
20 is the body weight of the mice described in FIG. 19 treated with the indicated TKIs.
21 is a result confirming the protein expression level of FYN by western blot when NVP-ADW742 (IGF1R inhibitor), gefitinib (EGFR inhibitor), and imatinib (ABL inhibitor) were treated.
22 is a quantitative real-time PCR result of PP2, NVP-ADW742 and gefitinib.
23 shows a decrease in CBL-b protein when treated with NVP-ADW742.
24 is a summary of the mechanism by which activated FYN has resistance to TKI by inducing cell survival and proliferation.

Unless otherwise specified, all numbers, values, and/or expressions expressing ingredients, reaction conditions, and amounts of ingredients used herein refer to a variety of measures that may occur in obtaining such values, among others, in which such numbers are inherently different. Since they are approximations reflecting uncertainty, it should be understood as being modified by the term “about” in all cases. Also, when numerical ranges are disclosed in this description, such ranges are continuous and inclusive of all values from the minimum to the maximum inclusive of the range, unless otherwise indicated. Furthermore, when such ranges refer to integers, all integers inclusive from the minimum to the maximum inclusive are included, unless otherwise indicated.

In this specification, when a range is described for a variable, the variable will be understood to include all values within the stated range including the stated endpoints of the range. For example, a range of “5 to 10” includes the values of 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. It will be understood to include any value between integers that are appropriate for the scope of the recited range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like. Also for example, ranges from "10% to 30%" include values of 10%, 11%, 12%, 13%, etc. and all integers up to and including 30%, as well as 10% to 15%, 12% to It will be understood to include any subranges such as 18%, 20% to 30%, etc., as well as any value between reasonable integers within the scope of the recited ranges, such as 10.5%, 15.5%, 25.5%, and the like.

Hereinafter, the present invention will be described in detail.

Tyrosine kinases are major regulators of cell proliferation and survival, and are being extensively studied as target proteins for cancer therapy. However, the efficacy of most tyrosine kinase inhibitors (TKIs) for the treatment of cancer is hampered by the emergence and limited efficacy of treatment-resistant cancer cells. Accordingly, the inventors of the present invention intend to provide a novel and innovative therapeutic method or pharmaceutical composition for the treatment of triple-negative breast cancer comprising a synergistic TKI combination. Accordingly, the inventors of the present invention performed a massively parallel combinatorial CRISPR screen to identify synthetic lethal drug tyrosine kinase pairs. Screen results were validated with a set of TKIs both in vitro and in vivo in a xenograft assay. Network analysis was utilized to newly identify FYN as a key molecule enhancing the effects of second TKIs such as NVP-ADW742 (IGF-1R inhibitor), gefitinib (EGFR inhibitor) and imatinib (ABL inhibitor). Simultaneous inhibition of FYN and IGF1R synergistically promotes apoptosis and cell cycle arrest. FYN expression is increased upon TKI treatment. That is, the present invention provides an effective combination TKI therapy for triple negative breast cancer with few options for targeted therapy.

TKIs are widely used in cancer treatment. However, therapy resistance often undermines the efficacy of TKIs. Combination therapies that simultaneously target multiple genes can combat tumor cells while exerting a synergistic effect by targeting both causative oncogenes and resistance mechanisms. The inventors of the present invention have discovered a combination of pairs of tyrosine kinases that exhibit synergistic apoptotic effects on triple negative breast cancer (TNBC) when simultaneously inhibited using combinatorial CRISPR screens. The inventors of the present invention identified FYN and IGF1R or EGFR as one of the target gene pairs showing a synergistic effect and verified the effectiveness. Synergy of the two combinations in tumor cell death and growth arrest was confirmed both in vitro and in vivo. In the present invention, in particular, the combination showing the synergistic effect as described above is superior to the simple sum of the effects of each existing anticancer agent, and exhibits a novel effect that can lower the risk of side effects of each anticancer agent.

The inventors of the present invention first selected 76 tyrosine kinases that can be inhibited by one or more drugs from the drug repurposing herb database as shown in Table 1 below.

