CN113667747A - Method for treating oral cancer - Google Patents

Method for treating oral cancer Download PDF

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CN113667747A
CN113667747A CN202010409087.3A CN202010409087A CN113667747A CN 113667747 A CN113667747 A CN 113667747A CN 202010409087 A CN202010409087 A CN 202010409087A CN 113667747 A CN113667747 A CN 113667747A
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methyl
amino
pyridin
cancer
pyrrolo
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杨佳郁
张凯评
谢佳训
吴治庆
欧阳俊男
刘巧柔
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Chang Gung University CGU
Chang Gung Memorial Hospital
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Chang Gung Memorial Hospital
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Abstract

Provided herein is a method for treating oral cancer in an individual. According to an embodiment of the present disclosure, the method comprises obtaining first and second biological samples from a diseased site and a non-diseased site, respectively, of the subject; measuring the expression level of CHEK1, PIK3CA, or PIK3CD in the two biological samples by qRT-PCR, respectively, to obtain a first and a second expression level; determining a ratio of the first expression level to the second expression level; and administering to the subject an effective amount of a checkpoint kinase 1 inhibitor when the ratio of CHEK1 is at least 1.7; or administering a phosphatidylinositol 3-kinase inhibitor when the ratio of PIK3CA is at least 2.4, or when the ratio of PIK3CD is at least 3.1.

Description

Method for treating oral cancer
Technical Field
The present disclosure relates generally to the field of cancer therapy. More particularly, the present disclosure relates to methods for treating oral cancer in an individual in need thereof.
Background
Cancer is a group of diseases characterized by abnormal cell growth, which may invade or spread to other parts of the body, and subsequently cause death in the affected individual. According to the report of the world health organization, 1810 ten thousands of new cancer cases and 960 ten thousands of cancer deaths occur in 2018 all over the world; cancer is the second leading cause of death worldwide, second only to cardiovascular disease. In taiwan, cancer was the first leading cause of death by 2018 for 37 consecutive years, totaling 48,784. In terms of mortality in taiwan 2018, the ten major cancer types are, in order, (1) tracheal cancer, bronchial cancer, and lung cancer; (2) liver cancer, gallbladder cancer, and biliary tract cancer; (3) colon and rectal cancers; (4) breast cancer in women; (5) oral cancer; (6) prostate cancer; (7) gastric cancer; (8) pancreatic cancer; (9) esophageal cancer; (10) cervical cancer.
Oral cancer refers to cancer that occurs on the inner membrane of the lips, mouth or upper throat, with Oral Squamous Cell Carcinoma (OSCC) accounting for 90% of all oral malignancies. In 2018, about 355,000 people worldwide developed oral cancer, resulting in 177,000 deaths, while in taiwan, oral cancer resulted in 3,027 deaths. The risk factors for oral cancer are mainly smoking and heavy drinking, although chewing areca and Human Papillomavirus (HPV) infection are reported to be one of the major predisposing factors. Management of OSCC is currently in early surgical resection followed by adjuvant therapy (e.g., radiation therapy) with or without chemotherapy. Among them, chemotherapy with Cisplatin (cissplatin) or Cetuximab (Cetuximab) are the current standard treatment for relapsed/metastatic OSCC. However, such treatments are not customized and often cause severe adverse effects to patients leading to a deterioration in their quality of life and often require rehabilitation to help them return to normal life.
In view of the above, there is a need in the art for a novel therapy that can precisely target oral cancer to improve the safety and effectiveness of treating oral cancer.
Disclosure of Invention
The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.
As embodied and broadly described herein, one aspect of the present disclosure relates to a kit for detecting the susceptibility of an individual suffering from oral cancer to a kinase inhibitor, comprising seq id no: 1 and 2, sequence number: 3 and 4, or sequence number: 5 and 6; the sequence numbers are as follows: 1 and 2 is used for detecting the expression level of the CHEK1 gene of the individual; the sequence numbers are as follows: 3 and 4 for detecting the expression level of the PIK3CA gene of the individual; the sequence numbers are as follows: 5 and 6 for detecting the expression level of the PIK3CD gene of the individual; and the kinase inhibitor is selected from the group consisting of: checkpoint kinase 1 (CHK 1) inhibitors, and phosphatidylinositol 3-kinase (PI 3K) inhibitors.
According to certain embodiments of the present disclosure, the kit of the present invention further comprises the sequence of seq id no: 7 and 8, the sequence numbers: 7 and 8 is used for detecting the expression level of the TBP gene of the individual.
According to certain embodiments of the present disclosure, the oral cancer is Oral Squamous Cell Carcinoma (OSCC).
An example of a CHK1 inhibitor suitable for use in the present disclosure may be
(S) -5- (3-Fluorophenyl) -N- (piperidin-3-yl) -3-ureidothiophene-2-carboxamide ((S) -5- (3-Fluorophenyl) -N- (piperidin-3-yl) -3-ureidothiophene-2-carboxamide) (AZD 7762);
4- (((3S) -1-Azabicyclo (2.2.2) oct-3-yl) amino) -3- (1H-benzimidazol-2-yl) -6-chloroquinolin-2 (1H) -one (4- (((3S) -1-Azabicyclo (2.2.2) oct-3-yl) amino) -3- (1H-benzimidazol-2-yl) -6-chloro quinolin-2(1H) -one) (CHIR-124);
laborab (Rabusertib) (LY 2603618);
proticit (preshasertib) (LY 2606368);
4- (2, 6-dichlorophenyl) -9-hydroxy-6- (3- (methylamino) propyl) pyrrolo [3,4-c ] carbazole-1, 3(2H,6H) -dione
(4-(2,6-dichlorophenyl)-9-hydroxy-6-(3-(methylamino)propyl)pyrrolo[3,4-c]carba zole-1,3(2H,6H)-dione)(PD-321852)；
(R) -2-amino-2-cyclohexyl-N- (2- (1-methyl-1H-pyrazol-4-yl) -6-oxo-5, 6-dihydro-1H- [1,2] diazepino [4,5,6-cd ] indol-8-yl) acetamide ((R) -2-amino-2-cyclohexyl-N- (2- (1-methyl-1H-pyrazol-4-yl) -6-oxo-5, 6-dihydro-1H- [1,2] diazepino [4,5,6-cd ] indol-8-yl) acetamide) (PF 477736);
(R) -6-Bromo-3- (1-methyl-1H-pyrazol-4-yl) -5- (piperidin-3-yl) pyrazolo [1,5-a ] pyrimidin-7-amine ((R) -6-Bromo-3- (1-methyl-1H-pyrazol-4-yl) -5- (piperdin-3-yl) pyrazolo [1,5-a ] pyrimidin-7-amine) (SCH 900776);
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) nicotinamide ((R) -N- (4- (3-aminoperidin-1-yl) -5-bromo-1H-pyro [2,3-b ] pyridin-3-yl) nicotinin amide);
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl isobutyramide ((R) -N- (4- (3-aminoperidin-1-yl) -5-bromo-1H-pyroro [2,3-b ] pyridin-3-yl isobutryamide);
(R) -N- (5-bromo-4- (3- (methylamino) piperidin-1-yl) -1H-pyrrolo [2,3-b ] pyridin-3-yl) nicotinamide ((R) -N- (5-bromo-4- (3- (methylamino) piperidin-1-yl) -1H-pyro [2,3-b ] pyridine-3-y l) nicotinamide);
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-methylnicotinamide ((R) -N- (4- (3-aminoperidin-1-yl) -5-bromo-1H-pyro [2,3-b ] pyridin-3-yl) -5-methylnicotinamide);
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) cyclopropanecarboxamide ((R) -N- (4- (3-aminoperidin-1-yl) -5-bromo-1H-pyro [2,3-b ] pyridin-3-yl) cyclopropanecarboxamide);
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -3-methylbutanamide ((R) -N- (4- (3-aminoperidin-1-yl) -5-bromo-1H-pyro [2,3-b ] pyridin-3-yl) -3-methylbutanamide); or
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -2-cyclopropylacetamide ((R) -N- (4- (3-aminoperidin-1-yl) -5-bromo-1H-pyro [2,3-b ] pyridin-3-yl) -2-cyclopropyracetamide).
In some embodiments, the CHK1 inhibitor is (S) -5- (3-fluorophenyl) -N- (piperidin-3-yl) -3-ureidothiophene-2-carboxamide;
porisin; or (R) -2-amino-2-cyclohexyl-N- (2- (1-methyl-1H-pyrazol-4-yl) -6-oxo-5, 6-dihydro-1H- [1,2] diazepino [4,5,6-cd ] indol-8-yl) acetamide.