BTK JAK1 ROS1 EPHB3 MET FYN EPHA10 INSRR KDR FGFR1 TYK2 EPHB6 FLT3 HCK EPHA3 LCK ABL1 FGFR4 BMX TNK1 KIT SRC EPHA4 PTK2B ALK LYN DDR1 TNK2 PDGFRB TXK EPHA5 MST1R EGFR SYK WEE1 ABL2 CSK CSF1R EPHA6 SRMS FGFR2 PDGFRA EPHB4 PTK6 RET INSR EPHA7 TEK FGFR3 AXL ITK PTK2 YES1 BLK EPHA8 NTRK1 IGF1R MERTK FES ERBB2 FLT1 EPHA2 EPHB1 NTRK2 JAK2 TYRO3 DDR2 ERBB3 FLT4 FGR EPHB2 NTRK3 JAK3 FRK EPHA1 ERBB4

Three guide RNAs were extracted from the list of Brunello sgRNAs optimized for Cas9-mediated knockout. 233 sgRNAs were cloned into this library, along with 228 (=76x3) sgRNAs targeting tyrosine kinase, and 5 sgRNAs included as control sgRNAs lacking the target sequence in the human genome.

A pairwise CombiGEM library that simultaneously expresses two sgRNAs was constructed using the iterative cloning technique. The resulting library enabled large-scale parallel screens of paired knockout phenotypes with 233x233 = 54,289 sgRNA pairs corresponding to 3,003 pairs of gene disruptions. The inventors of the present invention validated the library using next-generation sequencing, and 99.5% (2,989/3,003) of gene pairs were represented by at least 6 pairs of sgRNAs with threshold reads greater than one millionth (Figure 1). 89.4% (2,689/3,003) of gene pairs were marked above the threshold with all possible sgRNA pairs in the library.

We infected MDA-MB-231 cells stably expressing Cas9 with a lentiviral library at an infection rate (Multiplicity of Infection, MOI) of 0.3 with an initial expected coverage of ~500 cells per sgRNA pair. Cells were then grown and maintained at an expected coverage of ˜1000 cells per sgRNA pair. Genomic DNA was harvested 3 days after infection (designated as [D0]) and 23 days after infection (D20) for PCR amplification of sgRNA pairs for NGS (Next Generation Sequencing) analysis.

Previous CombiGEM screen approaches of continuous stretch sequencing of barcodes to sgRNAs could not accurately represent sgRNA combinations expressed in cells due to uncoupling of sgRNAs with barcodes during lentiviral replication and reverse transcription. Indeed, the rate of separation of barcodes from sgRNAs increased with the distance between them, approaching ~35% in the pairwise screen (Fig. 2). To solve this problem, we directly sequenced two sgRNA sequences instead of barcodes using NGS. The occurrence of each sgRNA pair in the NGS data was counted and the normalized log2-fold change in counts between samples on days 20 and 0 was calculated as the growth phenotype score Z. We found that the growth phenotype score Z was correlated between two different permutations of the same pair of sgRNAs (e.g., sgRNA-A + sgRNA-B and sgRNA-B + sgRNA-A) (Fig. 3). ). Therefore, we added counts for both permutations of sgRNA pairs and recalculated the growth phenotype score Z.

The inventors of the present invention have a gene interaction score (GI) that compares growth phenotype scores Z by disruption of a pair of genes (Z _{A + B} , observed Z scores) and disruption of each gene within a pair (Z _{A + Con} By calculating a simple sum of the Z scores by + Z _{B + Con} , the expected Z score), we tried to find pairs of gene deletions that kill cells synergistically. The Z scores of the three biological replicates were averaged to calculate the GI score (Figure 4). The GI score is calculated at both the sgRNA level and the gene level, and is the Z score for all sgRNA pairs targeting the same gene pair to calculate the gene interaction score (GI) at the gene level. We confirmed that different independent sgRNA pairs targeting the same set of genes confer well-relevant changes in growth phenotype (Fig. 5). As can be seen in Figure 6 for the gene level and Figure 7 for the sgRNA level analysis, the predicted and observed Z scores for each gene (or sgRNA) pair were closely correlated, suggesting that most combinations of gene deletions are cancerous. It has been shown to have an additive effect on apoptosis. The GI score was calculated as the deviation of the observed Z score from the second fit of the predicted Z score plot. As mentioned earlier, gene interaction scores tend to scatter more when Z scores are low, raising concerns that GI scores may be overestimated in gene/sgRNA pairs with strong Z scores. The standard deviation of the nearest 200 nearest-neighbor GI scores with respect to the expected Z-scores increased significantly at the consistently low Z-scores (Fig. 8). Therefore, the GI score was normalized by dividing the raw gene interaction score by the standard deviation of the GI score of the 200 nearest neighbors in terms of the expected Z score (Fig. 6b).

The inventors of the present invention selected putative synergistic therapeutic target genes based on several criteria. A p-value by RIGER analysis with a GI score cutoff of <-2 at the gene level and a GI score at the sgRNA level below 0.01 was chosen to ensure a synergistic killing effect. In addition, a Z score cutoff of <-5 was applied to identify gene disruption pairs that significantly killed cells (Fig. 6c). The inventors of the present invention confirmed the destruction of 30 pairs of tyrosine kinases causing synthetic lethality as shown in Table 2.

Z Norm GI -log10 RIGER Z -log10 RIGER GI ABL2, TEK -8.57859 -5.048815694 1.418505458 2.474566447 BTK, ITK -5.24425 -4.219102437 0.600153287 2.800519085 FRK, DDR2 -6.64997 -4.185161787 0.968187729 2.37716452 YES1, SRC -13.0514 -3.946109345 5.698970004 2.995248844 FGFR2, ERBB2 -6.58267 -3.80549129 0.829151796 2.418391634 JAK1,FRK -6.55692 -3.701294557 0.787546039 2.808828544 ITK, TEK -6.09158 -3.578261634 0.4165745 2.723078868 FLT3, FYN -8.78937 -3.540615669 3.608535588 2.935916564 FRK, EPHA5 -5.06546 -3.434318577 0.192329699 2.387534036 FGFR2, EPHA10 -6.27611 -3.418585737 0.622147581 3.527828853 ABL1,FRK -5.40273 -3.38240008 1.319211388 2.620150821 FGFR2,PTK6 -7.05704 -3.360800745 0.666552726 3.439137305 EGFR, FYN -6.59527 -3.356498167 0.549135308 2.142305742 FGFR2, FGFR4 -6.5895 -3.333429846 0.906228219 2.005023326 TYRO3,FRK -5.49712 -3.266015356 0.20259395 2.657577319 FLT4, TEK -6.07012 -3.204521109 0.490259984 2.444663672 FYN, TEK -6.79605 -3.00623154 1.310513552 2.138286412 KDR, TEK -5.23021 -3.000230239 0.212043877 2.189767482 PDGFRA,FRK -6.39186 -2.993401007 0.80161787 2.047401265 SYK, TEK -5.00545 -2.922204934 0.137808969 2.58686795 FGFR2, PDGFRA -7.1959 -2.841551035 1.462306806 4.600326279 FGFR2, JAK3 -7.18958 -2.715749183 1.013496985 2.37788964 ABL1, TEK -5.26102 -2.698705947 0.346305205 2.019905686 IGF1R, FYN -7.55965 -2.692806504 1.208730519 2.18809002 FYN, ABL2 -8.87645 -2.664083726 3.60502328 3.620331966 PTK2, PTK2B -13.5103 -2.478985633 7 2.356744775 TEK, NTRK1 -5.34841 -2.410330234 0.162032982 2.609241471 TYK2,TEK -5.9401 -2.297979882 0.607830851 2.220981028 TYRO3,TEK -5.24389 -2.225595555 0.108351056 2.007976972 ABL1, PDGFRA -6.2016 -2.115228956 1.163865851 2.304168227

The SRC-YES pair is one of the strongest synthetic lethal gene pairs. SRC-YES belongs to the same tyrosine kinase family and is known to have overlapping functions. Their simultaneous destruction is expected to result in synergistic lethality, and the fact that this pair is most effective on screen indicates that there are other synergistic effects as well. Several gene pairs carried genes (eg, FGFR2) known to be expressed at very low levels in MDA-MB-231 cells. Although these genes can be induced when the other pair of tyrosine kinases are disrupted, the inventors of the present invention have mainly focused on the gene pair known through previous reports in which both genes are expressed in breast cancer cells.