An example of a PI3K inhibitor suitable for use in the present disclosure may be
(2S) -N1- [5- (2-tert-butyl-4-thiazolyl) -4-methyl-2-thiazolyl ] pyrrolidine-1, 2-dicarboxamide ((2S) -N1- [5- (2-tert-butyl-4-thiazolyl) -4-methyl-2-thiazolyl ] pyrolidine-1, 2-dicarboxamide) (A66);
(Z) -5- ((5- (4-fluoro-2-hydroxyphenyl) furan-2-yl) methylene) thiazolidine-2,4-dione ((Z) -5- ((5- (4-fluoro-2-hydroxyphenyl) furan-2-yl) methyl) methylene) thiazolidine-2,4-dione) (AS-252424);
5- (2,2-Difluoro-benzo [1,3] dioxolan-5-ylmethylene) -thiazolidine-2,4-dione (5- (2,2-Difluoro-benzo [1,3] dioxol-5-ylmethylene) -thiazolidine-2,4-dione) (AS-604850);
(R) -2- (1- (7-methyl-2-morpholinyl-4-oxo-4H-pyrido [1,2-a ] pyrimidin-9-yl) ethylamino) benzoic acid ((R) -2- (1- (7-methyl-2-morpholino-4-oxo-4H-pyrido [1,2-a ] pyrimidin-9-yl) ethyl lam ino) benzoic acid) (AZD 6482);
2-amino-N- [7-methoxy-8- (3-morpholinopropoxy) -2,3-dihydroimidazo [1,2-c ] quinazoline (2-amino-N- [7-methoxy-8- (3-morpholinopropoxy) -2, 3-dihydroimidazoie [1,2-c ] quinazolin) (BAY 80-6945);
copanlisib (Copanlisib) (BAY 80-6946);
8- (6-methoxypyridin-3-yl) -3-methyl-1- (4- (piperazin-1-yl) -3- (trifluoromethyl) phenyl) -1H-imidazo [4,5-c ] quinolin-2(3H) -one maleate (8- (6-methoxypyridin-3-yl) -3-methyl-1- (4- (piperazin-1-yl) -3- (trifluoromethyl) phenyl) -1H-imidozo [4,5-c ] quinolin-2(3H) -one maleate) (BGT 226);
buparville (Buparlisib) (BKM 120);
abacisib (Alpelisib) (BYL 719);
(5E) -5- { [5- (4-fluorophenyl) furan-2-yl ] methylene } -1,3-thiazolidine-2,4-dione ((5E) -5- { [5- (4-fluorophenyl) furan-2-yl ] methylidene } -1,3-thiazolidine-2,4-dione) (CAY 10505);
5- (2-Amino-8-fluoro- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) -N-tert-butylpyridine-3-sulfonamide (5- (2-Amino-8-fluoro- [1,2,4] triazo [1,5-a ] pyridin-6-yl) -N-tert-butyl pyrindine-3-sulphonamide) (CZC 24832);
duveliiib (IPI-145);
2- ((6-amino-9H-purin-9-yl) methyl) -5-methyl-3-o-tolylquinazolin-4(3H) -one (2- ((6-amino-9H-purin-9-yl) methyl) -5-methyl-3-o-tolylquinazolin-4(3H) -one) (IC-87114);
idelalisib (GS-1101, CAL-101);
serratia chromocor (Serabelisib) (INK 1117);
taselisib (Taselisib) (GDC-0032);
pituitib (Pictilisib) (GDC-0941);
apigliceos (Apitolisib) (GDC-0980);
(Z) -5- ((4- (pyridin-4-yl) quinolin-6-yl) methylene) thiazolidine-2,4-dione ((Z) -5- ((4- (pyridin-4-yl) quinolin-6-yl) methyl) thiazolidine-2,4-dione) (GSK 1059615);
omipalisib (GSK 2126458);
2-methyl-1- (2-methyl-3- (trifluoromethyl) benzyl) -6-morpholinyl-1H-benzo [ d ] imidazole-4-carboxylic acid (2-methyl-1- (2-methyl-3- (trifluoromethyl) benzyl) -6-morpholino-1H-benzo [ d ] imidazole-4-carboxylic acid) (GSK 2636771);
6- [5- [ (phenylsulfonyl) amino ] -3-pyridyl ] -imidazo [1,2-a ] pyridine-3-carboxylic acid, ethyl ester (6- [5- [ (phenylsulfonyl) amino ] -3-pyridyl ] -imidozo [1,2-a ] pyridine-3-carboxylic acid, ethyl ester) (HS-173);
2-amino-N, N-dimethyl-5- (3- (2-methylpyridin-4-yl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) pyridine-3-sulfonamide (2-amino-N, N-dimethyl-5- (3- (2-methylpyridin-4-yl) -1H-pyrolo [2,3-b ] pyridin-5-yl) pyridine-3-sulfonamide) (HS-527);
2- (4-Morpholinyl) -8-phenyl-4H-1-benzopyran-4-one (2- (4-morpholino) -8-phenyl-4H-1-benzopyran-4-one) (LY 294002);
datolisib (Dactlisib) (NVP-BEZ 235);
pirifocine (Perifosine);
2-Amino-8- [4- (2-hydroxyethoxy) cyclohexyl ] -6- (6-methoxypyridin-3-yl) -4-methylpyrido [2,3-d ] pyrimidin-7-one (2-Amino-8- [4- (2-hydroxyethoxy) cyclohexyl ] -6- (6-methoxypyridin-3-yl) -4-meth ylpyrido [2,3-d ] pyrimidin-7-one) (PF-04691502);
gedatolisib (Gedatolisib) (PF-05212384);
3- (4-morpholinopyrido [ 3', 2': 4,5] furo [3,2-d ] pyrimidin-2-yl) phenol (3- (4-morpholinopyrido [ 3', 2': 4,5] furo [3,2-d ] pyrimidin-2-yl) phenol) (PI-103);
2-methyl-5-nitro-2- [ (6-bromoimidazo [1,2-a ] pyridin-3-yl) methylene ] -1-methylhydrazide-benzenesulfonic acid (2-methyl-5-nitro-2- [ (6-Bromoidazo [1,2-a ] pyridine-3-yl) methyl ] -1-methyl-drazide-benzosulfonic acid) (PIK-75);
n- (2,3-dihydro-7,8-dimethoxyimidazo [1,2-c ] quinazolin-5-yl) -3-pyridinecarboxamide (N- (2,3-dihydro-7, 8-dimethoxyimidozo [1,2-c ] quinazolin-5-yl) -3-pyridinecarboxamide) (PIK-90);
2- [ (4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl ] -5-methyl-3- (2-methylphenyl) -4(3H) -quinazolinone (2- [ (4-amino-1H-pyrazolo [3,4-d ] pyrimid-1-yl) methyl ] -5-methyl-3- (2-methyl nyl) -4(3H) -quinazone) (PIK-293);
2- [ [4-amino-3- (3-hydroxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl ] methyl ] -5-methyl-3- (2-methylphenyl) -4(3H) -quinazolinone (2- [ [4-amino-3- (3-hydroxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl ] methyl ] -5-methyl-3- (2-methylphenenyl) -4(3H) -quinazone) (PIK-294);
1- (4- (3-ethyl-7-morpholinyl-3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-yl) phenyl) -3- (4- (4-methylpiperazine-1-carbonyl) phenyl) urea (1- (4- (3-ethyl-7-morpholino-3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-yl) phenyl) -3- (4- (4-methylpiperazine-1-carbonyl) phenyl) urea) (PKI-402);
sonolisse (Sonolisib) (PX-866);
(S) -2- (2- (2-Methylindolin-1-yl) -2-oxoethyl) -6-morpholinylpyrimidin-4 (3H) -one ((S) -2- (2- (2-Methylindolin-1-yl) -2-oxoethyl) -6-morpholinopyrim idin-4(3H) -one) (SAR 260301);
pitalia (pilalaisib) (SAR 2457408);
(2S) -2- [ [ (2S) -3-Carboxy-2- [ [2- [ [ (2S) -5- (diaminomethyleneamino) -2- [ [4-oxo-4- [ [4- (4-oxo-8-phenylchromen-2-yl) morpholin-4-ium-4-yl ] methoxy ] butanoyl ] amino ] pentanoyl ] amino ] acetyl ] amino ] propanoyl ] amino ] -3-hydroxypropionate ((2S) -2- [ [ (2S) -3-Carboxy-2- [ [2- [ [ (2S) -5- (diaminomethylideneamino) -2- [ [4-oxo-4- [ [4- (4-oxo-8-phenylchromen-2-yl) morpholinon -4-ium-4-yl ] methoxy ] butanoyl ] amino ] pentanoyl ] amino ] acetyl ] amino ] propanoyl ] amino ] -3-hydroxypropanoate (SF 1126);
3- (2,4-Diaminopteridin-6-yl) phenol (3- (2, 4-diamminetripteridin-6-yl) phenol) (TG 100713);
umbraliella (Umbralisib) (TGR-1202);
5- (9-Isopropyl-8-methyl-2-morpholinyl-9H-purin-6-yl) pyrimidin-2-amine (5- (9-isoproyl-8-methyl-2-morpholino-9H-purin-6-yl) pyrimidin-2-amine) (VS-5584);
voxtalisib (Voxtalisib) (XL 765); or
4,4 '- (6- (2- (Difluoromethyl) -1H-benzo [ d ] imidazol-1-yl) -1,3,5-triazine-2,4-diyl) dimorpholine (4, 4' - (6- (2- (Difluoromethyl) -1H-benzol [ d ] imidozol-1-yl) -1,3,5-triazine-2,4-diyl) di morphine) (TK ZS 474).