Next, candidate synthetic lethal tyrosine kinase knockout pairs were validated. Synergistic target gene pairs were validated with sgRNA pairs missing. As previously reported, we stably expressed GFP or mCherry along with sgRNA in MDA-MB-231 cells expressing Cas9, resulting in a mixture of cells deficient in one or both genes in a synergistic target gene pair. was created. The inventors of the present invention confirmed efficient gene editing by sgRNAs used for confirmation by T7 endonuclease assay (FIG. 9). Consistent with the CRISPR screen results, many of the synergistic target gene deletion combinations induced more cell death than predicted by the Bliss independent model ( FIGS. 10 and 11 ). The relative viability and synergistic killing rate of double knockout cells correlated with what was observed in the screen ( FIG. 12 ).

We found that several validated synergistic target gene pairs include FYN (eg, FYN + IGF1R, FYN + EGFR and FYN + ABL2). Indeed, network analysis of 30 candidate synergistic tyrosine kinase pairs revealed that FYN was one of the key nodes found to make synergistic target gene pairs with 5 genes (Fig. 13). We tested whether simultaneous inhibition of FYN and other kinases by TKI combination could inhibit cancer cell growth. Although PP2 is known as a broad-spectrum SRC family kinase inhibitor, it is the most potent against FYN among other SRC family kinases. Therefore, PP2 was treated with NVP-ADW742 (IGF1R inhibitor), gefitinib (EGFR inhibitor) or imatinib (ABL inhibitor) at various concentrations. Interestingly, all TKI combinations synergistically killed MDA-MB-231 cells (Figure 14). The combination of PP2 + NVP-ADW742 and PP2 + gefitinib synergistically killed the other triple negative breast cancer cell line HCC38 ( FIG. 15 ). The dose-response curves showed that co-treatment with PP2 reduced the IC50 of TKIs by 51-81%, making PP2-treated cancer cells sensitive to the tested TKIs (Figure 14). We next tested whether apoptosis and cell proliferation were affected by the TKI combination. As expected, the combination of PP2 + NVP-ADW742 and PP2 + gefitinib synergistically induced apoptosis while inhibiting cell growth (Fig. 16).

The finding that FYN inhibition can synergize with several other TKIs suggests that FYNs may be involved in mechanisms of resistance to TKI therapy in general. Consistent with this, FYN protein levels were increased upon TKI treatment, suggesting that upregulation of FYN may provide a compensatory survival signal in TKI-treated cells (Fig. 31). However, there was no difference in the expression level of FYN mRNA level by TKI treatment ( FIG. 32 ), and it was speculated that the protein expression level of FYN was regulated by the regulatory mechanism after transcription. In fact, it was confirmed that c-CBL and CBLB genes, which help ubiquitination and degradation of FYN protein, were decreased by TKI treatment (FIG. 33). This result means that the treatment of TKI reduces c-CBL and CBLB gene expression to stabilize the FYN protein, thereby increasing the expression level, and this increased FYN confer resistance to TKI. Therefore, this shows a molecular mechanism by which FYN inhibition can enhance TKI-induced apoptosis by lowering resistance to TKI (FIG. 34).

We also tested the in vivo efficacy of simultaneous inhibition of FYN and IGF1R using the MDA-MB-231 xenograft model. A similar synergy in cell death by treatment with saracatinib and NVP-ADW742 was confirmed in vitro ( FIG. 17 ). Surprisingly, treatment with saracatinib and NVP-ADW742 synergistically reduced tumors, whereas single agents were ineffective in slowing tumor growth ( FIGS. 18 and 19 ). All groups showed minimal changes in body weight, indicating that overall animal health was not affected by drug treatment (Figure 20).

Hereinafter, various aspects of the present invention will be described.

One aspect of the present invention is a first agent that inhibits the activity of FYN kinase; and

For the prevention, treatment or alleviation of cancer comprising; a second agent comprising at least one selected from an agent for inhibiting the activity of IGR-1R kinase, an agent for inhibiting the activity of EGFR kinase, and an agent for inhibiting the activity of ABL kinase A pharmaceutical composition is provided.