In some embodiments, the PI3K inhibitor is abacteriol;
a Pituity plug; or (Z) -5- ((4- (pyridin-4-yl) quinolin-6-yl) methylene) thiazolidine-2, 4-dione.
According to some embodiments of the disclosure, the subject is a human.
Many of the attendant features and advantages of this disclosure will be better understood by reference to the following detailed description considered in connection with the accompanying drawings.
Drawings
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:
fig. 1A-1D depict the expression of CHEK1, PIK3CA, and PIK3CD in OSCC according to a working example of the present disclosure. FIGS. 1A-1C illustrate the expression of CHEK1 (FIG. 1A), PIK3CA (FIG. 1B), and PIK3CD (FIG. 1C) in 32 pairs of matched OSCC patients by qRT-PCR analysis. Expression levels of these genes were determined by 2-ΔΔCtMethods a TATA-box binding protein (TBP) was determined as an internal control. FIG. 1D is a dot plot depicting the ratio of gene expression in tumor tissue of OSCC patients to gene expression in adjacent normal tissue.
Fig. 2A-2F depict the therapeutic effect of CHK1 inhibitors on OSCC. Fig. 2A is a graph depicting the dose-dependent cytotoxic effects of various CHK1 inhibitors (including PF477736, AZD7762, and LY2606368) on OSCC cells SAS and OEC-M1, respectively, as determined by the MTT assay. FIGS. 2B-2C show the dose-dependent cytotoxic effect of PF477736 on OSCC cells, as determined by colony-forming assay (fig. 2B) and by membrane-linked protein V staining (Annexin V staining) (fig. 2C). Figure 2D illustrates the effect of CHK1 inhibitors on UV-induced CHK1 phosphorylation. FIG. 2E depicts the dose-dependent cytotoxic effect of cisplatin on OSCC cells SAS, OC3 or OEC-M1 as determined by MTT assay. FIG. 2F is a bar graph depicting the effect of PF477736 and cisplatin on SAS or OC3 cells as determined by the MTT assay. Data were repeated in three separate experiments. Results are expressed as mean ± s.e.m. Statistical analysis between the two groups was performed using Student's t-test (Student's t-test). P < 0.05; p < 0.01; p < 0.001.
Figures 3A-3G depict the therapeutic effect of PI3K inhibitors on OSCC. FIG. 3A is a line graph depicting the dose-dependent cytotoxic effect of PI3K inhibitors (including BYL719, GDC-0941, and GSK1059615) on OSCC cells SAS or OEC-M1 as determined by MTT assay. FIGS. 3B-3C are bar graphs showing the dose-dependent cytotoxic effect of BYL719 on OSCC cells, as determined by colony formation assay (FIG. 3B) and annexin V staining (FIG. 3C). Fig. 3D depicts the effect of PI3K inhibitors on the phosphorylation of AKT. Figure 3E depicts the effect of BYL719 and PF477736 on SAS cells as determined by MTT assay. Fig. 3F is a bar graph depicting apoptotic cells in fig. 3F as determined by membrane-associated protein V staining. FIG. 3G depicts the effect of GDC-0941 and AZD7762 on SAS cells as determined by MTT assay. Data were repeated in three separate experiments. Results are expressed as mean ± s.e.m. Statistical analysis between the two groups was performed using student t-test. P < 0.05; p < 0.01; p < 0.001.
Figures 4A-4D are line graphs depicting the effect of CHK1 inhibitor PF477736 and PI3K inhibitor BYL719 on OSCC xenografts. SAS cells were injected subcutaneously into NOD/SCID mice until tumor volumes reached 300-3At the time, the mice were randomly divided into several groups. The transplanted mice were administered vehicle control, 10 or 20mg/kg PF477736 (FIG. 4A), 25 or 50mg/kg BYL719 (FIG. 4B), PF477736(20mg/kg) plus BYL719(50mg/kg) (FIG. 4C), and 5mg/kg cisplatin (FIG. 4D). Tumor volumes were measured and calculated at the indicated times. Data are expressed as mean ± s.e.m. Statistical analysis between groups was performed using student's t-testIn (1). P<0.05；**p<0.01； ***p<0.001.
Figures 5A-5E show the effect of CHK1 inhibitor PF477736 in combination with cisplatin or PI3K inhibitor BYL719 on tumor growth in an OSCC patient-derived xenograft (PDX) model. Figure 5A provides results for three OSCC PDX mice (patient #1PDX, patient #2PDX and patient #3PDX) treated with vehicle control, cisplatin alone, PF477736 alone, and PF477736 plus cisplatin. FIG. 5B is a representative photograph of hematoxylin-eosin (H & E) and Immunohistochemistry (IHC) staining (Ki-67; upper panel); the line graph depicts the corresponding quantitative results from the patient #2PDX model (lower panel). Figure 5C is a line graph depicting the respective changes in tumor volume for patient #1PDX and patient #2PDX models treated with vehicle control, BYL719 alone, PF477736 alone, or a combination of PF477736 and BYL 719. FIG. 5D provides the results of the patient #2PDX model determined by IHC staining (Ki-67; upper panel), and the corresponding quantitative results (lower panel). Fig. 5E is a line graph depicting the change in body weight of the patient #2PDX model after treatment. Tumor volume was measured twice weekly and mice were monitored for 3 weeks. Data are expressed as mean ± s.e.m. Statistical analysis between the two groups was performed using student's t-test. P < 0.05; p < 0.01.
Detailed Description
The detailed description provided below in connection with the appended drawings is intended as a description of embodiments of the present invention and is not intended to represent the only forms in which the embodiments of the present invention may be constructed or utilized. The description sets forth the functions of the embodiments and the sequence of steps for constructing and operating the embodiments. However, the same or equivalent functions and sequences may be accomplished by different embodiments.
I. Definition of
For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless defined otherwise herein, scientific and technical terms used in the present disclosure shall have the meanings that are commonly understood and used by those of ordinary skill in the art. In addition, unless the context requires otherwise, it should be understood that singular terms shall include the plural form of the item and that plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Also, as used herein and in the claims, the terms "at least one" (an) and "one or more" (a) have the same meaning and include one, two, three or more. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" (about) generally refers to within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean, as considered by one of ordinary skill in the art. Except as may be the case in the operating/working examples, or otherwise expressly specified, all numerical ranges, amounts, values and percentages herein disclosed, for example, for terms such as amounts of materials, durations, temperatures, operating conditions, quantitative ratios, and the like, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that may vary depending upon the desired properties. In any event, each numerical parameter should at least be construed as a numerical value given by the number of reported significant digits and by applying ordinary rounding techniques.
As used herein, the term "treatment" (or treating) may refer to a cure or palliative measure. In particular, the term "treating" as used herein means applying the methods of the invention to an individual suffering from cancer, a symptom associated with cancer, a disease or disorder secondary to cancer, with the purpose of partially or completely alleviating, ameliorating, reducing, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing the incidence of one or more symptoms or features thereof.
The terms "cancer" (cancer) and "tumor" (tumor) are used interchangeably in this disclosure and preferably refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer in this regard includes metastatic cancer and/or drug resistant cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include bladder cancer, biliary tract cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, epidermal cancer, gastric cancer, gastrointestinal stromal tumor, glioblastoma, glioma, hepatocellular cancer, non-Hodgkin's lymphoma, Kaposi's sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, pancreatic cancer, prostate cancer, retinoblastoma, ovarian cancer, renal cell cancer, spleen cancer, squamous cell carcinoma (e.g., oral squamous cell carcinoma, epithelial squamous cell carcinoma), thyroid cancer, or thyroid follicular cancer. According to a specific working example, the cancer is oral cancer; the cancer is oral cancer. More specifically, OSCC.
The term "subject" or "patient" refers to an animal, including a human species, that can be treated by the methods of the present disclosure. The term "individual" or "patient" means both male and female unless one gender is specifically indicated. Thus, the term "individual" or "patient" includes any mammal that may benefit from treatment for cancer. Examples of "subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and birds. In an exemplary embodiment, the individual is a mouse. In another exemplary embodiment, the subject is a human.
The term "lesion" (precision) refers to any pathological or traumatic discontinuity or partial loss of function of a tissue. For the purposes described herein, focal (localized) lesions are of most interest, i.e., lesions that can be visually attributed to localized regions, such as those located in the oral cavity. As described herein, a lesion is associated with hyperproliferative cell division, which may be malignant, benign, or a precancerous lesion in between after biopsy analysis, where "precancerous lesion" (premalignant) refers to a benign lesion that is evolving into a malignant lesion. An individual with a pathology may be susceptible or predisposed to a disease (e.g., cancer), and may not have been diagnosed as having the disease. As used herein, the term "susceptible" refers to a predisposition of an individual to develop a condition (e.g., a trait, phenotype, or disease), or to be less resistant to a condition than the average individual.
As used herein, the term "biomarker" refers to a gene whose level of expression, as measured using the gene product.
As used herein, the term "at least" refers to a non-strict inequality with an unequal comparison between two numbers. For example, the term "X is at least Y" (X is at least Y) means that X is greater than or equal to Y, or X is not less than Y.
The terms "administration," "administering," or "administration" (administered, or administration) are used interchangeably herein to refer to a means of administering an anti-cancer treatment as described in the present disclosure to a subject in need thereof.
As used herein, the term "effective amount" (an effective amount) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result with respect to the treatment of cancer. For example, in the treatment of cancer, an agent that reduces, prevents, delays or inhibits or prevents the expression of the CHEK1 gene or the PIK3CA gene (i.e., a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor) may be effective in preventing cancer cell spreading and/or growth. The effective amount of the agent does not necessarily cure the disease or condition, but will provide treatment of the disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the symptoms of the disease or condition are ameliorated. The specific effective or sufficient amount will vary depending on factors such as: the particular condition being treated, the physical condition of the patient (e.g., the patient's weight, age, or sex), the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the particular formulation employed, among other things. For example, an effective amount can be expressed as the total mass of the active agent (e.g., in grams, milligrams, or micrograms) or the ratio of the mass of the active agent to the body weight, e.g., in milligrams per kilogram (mg/kg). The effective amount may be divided into one, two or more doses in a suitable form to be administered once, twice or more over a specified period. Preferably, an effective amount refers to a Human Equivalent Dose (HED), which is the maximum safe dose for a human subject. HED can be calculated according to the Industrial guidelines issued by the U.S. Food and Drug Administration, FDA entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for treatment of Adult Healthy Volunteers" Estimating the Maximum Safe Starting Dose for human subjects.
The term "radiotherapy" (also known as radiotherapy) refers to the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that damages or destroys cells located in the area to be treated ("target tissue") by damaging their genetic material, thereby preventing these cells from continuing to grow. Radiation often causes damage to both cancer and normal cells, the latter being more likely to resume normal operation. In some embodiments, radiation therapy is used to treat localized solid tumors, such as cancers of the skin, tongue, larynx, brain, breast, prostate, colon, uterus, and/or cervix. In some embodiments, radiation therapy is also used to treat leukemia and lymphoma.
As used herein, the term "chemotherapy" (chemotherapy) is directed to the administration of one or more chemotherapeutic agents to a patient in need thereof with the purpose of reducing, preventing, alleviating, limiting and/or delaying the growth or metastasis of a tumor in the patient, or directly killing tumor cells by tumor necrosis or apoptosis or any other mechanism. As used herein, the term "chemotherapeutic agent" refers to any chemical substance known to a clinical practitioner of ordinary skill in the art for treating or ameliorating cancer and/or as an inducer of apoptosis in a patient.
Description of the invention
The present disclosure is based, at least in part, on the following findings: genes CHEK1, PIK3CA and/or PIK3CD are differentially expressed between tumor tissue and matched adjacent normal tissue in OSCC patients. Thus, administration of a kinase inhibitor (e.g., checkpoint kinase 1 inhibitor or phosphatidylinositol 3-kinase inhibitor) to a patient according to genes differentially expressed in the patient (i.e., CHEK1, PIK3CA, and/or PIK3CD) would improve the therapeutic efficacy of the kinase inhibitor.
1. Methods of treating cancer
Accordingly, a first aspect of the present disclosure relates to a method for treating cancer in an individual. The method comprises the following steps:
(a) obtaining first and second biological samples from a diseased site and a non-diseased site, respectively, of the individual;
(b) measuring the expression level of a biomarker in the first and second biological samples by qRT-PCR, respectively, to obtain a first expression level and a second expression level, wherein the biomarker is a CHEK1 gene, a PIK3CA gene, or a PIK3CD gene;
(c) determining a ratio of the first expression level to the second expression level; and
(d) when the biomarker is the CHEK1 gene and the ratio determined in step (c) is at least 1.7; or when the biomarker is the PIK3CA gene and the ratio determined in step (c) is at least 2.4; or when the biomarker is the PIK3CD gene and the ratio determined in step (c) is at least 3.1, administering to the individual an effective amount of a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor.
According to preferred embodiments of the present disclosure, the methods of the present invention may be used to treat cancer as described above. Preferably, the methods of the invention are useful for treating oral cancers such as tumors of the bottom of the mouth, gum cancer, lip cancer, primary salivary gland cancer, oropharyngeal cancer, OSCC, other oral cancers, jaw cancer, and tongue cancer. In some specific embodiments, the cancer treatable by the methods of the invention is OSCC.
According to some embodiments of the present disclosure, the subject treatable by the methods of the invention is as described above. In some embodiments, the individual who will benefit from the methods of the invention is a mouse. In other embodiments, the subject suitable for treatment by the methods of the invention is a human.
First, paired samples (biological samples taken from a diseased site and a non-diseased site of an individual, respectively) are referred to as first and second biological samples (step (a)). Since the first biological sample is collected from the diseased site, the second biological sample is collected from a normal tissue and may be any of skin epithelium, nasal mucosa, oral mucosa, buccal epithelium, jaw epithelium, sublingual epithelium, submucosa, rectal epithelium, vaginal epithelium, intrathecal tissue, intramuscular tissue, intravenous tissue, ligament or tendon. Preferably, the second biological sample is the oral mucosa adjacent to the lesion. According to alternative embodiments of the present disclosure, the first biological sample and the second biological sample may be from different individuals. For example, the first biological sample is from a diseased site of an individual associated with cancer, while the second biological sample is from a non-diseased site of a healthy donor.
Then, both the first and second biological samples are subjected to gene expression analysis for a specific biomarker (step (b)). The biomarker to be detected in the method of the invention (i.e. in step (b)) may be the CHEK1 gene or the PIK3CA gene, or optionally, PIK3 CD. In some embodiments, the respective levels of the CHEK1 gene in the first and second biological samples are determined. In other embodiments, the respective levels of PIK3CA gene in the first and second biological samples are determined. Alternatively, in other embodiments, the respective levels of PIK3CD gene in the first and second biological samples are determined. Techniques suitable for assessing gene expression are well known in the art, including but not limited to: whole genome expression profiling using Expressed Sequence Tag (EST) analysis, Sequence Analysis of Gene Expression (SAGE), DNA microarray, Massively Parallel Signature Sequencing (MPSS), RNA sequencing (RNA-seq), qRT-PCR, digital polymerase chain reaction (dPCR), two-dimensional gel electrophoresis (2-D electrophoresis), tissue array, Immunohistochemistry (IHC) staining, and the like. In some embodiments, gene expression is analyzed by RNA-seq. In other embodiments, gene expression is analyzed by qRT-PCR. In other embodiments, IHC staining is used. Preferably, in the method of the invention, gene expression is analyzed by qRT-PCR.
Alternatively or optionally, the levels of the biomarkers determined may be normalized in order to minimize individual variation from one individual to another. As used herein, the term "normalized" when applied to a gene refers to a normalized level of a gene product, e.g., a normalized value determined for the RNA expression level of the gene or for the polypeptide expression level of the gene. Typically, normalization refers to dividing the value of a biomarker in a sample by the value of a housekeeping gene. A "biomarker" (biomarker) is defined as a laboratory measurement that reflects the activity of a disease process, in other words, a biomarker is quantitatively related (forward or reverse) to the progression of the disease. "housekeeping gene" (housekeeping gene) refers to a gene that is constitutively expressed at a relatively constant level under many or all known conditions, because it encodes a protein that is constantly required by the cell, and thus, it is essential to the cell and always present in any situation. Its expression is assumed to be unaffected by the experimental conditions. The proteins it encodes are often involved in essential functions required for cell nutrition or maintenance. According to embodiments of the present disclosure, the housekeeping gene may be any one of β -Actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β -Glucuronidase (GUSB), hydroxymethylcholine synthase (HMBS), hypoxanthine guanine phosphoribosyltransferase 1(HPRT1), TATA box binding protein (TBP), or 18S ribosomal rna (rrna). In a particular embodiment, the housekeeping gene is TBP.
After determining and optionally normalizing the gene expression levels (e.g., CHEK1, PIK3CA, or PIK3CD) in the first and second biological samples, the relative expression of a biomarker (e.g., CHEK1, PIK3CA, PIK3CD, or a combination thereof) between the first biological sample and the second biological sample (i.e., a lesion and a normal site) is then determined (i.e., step (c)) by calculating the ratio of the first expression level (i.e., the level of the biomarker in the first biological sample) to the second expression level (i.e., the level of the same biomarker in the second biological sample) by dividing the first expression level by the second expression level.