In one aspect of the present invention, the second agent provides a pharmaceutical composition for preventing, treating or alleviating cancer, which is an agent that inhibits the activity of IGR-1R kinase.

In one aspect of the present invention, the second agent provides a pharmaceutical composition for preventing, treating or alleviating cancer, which is an agent that inhibits the activity of EGFR kinase.

In one aspect of the present invention, the second agent provides a pharmaceutical composition for preventing, treating or alleviating cancer, which is an agent that inhibits the activity of ABL kinase.

In one aspect of the present invention, the first agent or the second agent are each independently at least one selected from a small molecule, a protein, a peptide, an RNA molecule, an endonuclease complex, and a DNA construct, prevention, treatment of cancer Or it provides a pharmaceutical composition for alleviation.

In one aspect of the present invention, the first agent and the second agent provide a pharmaceutical composition for preventing, treating or alleviating cancer, each of which is a kinase inhibitor that inhibits kinase activity with small molecules.

In one aspect of the present invention, the first agent is a small molecule FYN kinase inhibitor, and the agent that inhibits the activity of IGR-1R kinase is a small molecule IGR-1R kinase inhibitor, and inhibits the activity of the EGFR kinase The agent is a small molecule EGFR kinase inhibitor, and the agent inhibiting the activity of ABL kinase is a small molecule ABL kinase inhibitor, and provides a pharmaceutical composition for preventing, treating or alleviating cancer.

In one aspect of the present invention, the FYN kinase inhibitor is 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4- d ]pyrimidine (PP2) or N- ( 5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methyl-1-piperazinyl)ethoxy]-5-[(tetrahydro- 2H -pyran-4 -yl)oxy]-4-quinazolinamine (Saracatinib), wherein the IGF-IR kinase inhibitor is 5-(3-phenylmethoxyphenyl)-7-[3-(pyrrolidin-1-ylmethyl)cyclo Butyl]pyrrolo[2,3-d]pyrimidin-4-amine (NVP-ADW742), wherein the EGFR kinase inhibitor is N- (3-chloro-4-fluoro-phenyl)-7-methoxy-6 -(3-morpholin-4-ylpropoxy)quinazolin-4-amine (gefitinib), wherein the ABL kinase inhibitor is 4-[(4-methylpiperazin- 1 -yl)methyl]-N- [4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]-phenyl]-benzamide (imatinib), provides a pharmaceutical composition for the prevention, treatment or alleviation of cancer.

In one aspect of the present invention, the composition is applied to a patient group having any one or more of FYN overexpression gene, IGF-1R overexpression gene, EGFR overexpression gene and ABL overexpression gene, preventing, treating or alleviating cancer pharmaceutical composition to provide.

In one aspect of the present invention, the DNA construct includes an endonuclease gene encoding a CRISPR-related (Cas) protein, and provides a pharmaceutical composition for preventing, treating or alleviating cancer.

In one aspect of the present invention, the cancer is breast cancer, it provides a pharmaceutical composition for preventing, treating or alleviating cancer.

In one aspect of the present invention, the cancer is triple negative breast cancer, it provides a pharmaceutical composition for the prevention, treatment or alleviation of cancer.

In one aspect of the present invention, the first agent and the second agent are administered in a weight ratio of 1 to 0.1 to 10, and provides a pharmaceutical composition for preventing, treating or alleviating cancer.

In one aspect of the present invention, the first agent is administered in an amount of 10 to 50 mg/kg, and the second agent is administered in an amount of 10 to 50 mg/kg, a pharmaceutical composition for preventing, treating or alleviating cancer to provide.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are only examples to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

Discovery of an effective tyrosine kinase inhibitory combination using the triple-negative breast cancer model cell line MDA-MB-231

To find an effective therapeutic target combination for triple-negative breast cancer, a triple-negative breast cancer cell line, MDA-MB-231 cells, was used. MDA-MB-231 is a highly metastatic triple-negative breast cancer cell line that is negative for estrogen receptor, progesterone receptor, and HER2. MDA-MB-231 Cas9 cells in which the Cas9 gene was stably expressed using a lentivirus were prepared.

In order to select tyrosine kinases to be screened, tyrosine kinases whose activity can be modulated by one or more drugs were found in the Drug Repurposing Hub ( https://clue.io/repurposing ), and a total of 76 types of tyrosine kinases were selected. (Table 1). A single guide RNA (sgRNA) capable of deleting the corresponding tyrosine kinase was selected from the Brunello Library, a human genome sgRNA library that has undergone optimization. (76 genes x 3 sgRNA/gene)+5 control sgRNA=233 Oligonucleotide DNA coding for sgRNA was chemically synthesized and cloned into a Lentiviral vector. This process is not a method of cloning each of 233 sgRNAs, but a pooled cloning technique that clones hundreds of sgRNAs at once in a mixed state. All combinations of 233 sgRNAs, that is, (233 x 233 = 54289) sgRNA libraries capable of tyrosine kinase combination deletions were constructed.

After constructing a lentivirus capable of expressing the sgRNA library, MDA-MB-231 Cas9 cells were infected at an infection rate of 0.3. Only successfully infected cells were obtained by treating MDA-MB-231 Cas9 cells infected with the Puromycin resistance gene of the library with Puromycin. After 3 days of infection with the sgRNA library lentivirus, the cells were collected, and 20 days after that (23 days after infection), the cells were collected again, and genomic DNA from each cell was extracted. And to find out which sgRNA each cell expresses, a part of the lentiviral genome including sgRNA was amplified using Polymerase Chain Reaction (PCR). The sgRNA sequence was found in the amplified DNA using next-generation sequencing using an Illumina HiSeq2000 instrument.

In order to find the combination of sgRNA that induced apoptosis of MDA-MB-231 cells, the frequency of each sgRNA combination was observed in the cells 3 days and 23 days after infection with the lentivirus. Under the assumption that all sgRNAs in the library efficiently deleted genes, if cell death increases when any two genes are deleted at the same time, cells with the combination of sgRNAs that delete the two genes die, and the frequency of the sgRNA combination becomes infected. There will be a gradual decrease between 3 days and 23 days after administration. The frequency change ratio of each sgRNA combination was calculated, the ratio was log-transformed, and the value was corrected with the mean and standard deviation of the control sgRNA combinations to derive the apoptosis induction score Z (sgRNA level Z score) of each sgRNA combination. . In addition, a Z score at the gene combination level was derived by averaging the Z scores of all sgRNA combinations capable of missing one gene combination.

In order to discover a synergistic sgRNA/gene combination that has a stronger apoptosis inducing ability than the simple sum of the apoptosis inducing ability caused by each sgRNA/gene deletion in the sgRNA/gene combination, the apoptosis inducing ability by each sgRNA/gene deletion The simple sum and the ability to induce apoptosis by the sgRNA/gene deletion combination were analyzed by scatter plots. As a result, most combinations of sgRNA/gene deletion induced only apoptosis equivalent to the simple sum of the apoptosis inducing capacity caused by each sgRNA/gene deletion, but few combinations of sgRNA/gene deletion caused more apoptosis than the simple sum. It deviated downward from the trend line of the scatter plot (Fig. 6a). The score calculated by how much the Z score of each sgRNA/gene deletion combination deviates from the scatter plot was named as a gene interaction [GI] score.

In order to derive a target gene for effective triple-negative breast cancer treatment, the following criteria were established from the Z and GI scores derived above to find a gene deletion combination that satisfies them all (FIG. 6c).

Criterion 1: Z<-5. This ensures that the combination of gene deletions results in sufficient apoptosis.

Criterion 2: GI<-2. This ensures that the combination of gene deletions causes more apoptosis than the simple sum of the apoptosis-inducing capacities of each gene deletion.

Criterion 3: RIGER GI p<0.01. This is a way of measuring statistical significance by observing the GI scores of all sgRNA combinations that induce the same gene deletion combination, thus ensuring that the GI scores of the gene deletion combinations are statistically significant.

Thirty gene combinations meeting the above three conditions (Table 2) were obtained (referred to as “candidate gene combinations” below), and among them, 8 gene combinations with reasonable grounds through previous studies through prior literature research synergistic apoptosis induction was verified.