Then, treating the individual suffering from OSCC based on the respective ratio of the biomarkers determined in step (c). In the methods of the invention, when the ratio of a particular biomarker (e.g., CHEK1, PIK3CA, or PIK3CD) is above a predetermined threshold, then the subject is administered a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor. For the biomarker CHEK1 gene, the predetermined threshold was 1.7; for the biomarker PIK3CA gene, the predetermined threshold was 2.4; for the biomarker PIK3CD gene, the predetermined threshold was 3.1. Thus, an OSCC individual may be treated with a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor if the ratio of the CHEK1, PIK3CA, and PIK3CD genes between the diseased site and the normal site is independently at least 1.7, 2.4, or 3.1.
Examples of CHK1 inhibitors suitable for use in the methods of the invention include, but are not limited to:
(S) -5- (3-fluorophenyl) -N- (piperidin-3-yl) -3-ureidothiophene-2-carboxamide (AZD 7762);
4- (((3S) -1-azabicyclo (2.2.2) oct-3-yl) amino) -3- (1H-benzoimidazol-2-yl) -6-chloroquinolin-2 (1H) -one (CHIR-124);
labertian (LY 2603618);
pleiotisine (LY 2606368);
4- (2, 6-dichlorophenyl) -9-hydroxy-6- (3- (methylamino) propyl) pyrrolo [3,4-c ] carbazole-1, 3(2H,6H) -dione (PD-321852);
(R) -2-amino-2-cyclohexyl-N- (2- (1-methyl-1H-pyrazol-4-yl) -6-oxo-5, 6-dihydro-1H- [1,2] diazepino [4,5,6-cd ] indol-8-yl) acetamide (PF 477736);
(R) -6-bromo-3- (1-methyl-1H-pyrazol-4-yl) -5- (piperidin-3-yl) pyrazolo [1,5-a ] pyrimidin-7-amine (SCH 900776);
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) nicotinamide;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl isobutyramide;
(R) -N- (5-bromo-4- (3- (methylamino) piperidin-1-yl) -1H-pyrrolo [2,3-b ] pyridin-3-yl) nicotinamide;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-methylnicotinamide;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) cyclopropanecarboxamide;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -3-methylbutanamide; or
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -2-cyclopropylacetamide.
In some embodiments, the CHK1 inhibitor used in the methods of the invention is (S) -5- (3-fluorophenyl) -N- (piperidin-3-yl) -3-ureidothiophene-2-carboxamide;
porisin; and (R) -2-amino-2-cyclohexyl-N- (2- (1-methyl-1H-pyrazol-4-yl) -6-oxo-5, 6-dihydro-1H- [1,2] diazepino [4,5,6-cd ] indol-8-yl) acetamide.
Examples of PI3K inhibitors suitable for use in the methods of the invention include, but are not limited to:
(2S) -N1- [5- (2-tert-butyl-4-thiazolyl) -4-methyl-2-thiazolyl ] pyrrolidine-1, 2-dicarboxamide (a 66);
(Z) -5- ((5- (4-fluoro-2-hydroxyphenyl) furan-2-yl) methylene) thiazolidine-2,4-dione (AS-252424);
5- (2,2-difluoro-benzo [1,3] dioxolan-5-ylmethylene) -thiazolidine-2,4-dione (AS-604850);
(R) -2- (1- (7-methyl-2-morpholinyl-4-oxo-4H-pyrido [1,2-a ] pyrimidin-9-yl) ethylamino) benzoic acid (AZD 6482);
2-amino-N- [7-methoxy-8- (3-morpholinylpropoxy) -2,3-dihydroimidazo [1,2-c ] quinazoline (BAY 80-6945);
copenexis (BAY 80-6946);
8- (6-methoxypyridin-3-yl) -3-methyl-1- (4- (piperazin-1-yl) -3- (trifluoromethyl) phenyl) -1H-imidazo [4,5-c ] quinolin-2(3H) -one maleate (BGT 226);
buparlys (BKM 120);
abacteriol (BYL 719);
(5E) -5- { [5- (4-fluorophenyl) furan-2-yl ] methylene } -1,3-thiazolidine-2,4-dione (CAY 10505);
5- (2-amino-8-fluoro- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) -N-tert-butylpyridine-3-sulfonamide (CZC 24832);
duoweili plug (IPI-145);
2- ((6-amino-9H-purin-9-yl) methyl) -5-methyl-3-o-tolylquinazolin-4(3H) -one (IC-87114);
idelalisib (GS-1101, CAL-101);
cerilide plug (INK 1117);
taseliser (GDC-0032);
petilips (GDC-0941);
apiglicen (GDC-0980);
(Z) -5- ((4- (pyridin-4-yl) quinolin-6-yl) methylene) thiazolidine-2,4-dione (GSK 1059615);
omimbalise (GSK 2126458);
2-methyl-1- (2-methyl-3- (trifluoromethyl) benzyl) -6-morpholinyl-1H-benzo [ d ] imidazole-4-carboxylic acid (GSK 2636771);
6- [5- [ (benzenesulfonyl) amino ] -3-pyridinyl ] -imidazo [1,2-a ] pyridine-3-carboxylic acid, ethyl ester (HS-173);
2-amino-N, N-dimethyl-5- (3- (2-methylpyridin-4-yl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) pyridine-3-sulfonamide (HS-527);
2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one (LY 294002);
datorrich plug (NVP-BEZ 235);
pirifoxine;
2-amino-8- [4- (2-hydroxyethoxy) cyclohexyl ] -6- (6-methoxypyridin-3-yl) -4-methylpyrido [2,3-d ] pyrimidin-7-one (PF-04691502);
jedalise plug (PF-05212384);
3- (4-morpholinopyrido [ 3', 2': 4,5] furo [3,2-d ] pyrimidin-2-yl) phenol (PI-103);
2-methyl-5-nitro-2- [ (6-bromoimidazo [1,2-a ] pyridin-3-yl) methylene ] -1-methylhydrazide-benzenesulfonic acid (PIK-75);
n- (2,3-dihydro-7,8-dimethoxyimidazo [1,2-c ] quinazolin-5-yl) -3-pyridinecarboxamide (PIK-90);
2- [ (4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl ] -5-methyl-3- (2-methylphenyl) -4(3H) -quinazolinone (PIK-293);
2- [ [4-amino-3- (3-hydroxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl ] methyl ] -5-methyl-3- (2-methylphenyl) -4(3H) -quinazolinone (PIK-294);
1- (4- (3-ethyl-7-morpholinyl-3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-yl) phenyl) -3- (4- (4-methylpiperazine-1-carbonyl) phenyl) urea (PKI-402);
sonolisai (PX-866);
(S) -2- (2- (2-methylindolin-1-yl) -2-oxoethyl) -6-morpholinylpyrimidin-4 (3H) -one (SAR 260301);
plausiplug (SAR 2454408);
(2S) -2- [ [ (2S) -3-carboxy-2- [ [2- [ [ (2S) -5- (diaminomethyleneamino) -2- [ [4-oxo-4- [ [4- (4-oxo-8-phenylchromen-2-yl) morpholin-4-ium-4-yl ] methoxy ] butanoyl ] amino ] pentanoyl ] amino ] acetyl ] amino ] propanoyl ] amino ] -3-hydroxypropionate (SF 1126);
3- (2,4-diaminopteridin-6-yl) phenol (TG 100713);
upaleo stopper (TGR-1202);
5- (9-isopropyl-8-methyl-2-morpholinyl-9H-purin-6-yl) pyrimidin-2-amine (VS-5584);
vodarriella (XL 765); and
4, 4' - (6- (2- (difluoromethyl) -1H-benzo [ d ] imidazol-1-yl) -1,3,5-triazine-2,4-diyl) dimorpholine (ZSTK 474).
In some embodiments, used in the methods of the invention is apentamide;
a Pituity plug; and
(Z) -5- ((4- (pyridin-4-yl) quinolin-6-yl) methylene) thiazolidine-2, 4-dione.
According to some embodiments of the disclosure, an OSCC individual is treated with a CHK1 inhibitor when the ratio of the CHEK1 gene between the lesion and the normal site is at least 1.7. In the case where the ratio of the CHEK1 gene is less than 1.7, the OSCC subject is treated (e.g., surgery, radiation therapy, chemotherapy, immunotherapy, or combination thereof) with an inhibitor of CHK 1. Alternatively or additionally, OSCC individuals are treated with PI3K inhibitors when the ratio of PIK3CA gene or PIK3CD gene between the lesion and the normal site is at least 2.4 or 3.1, respectively. In the case where the ratio of PIK3CA gene or PIK3CD gene is less than 2.4 or 3.1, respectively, the OSCC individual is treated with an inhibitor other than PI 3K. In some embodiments, OSCC individuals with a CHEK1 alone ratio of at least 1.7 can exhibit a good response to a combination therapy of a CHK1 inhibitor (e.g., PF477736) plus a chemotherapeutic agent (e.g., cisplatin). In other embodiments, OSCC individuals having a CHEK1 ratio of at least 1.7 and a PIK3CA and/or PIK3CD ratio of at least 2.4 and/or 3.1 may exhibit a good response to a combination therapy of a CHK1 inhibitor (e.g., PF477736 or AZD7762) plus a PI3K inhibitor (e.g., BYL719 or GDC-0941).
Alternatively or optionally, the method of the invention further comprises administering to the individual, prior to, simultaneously with, or after the above-described treatment step (i.e., step (d)), an additional anti-cancer treatment, e.g., surgery, radiation therapy, chemotherapy, immunotherapy, or a combination thereof. Combination therapy refers to a combination of at least two of the above listed anti-cancer therapies, e.g., a combination of surgery and radiation therapy, a combination of surgery and chemotherapy, and the like.
The chemotherapy is administered by administering a chemotherapeutic agent to a subject in need thereof to alleviate or ameliorate the symptoms of the cancer. Examples of chemotherapeutic agents suitable for use in the present methods include, but are not limited to, alkylating agents, platinum drugs, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, and/or enzyme inhibitors. Exemplary alkylating agents include, but are not limited to, nitrogen mustard hydrochloride (Chlormethine), Ifosfamide (Ifosfamide), Trofosfamide (Trofosfamide), Melphalan (Melphalan), Prednimustine (Prednimustine), Bendamustine (Bendamustine), Uramustine (Uramustine), Carmustine (Carmustine), Semustine (Semustine), Fotemustine (Fotemustitine), Nimustine (Nimustine), ramustine (Ranimustine), Streptozocin (Streptozocin), Mannosulfan, Trioslfan (Treosulfan), carboquinone (Carbouuuqutepa), Thiotepa (Thioqtepa), triimine (Triaziuuquinone), and triethylmelamine (Triethylelene). Examples of platinum drugs suitable for use in the methods of the invention are Carboplatin (Carboplatin), cisplatin, Dicycloplatin (Dicycloplatin), Nedaplatin (Nedaplatin), Oxaliplatin (Oxaliplatin) or Satraplatin (Satraplatin).
In some embodiments, the chemotherapeutic agent used in the methods of the invention is an antimetabolite, for example, Aminopterin (Aminopterin), Hydroxyurea (Hydroxyurea), Methotrexate (Methotrexate), Pemetrexed (Pemetrexed), Pralatrexate (Pralatrexate), Raltitrexed (polytexed), Pentostatin (pentastatin), Cladribine (Cladribine), Clofarabine (Clofarabine), Fludarabine (Fludarabine), Nelarabine (nerabadine), thioguanine (tioguanadine), Mercaptopurine (meaptourine), Fluorouracil (Fluorouracil), Capecitabine (Capecitabine), fluoroflurouridine (doxifludine), gafur-gardine (Tegafur), carmofluorine (Carmofur), Fluorouracil (carmustine), Cytarabine (Cytarabine), Gemcitabine (doxycycline), or doxycycline (doxycycline). The antitumor antibiotic may be Doxorubicin (Aclarubicin), Daunorubicin (Daunorubicin), Doxorubicin (Doxorubicin), Epirubicin (Epirubicin), Idarubicin (Idarubicin), Actinomycin (Actinomycin), Bleomycin (Bleomycin), Actinomycin d (dactinomycin), Mitomycin (Mitomycin), or Plicamycin (Plicamycin). Likewise, the topoisomerase inhibitor may be Camptothecin (Camptothecin), kexitecan (Cositecan), Belotecan (Belotecan), gemmacecan (Gimatecan), Irinotecan (Exatecan), Irinotecan (Irinotecan), Lurtotecan (Lurtotecan), ceritin (Silatecan), Topotecan (Topotecan), Rubitecan (Rubitecan), Etoposide (Etoposide) or Teniposide (Teniposide).
In addition, mitotic inhibitors suitable for use in the methods of the invention are Vinblastine (Vinblastine), Vincristine (vinristine), Vinflunine (Vinflunine), Vindesine (Vindesine), Vinorelbine (Vinorelbine), Cabazitaxel (Cabazitaxel), Docetaxel (Docetaxel), Larotaxel (Larotaxel), Ortataxel (Orotaxel), Paclitaxel (Paclitaxel), texaxel (Teetaxel) or Ixabepilone (Ixabepilone). Further, the enzyme inhibitor may be Tipifarnib (Tipifarnib), abbemaciclib (abemcilib), avocadi (Alvocidib), Palbociclib (Palbociclib), ribbociclib (Ribociclib), cericnib (selicinib), Bortezomib (Bortezomib), Carfilzomib (Carfilzomib), ixazomi (Ixazomib), Anagrelide (Anagrelide), thiazolecarboxamide nucleoside (Tiazofurin), Masoprocol (Masoprocol), nilapanib (Niraparib), Olaparib (Olaparib), lucapanib (Rucaparib), Belinostat (Belinostat), binostat (panostat), binopastat (panobist), mitosin (romisin), ritrin (rolistat (voraci), or etisoprocalcin.
According to some embodiments of the present disclosure, the chemotherapeutic agent used herein may be bleomycin, carboplatin, capecitabine, cisplatin, docetaxel, 5-FU, hydroxyurea, methotrexate, and paclitaxel. In a working embodiment of the present disclosure, the chemotherapeutic agent is cisplatin.
Immunotherapy refers to the killing of cancer cells by their immune system. More specifically, by activating the patient's own immune system, it is made to attack the malignant cells that cause the disease. In some embodiments, the immunotherapy is associated with the treatment of cancer using an immune checkpoint inhibitor as an immunotherapeutic. Exemplary immunotherapeutic agents include, but are not limited to, Pembrolizumab, Nivolumab, cimiciprimab (cemipimab), stivazumab (Spartalizumab), carpriclizumab (Camrelizumab), certilizumab (sintillizumab), tirezilizumab (tisslelizumab), terlipril mab (toriplalimab), Nivolumab, atelizumab (Atezolizumab), everolizumab (avelizumab), duruzumab (duvalumab), and Ipilimumab (Ipilimumab).
The effective amount of a therapeutic agent (e.g., a CHK1 inhibitor, a PI3K inhibitor, a chemotherapeutic agent, etc.) in the methods of the invention can vary depending on a number of factors, such as the particular condition being treated, the severity of the condition, the patient's personal parameters (including age, physical condition, size, sex, and weight), the duration of the treatment, the nature of concurrent therapy (if any), the particular route of administration, and the like, within the knowledge and expertise of the health care practitioner.
To elicit a therapeutic effect in a human, a therapeutic agent (e.g., a CHK1 inhibitor, a PI3K inhibitor, a chemotherapeutic agent, etc.) in the methods of the invention is administered to a human at a dose of about 10 μ g/kg to 50mg/kg (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 μ g/kg, or 1,2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50mg/kg) of body weight); preferably, the therapeutic agent in the methods of the invention is administered to the subject in an amount of about 50 μ g/kg to 10mg/kg body weight; more preferably, the therapeutic agent in the methods of the invention is administered to the subject at a dose of about 0.5mg/kg to about 5mg/kg of body weight. According to one working example, the therapeutic agent is the chemotherapeutic agent cisplatin, at a dose of about 0.5mg/kg body weight, to elicit a therapeutic effect (e.g., reduce tumor volume) in an individual. According to another working example, the therapeutic agent is the CHK1 inhibitor PF477736 in a dose of about 1 or 2mg/kg body weight to elicit a therapeutic effect in an individual. According to another working example, the therapeutic agent is the PI3K inhibitor BYL719 at a dose of about 2.5 or 5mg/kg body weight to elicit a therapeutic effect in an individual.
In the present methods of the invention, an effective amount of a therapeutic agent (e.g., CHK1 inhibitor, PI3K inhibitor, chemotherapeutic agent, etc.) can be administered in a single dose (e.g., a single intraperitoneal injection dose) or in multiple doses (e.g., multiple intraperitoneal injection doses). In certain embodiments, when multiple doses are administered to a subject, the frequency of administration of the multiple doses to the subject is three doses a day, two doses a day, one dose a day, five doses a week, four doses a week, three doses a week, two doses a week, one dose every other week, one dose a month, or one dose every other month. In certain embodiments, multiple doses are administered to the subject at a frequency of five doses per week. In certain embodiments, the multiple doses are administered to the subject at a frequency of four doses per week. In certain embodiments, the frequency of administration of multiple doses to a subject is two doses per week.
In certain embodiments, when multiple doses are administered to a subject, the duration between the first and last doses of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject. In certain embodiments, the duration between the first and last dose of the plurality of doses is three months, six months, or one year. In certain embodiments, the duration between the first and last dose of the plurality of doses is the lifetime of the individual. In a specific embodiment, the duration between the first and last dose of the plurality of doses is four weeks.
In the methods of the invention, therapeutic agents suitable for use in the methods of the invention may be administered by routes known to those skilled in the art, including oral, intracranial, intraspinal, intrathecal, intramedullary, intracerebroventricular, intravenous, intraarterial, intracardiac, intradermal, subcutaneous, transdermal, intraperitoneal, and intramuscular routes.