Verification of synergistic apoptosis induction using CRISPR-Cas9 and sgRNA

A lentivirus was constructed to express two sgRNAs capable of deleting a candidate gene combination together with GFP and mCherry fluorescent protein, respectively, and the MDA-MB-231 Cas9 cells were infected with an MOI of 0.3 to 0.5. In this case, MDA-MB-231 Cas9 cells not expressing sgRNA, expressing each sgRNA, or expressing both sgRNAs proliferate in a mixed state. In addition, the expression of sgRNA in each cell can be confirmed by whether or not the fluorescent protein is expressed. For the same reason as the CRISPR screen above, the frequency of cells expressing both GFP/mCherry fluorescent protein decreases over time because whether or not apoptosis of cells is changed due to gene deletion by sgRNA. In this way, when each gene in the candidate gene combination is deleted, and when both genes are deleted, the frequency of apoptosis was observed using flow cytometry, and as a result, most of the candidate gene combinations induce synergistic apoptosis. was confirmed ( FIGS. 10 and 11 ).

Of particular note, it was observed that gene combinations consisting of a combination of FYN and other tyrosine kinases such as FYN-IGF1R, FYN-EGFR, and FYN-ABL2 frequently appear among the verified gene combinations (FIG. 13). Through network analysis of the CRISPR screen, it was confirmed that FYN can induce apoptosis synergistically with several other tyrosine kinases. Consistent with this, it was confirmed through western blot that the protein expression level of FYN increased when NVP-ADW742 (IGF1R inhibitor), gefitinib (EGFR inhibitor), and imatinib (ABL inhibitor) were treated ( FIG. 21 ), which was the mRNA level was confirmed by quantitative real-time PCR (FIG. 22). Interestingly, it was confirmed that the protein of CBL-b was decreased when NVP-ADW742 was treated (FIG. 23). Since FYN is known to be degraded by ubiquitination by proteins such as c-CBL and CBL-b, a decrease in CBL-b may inhibit degradation of FYN protein and thus induce protein stabilization. Therefore, it can be inferred that the treatment of multiple TKIs induces a decrease in c-CBL or CBL-b expression, thereby increasing the protein expression of FYN. (Fig. 24).

FYN inhibitors PP2, NVP-ADW742 (IGF1R inhibitor), and gefitinib (EGFR inhibitor) were added to the MDA-MB-231 cell line to verify the synergistic killing of cells caused by a defective combination of FYN and other tyrosine kinases using a tyrosine kinase inhibitor. ), and imatinib (an ABL inhibitor) were treated together to confirm that apoptosis occurred synergistically using the MTT assay. As a result, it was confirmed that the combination of the tyrosine kinase inhibitor could synergistically kill MDA-MB-231 cells ( FIG. 14 ). This synergistic apoptosis was also observed in the other triple-negative breast cancer cell line HCC38 (FIG. 15). When treated with PP2 and NVP-ADW742 or gefitinib, it was confirmed that the dead cells stained with Ethidium Dimer increased synergistically using Calcein-AM-Ethidium Dimer (live-dead) staining, and cells using BrdU staining It was confirmed that the proliferation was also synergistically reduced (FIG. 16).

Finally, in order to confirm the death of cancer by double inhibition of FYN and IGF1R at the individual level, MDA-MB-231 xenograft was generated in nude mice and the proliferation of cancer was observed by inhibiting each or both of FYN and IGF1R at the same time. In this case, saracatinib was used, not PP2, which was used in the previous cell culture experiment, because it was difficult to administer a sufficiently large amount in the mouse experiment due to the low solubility of PP2 in aqueous solution. It was confirmed by MTT analysis that saracatinib also causes apoptosis when treated with NVP-ADW742 like PP2 ( FIG. 17 ). After MDA-MB-231 xenograft was generated in nude mice, 50 mg of saracatinib per 1 kg of body weight and/or 20 mg of NVP-ADW742 were dissolved in Phosphate buffered saline + 5% DMSO and administered by intraperitoneal injection daily for 2 weeks. It was confirmed that a stronger cancer tissue proliferation inhibitory ability than that of each of the kinase inhibitors administered ( FIGS. 18 and 19 ). In addition, the mice to which the two inhibitors were administered did not show a significant difference in body weight, confirming that the treatment of triple-negative breast cancer using the two inhibitors could be a safe and effective cancer treatment ( FIG. 20 ).