It is to be understood that the present disclosure also includes a method for predicting the likelihood that an individual having oral cancer will exhibit a beneficial response to a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor. The method comprises the following steps:
(a) measuring the expression level of a biomarker in a first and second sample obtained from the individual by qRT-PCR, respectively, to obtain a first expression level and a second expression level of the biomarker, wherein the biomarker is the CHEK1 gene, the PIK3CA gene, or the PIK3CD gene, and the first and second samples are a cancerous sample and a non-cancerous sample, respectively;
(b) determining a ratio of the first expression level to the second expression level; and
(c) determining the likelihood that the individual will exhibit a beneficial response to a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor based on the ratio determined in step (b),
wherein:
the individual may exhibit a beneficial response to a checkpoint kinase 1 inhibitor if the biomarker is the CHEK1 gene and the ratio is at least 1.7, or at least 2.4 if the biomarker is the PIK3CA gene and the ratio is at least 2.4; or the biomarker is the PIK3CD gene and the ratio determined in step (c) is at least 3.1, then the individual may exhibit a beneficial response to a phosphatidylinositol 3-kinase inhibitor.
It is to be understood that the present disclosure also includes kits for detecting a response to a kinase inhibitor in an individual having oral cancer. The kit comprises the following components in sequence number: 1 and 2, sequence number: 3 and 4 or sequence number: 5 and 6; wherein, the sequence number is: 1 and 2 are used for detecting the expression level of the CHEK1 gene; sequence number: 3 and 4 are used for detecting the expression level of the PIK3CA gene; sequence number: 5 and 6 are used for detecting the expression level of the PIK3CD gene; and the kinase inhibitor is selected from the group consisting of a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor. Alternatively, the kit further comprises the sequence of seq id no: 7 and 8, wherein the sequence numbers: the primer pairs of 7 and 8 were used to detect the expression level of the TBP gene.
The following examples are provided to illustrate certain aspects of the present invention and to assist those skilled in the art in carrying out the invention. These examples are in no way to be construed as limiting the scope of the invention in any way. Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present invention to its fullest extent. All publications cited herein are incorporated by reference in their entirety.
Examples
Materials and methods
1. Patient and clinical specimens
The study was enrolled in a group of patients and was approved by the institutional review board of the ChangG memorial Hospital. All participants had written informed consent prior to completion of sample collection. This group included 32 untreated OSCC patients with clinical characteristics as listed in table 1.
Table 132 clinical characteristics of OSCC patients
Figure RE-GDA0002633258190000231
Figure RE-GDA0002633258190000241
2. Cell line
OSCC cell lines including SAS (A), (B), (C) and (C) were used in the present study
Figure BDA0002492457090000242
64403TM) OEC-M1(CVCL _6782) and OC3(CVCL _ D859). SAS cells were maintained in Dulbecco's modified Eagle's Medium (DMEM; Gibco, USA) supplemented with 100U/ml penicillin and 100. mu.g/ml streptomycin (Gibco, USA) and 10% fetal bovine serum (FBS; Gibco, USA). OEC-M1 cells were maintained in Roswell Park Memorial center (Roswell Park Memori Institute)1640 medium (RPMI 1640; U.S.A. Thermo Fisher Scientific), OC3 cells were maintained in DMEM (U.S. Gibco) containing keratinocyte serum-free medium (KSFM; U.S. Gibco); both were supplemented with 10,000U/ml penicillin and 10,000. mu.g/ml streptomycin (U.S. Gibco) and 10% FBS (U.S. Gibco). All cell lines were maintained at 37 ℃ with 5% CO2Is in the humid air.
3. Drugs, chemicals and antibodies
The CHK1 inhibitors PF477736 (Sigma-Aldrich, USA), AZD7762 (Selleck Chemicals, Canada) and LY2606368 (MedChem Express, USA); and the PI3K inhibitors GDC-0941 (Cayman Chemical, USA), GSK1059615 (Cayman Chemical, USA) and BYL719 (Novartis Pharma AG, Switzerland) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) and stored at-20 ℃. Cisplatin (Sigma-Aldrich, USA) was dissolved in 1 XPBS and stored at 4 ℃. All antibodies used in the immunoblot assay were: anti-CHK 1, anti-p-CHK 1-Ser296, anti-AKT, anti-p-AKT-Ser 473, anti-Ki-67 (Cell Signaling Technology); anti-GAPDH (biowork, usa); HRP-conjugated goat anti-rabbit IgG (PerkinElmer, usa); and HRP conjugated goat anti-mouse IgG (PerkinElmer, usa).
RNA extraction and qRT-PCR
Total RNA from paired OSCC tumor tissue and surrounding normal tissue was extracted using TRIzol reagent (Gibco BRL) and mass and quantity confirmed using an Agilent 2100 bioanalyzer (Agilent Technologies, canada, santa clara). For qRT-PCR, first strand complementary DNA was synthesized from 1. mu.g total RNA using random hexamers (GeneDirex, Germany) and SuperScript III RT (Invitrogen, USA), and qRT-PCR analysis was performed using a MicroAmp Fast 96-well reaction plate (0.1mL) (Applied Biosystems, USA). The primers used in qRT-PCR are listed in Table 2. The average cycle threshold (Ct) for each replicate measurement was calculated. To determine the normalization value, tumor tissue and adjacent normal tissue are compared 2-ΔCtThe value is obtained. Δ Ct ═ CtTarget–CtTBP。
TABLE 2 qRT-PCR primers
Figure BDA0002492457090000251
Figure BDA0002492457090000261
5. Cell viability assay
For cell viability assays, approximately 5X 104Individual cells were seeded into each well of a 96-well plateAnd treated with vehicle control or inhibitor at the indicated concentrations for 48 hours in complete medium before performing MTT viability assays. The absorbance was read in a multi-well spectrophotometer (SpectraMax M2; Molecular Devices, USA) at 540 nm. All samples were replicated three times. For apoptosis assays, cells were plated in 6-well dishes and treated with vehicle control or various inhibitors at the indicated concentrations for 48 hours. Cells were then harvested, washed with PBS, and stained with 2 μ l FITC-conjugated annexin V antibody (Invitrogen, usa) and 50 μ g/ml propidium iodide (propidium iodide, Sigma-Aldrich, usa), followed by flow cytometry analysis (Attune NxT, Invitrogen, usa) and data analysis with FlowJo software (Tree Star, Inc.).
6. Immunoblotting
After treatment, the cells were lysed by incubation with RIPA lysis buffer (1% NP-40, 20mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM Na3VO4, 5mM EDTA (pH 8.0), 10% glycerol, PMSF, and 0.2% protease inhibitor) on ice for 15-30 minutes, followed by centrifugation at 15,000 Xg for 20 minutes at 4 ℃. Equal amounts of protein from each sample were fractionated by SDS-PAGE and transferred to PVDF membrane, which was incubated with the appropriate primary antibody. Proteins were detected with HRP conjugated secondary antibody and ECL substrate (PerkinElmer, usa).
7. Immunohistochemical staining
Immunohistochemical staining was performed using an immunohistochemical staining instrument (Bond, Leica BioSystems). Tissue sections were recovered using a Bond-max automated immunostaining instrument (Leica BioSystems) and stained with anti-Ki-67 antibody (mouse monoclonal antibody against Ki-67, Cell Signaling). A Polymer Detection system (Bond Polymer reference Detection, Leia BioSystems) was used to reduce non-specific staining. The tissue sections were developed with 3,3 '-diaminobenzidine (3, 3' -diaminobenzidine) reagent as the color reagent and hematoxylin as the counterstain.
8. Animal experiments
NOD/SCID mice and NOD. Cg-Prkdcscid II2rgtm1Wjl/SzJ (NSG) mice were housed in the animal facility of the university of ChangG. For cell line-derived xenografts derived from SAS cell linesderived xenograft, CDX) animal model, SAS cells (5X 10/ml) were prepared in serum-free DMEM6Individual cells). SAS cell suspensions (100 μ l) were injected subcutaneously into NOD/SCID mice (6-8 weeks) and tumors were established after 3-4 weeks. Once the xenograft reaches 300-3Mice were randomly assigned to treatment or control groups. PF477736 was used at a dose of 10 or 20mg/kg and was administered intraperitoneally 4 days per week. BYL719 was used at 25 or 50mg/kg dose, administered orally 5 days per week. Cisplatin was administered intraperitoneally at a dose of 5mg/kg 2 days per week. During the course of treatment (two to three weeks), tumor size was monitored twice weekly and tumor volume was calculated.