In the above, embodiments of the present invention have been described with reference to the accompanying drawings, but those of ordinary skill in the art to which the present invention pertains can practice the present invention in other specific forms without changing its technical spirit or essential features. You will understand that there is Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (14)

a first agent that inhibits the activity of FYN kinase; and
For the prevention, treatment or alleviation of cancer including; a second agent comprising at least one selected from an agent for inhibiting the activity of IGR-1R kinase, an agent for inhibiting the activity of EGFR kinase, and an agent for inhibiting the activity of ABL kinase pharmaceutical composition.
The method of claim 1,
The second agent is an agent that inhibits the activity of IGR-1R kinase, a pharmaceutical composition for the prevention, treatment or alleviation of cancer.
The method of claim 1,
The second agent is an agent that inhibits the activity of EGFR kinase, a pharmaceutical composition for the prevention, treatment or alleviation of cancer.
The method of claim 1,
The second agent is an agent that inhibits the activity of ABL kinase, a pharmaceutical composition for preventing, treating or alleviating cancer.
The method of claim 1,
The first agent or the second agent is each independently at least one selected from a small molecule, a protein, a peptide, an RNA molecule, an endonuclease complex, and a DNA construct, a pharmaceutical composition for preventing, treating or alleviating cancer.
6. The method of claim 5,
The first agent and the second agent are each a kinase inhibitor that inhibits kinase activity with a small molecule, a pharmaceutical composition for the prevention, treatment or alleviation of cancer.
7. The method of claim 6,
The first agent is a small molecule FYN kinase inhibitor,
The agent inhibiting the activity of the IGR-1R kinase is a small molecule IGR-1R kinase inhibitor,
The agent inhibiting the activity of the EGFR kinase is a small molecule EGFR kinase inhibitor,
The agent inhibiting the activity of ABL kinase is a small molecule ABL kinase inhibitor,
A pharmaceutical composition for preventing, treating or alleviating cancer.
8. The method of claim 7,
The FYN kinase inhibitor is 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4- d ]pyrimidine (PP2) or N- (5-chloro-1,3- Benzodioxol-4-yl)-7-[2-(4-methyl-1-piperazinyl)ethoxy]-5-[(tetrahydro- 2H -pyran-4-yl)oxy]-4- quinazolinamine (Saracatinib),
The IGF-IR kinase inhibitor is 5-(3-phenylmethoxyphenyl)-7-[3-(pyrrolidin-1-ylmethyl)cyclobutyl]pyrrolo[2,3-d]pyrimidine-4- amine (NVP-ADW742),
The EGFR kinase inhibitor is N- (3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (gefitinib) is,
The ABL kinase inhibitor is 4-[(4-methylpiperazin- 1 -yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]- phenyl]-benzamide (imatinib) phosphorus,
A pharmaceutical composition for preventing, treating or alleviating cancer.
The method of claim 1,
The composition is applied to a patient group having any one or more of FYN overexpression gene, IGF-1R overexpression gene, EGFR overexpression gene and ABL overexpression gene, preventing, treating or alleviating cancer pharmaceutical composition.
6. The method of claim 5,
The DNA construct comprises an endonuclease gene encoding a CRISPR-related (Cas) protein, a pharmaceutical composition for preventing, treating or alleviating cancer.
The method of claim 1,
The cancer is breast cancer, preventing, treating or alleviating cancer pharmaceutical composition.
The method of claim 1,
The cancer is triple negative breast cancer, preventing, treating or alleviating cancer pharmaceutical composition.
The method of claim 1,
The first agent and the second agent are administered in a weight ratio of 1 to 0.1 to 10, a pharmaceutical composition for preventing, treating or alleviating cancer.
The method of claim 1,
The first agent is administered in an amount of 10 to 50 mg/kg,
The second agent is administered in an amount of 10 to 50 mg / kg,
A pharmaceutical composition for preventing, treating or alleviating cancer.

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