For the OSCC PDX animal model, three newly diagnosed patients were recruited and written consent was obtained according to the protocol approved by the institutional review board of the longhept commemorative hospital. Tumor explants of the patient were prepared from surgical specimens and transplanted into NSG mice within 2 hours. Briefly, fresh tumor tissue was rinsed in PBS (Gibco, USA) containing an antibiotic-antifungal solution, and then cut into small pieces (. about.1 mm)3). The left flank of each NSG mouse was subcutaneously transplanted with 50-100mg of tumor. When the size of the tumor reaches 400-500mm3At this time, animals were randomly divided into control groups and different treatment groups. The treatment for each drug was the same as that used in the SAS CDX model described above. For the combination therapy, cisplatin was used at a dose of 5mg/kg in part of the combination therapy with cisplatin and PF477736, administered intraperitoneally 2 days a week for 1 week; PF477736 was also administered at a dose of 20mg/kg intraperitoneally 4 days a week for 1 week; and in the combined treatment fraction of PF477736 and BYL719, PF477736 is administered at a dose of 20mg/kg, 4 days per week for 1 week intraperitoneally; while BYL719 was used at a dose of 50mg/kg, 5 days per week for 2 weeks. Tumors were measured with calipers and the volume was calculated as 1/2 x length x width2. In total, the response of the three PDX models to monotherapy or combination therapy was evaluated. All animal experiments were approved by the animal care committee of the university of longg-g.
9. Statistical analysis
Patient characteristics were stratified by various clinico-pathological factors and calculated by chi-square test. All statistical data are expressed as mean ± s.e.m. Comparisons between groups were performed on both groups by student's t-test or Mann-Whitney U-test. All statistical tests were performed using SPSS software version 12.0 (SPSS corporation, chicago, il), or Prism. A p-value <0.05 is considered statistically significant.
Example 1 genes differentially expressed in OSCC
To understand genes differentially expressed in OSCC, a comprehensive transcriptome study method RNA-seq was deployed to analyze matched tumor tissue and adjacent normal tissue from 32 OSCC patients (data not shown).
From the data obtained from the foregoing method, three genes were found: CHEK1, PIK3CA, and PIK3CD had differential expression in OSCC. The expression of these genes in the 32-paired OSCC population was further confirmed by qRT-PCR. As shown in fig. 1A, CHEK1 expression was increased in OSCC compared to surrounding normal tissue. Similar results were found in PIK3CA and PIK3CD (fig. 1B-1C). The gene expression changes in each paired OSCC sample were determined and set to define a threshold for high expression, with a threshold of 1.7 for CHEK1, 2.4 for PIK3CA, and 3.1 for PIK3CD (fig. 1D).
Taken together, these findings indicate that CHEK1, PIK3CA and PIK3CD are significantly upregulated in OSCC patients.
Example 2 inhibition of the effects of CHK1 and PI3K on OSCC in vitro and in vivo
2.1 inhibition of CHK1 Effect on OSCC in vitro
To evaluate the feasibility of inhibiting CHK1 and PI3K as antitumor approaches, several small molecule inhibitors of CHK1 and PI3K were tested in this example. FIG. 2A provides results of cell viability of specific CHK1 inhibitors (including PF477736, AZD7762, and LY2606368) on OSCC cell lines (including SAS and OEC-M1). All tested CHK1 inhibitors showed dose-dependent inhibition on SAS and OEC-M1 cells (fig. 2A). Half maximal Inhibitory Concentration (IC) of PF477736 in SAS cells50) In the range of 120 to 400nM, and OEC-M1 cells50The concentration was 10. mu.M (graph)2A) In that respect In addition, the colony forming ability of SAS and OEC-M1 cells was examined after treatment with PF477736, and it was shown that PF477736 significantly impaired the colony forming ability of both cells (FIG. 2B). Furthermore, PF477736 significantly exacerbated apoptosis in three OSCC cells including SAS, OC3 and OEC-M1 cells as assessed by annexin V and PI staining (FIG. 2C). At the molecular level, the biochemical role of three CHK1 inhibitors in SAS cells was characterized. DNA damage was first induced by UV irradiation and then CHK1 inhibitor was applied to the damaged cells. Thereafter, phosphorylation of CHK1 and total CHK1 at Ser296 was analyzed by immunoblotting (fig. 2D). The results show that CHK1 inhibitors (i.e., PF477736, AZD7762, and LY2606368) were able to specifically reduce phosphorylation of CHK1 after UV irradiation, indicating that CHK1 activity was severely inhibited after CHK1 inhibitor treatment.
To evaluate combination therapy with CHK1 inhibitors and current standard chemotherapy in the treatment of OSCC, PF477736 and cisplatin were chosen as examples as part of the CHK1 inhibitor and chemotherapy, respectively, of the combination therapy in this study. First, the IC of cisplatin was determined for treatment of three OSCC cells (SAS, OC3 and OEC-M1 cells)50In the range of 2.5 to 5. mu.M (FIG. 2E). Next, the efficacy of the combination therapy was evaluated by examining the ability to inhibit cell proliferation. As shown in figure 2F, the combination treatment of PF477736 and cisplatin synergistically blocked proliferation of SAS and OC3 cells compared to control or monotherapy.
Taken together, these results demonstrate that CHK1 inhibitors, alone or in combination with chemotherapy (e.g., cisplatin), are effective in OSCC to slow cell proliferation, drive cell cycle arrest, and trigger cell death.
2.2 inhibition of PI3K Effect on OSCC in vitro
In this study, the efficacy of PI3K inhibitors (e.g., BYL719, GDC-0941, and GSK1059615) on eliminating OSCC cell lines (including SAS, OEC-M1, and OC3) was examined. First, a cell proliferation assay was performed after treatment with PI3K inhibitors (BYL719, GDC-0941, and GSK1059615), and the results are shown in FIG. 3A. The results show that SAS and OEC-M1 treated with PI3K inhibitor compared to those treated with vehicle controlThe cells showed a clear reduction in proliferation (fig. 3A). IC of BYL719 in SAS, OC3 and OECM1 cell lines50The range was 8 to 20. mu.M (FIG. 3A). Another measure of anti-cancer activity was determined by studying the colony forming ability of SAS and OEC-M1 cells treated with BYL 719. As shown in fig. 3B, the colony forming ability of the treated cells showed dose-dependent impairment. With respect to apoptosis, OSCC cells treated with BYL719 showed a gradual increase in apoptosis with increasing BYL719 concentration (fig. 3C). The effect of PI3K inhibitors on the PI3K/AKT/mTOR signaling pathway was also investigated. According to fig. 3D, PI3K inhibitors (i.e., BYL719, GDC-0941, and GSK1059615) specifically inhibited phosphorylation of AKT after UV irradiation, thereby attenuating the PI3K/AKT/mTOR signaling pathway after treatment.
The efficacy of PI3K inhibitors in combination with CHK1 inhibitors as combination therapies for the treatment of cancer was evaluated in this study. As an example, PF477736 and BYL719 were used as CHK1 inhibitor and PI3K inhibitor, respectively, in the following experiments. Results of cell proliferation following administration of the combination therapy are provided in fig. 3E, showing that the combination therapy synergistically prevented proliferation in SAS cells compared to control or monotherapy. In addition, combination therapy significantly increased the number of apoptotic cells in SAS cells (FIG. 3F). Similar results were found in the inhibition of cell proliferation using combination therapy, with the CHK1 inhibitor and the PI3K inhibitor being AZD7762 and GDC-0941, respectively (fig. 3G).
Taken together, these findings indicate that PI3K inhibitors have good anti-cancer activity, and combination therapy using PI3K and CHK1 inhibitors in combination can help to treat cancer.
2.3 Effect of combination therapy on OSCC in vivo
The data described above have prompted the inventors to further investigate the antitumor activity of CHK1 and PI3K inhibitors alone or in combination in a preclinical model of OSCC in vivo.
For the purpose of concept verification, an OSCC in vivo model (hereinafter referred to as OSCC CDX) was prepared using SAS cells. Details of the preparation of OSCC CDX are as described in materials and methods. Treatment with PF477736 at a concentration of 10 or 20mg/kg hardly inhibited the growth of the tumors compared to the control group (fig. 4A, 10mg/kg vs. control, p 0.083; 20mg/kg vs. control, p 0.081). For BYL719, treatment at 50mg/kg dose significantly inhibited tumor growth compared to the control group (fig. 4B, 25mg/kg vs. control, p 0.508; 50mg/kg vs. control, p 0.011). Furthermore, co-treatment with PF477736(20mg/kg) and BYL719(50mg/kg) resulted in effective impairment of tumor growth (fig. 4C, combined treatment vs. control, p < 0.01). The data indicate that PF477736 in combination with BYL719 showed enhanced therapeutic effect; the tumor volume in the combination treatment group was much smaller. Combined treatment with PF477736 and BYL719 demonstrated superior efficacy in compromising tumor growth in the OSCC CDX model compared to the current in vivo standardized chemotherapy cisplatin (fig. 4D, 5mg/kg vs. control, p ═ 0.03) that could exhibit the ability to inhibit tumor growth.
Example 3 inhibition of the effects of CHK1 and PI3K on the OSCC PDX model
CHK1 and PI3K inhibitors were further evaluated as a means of controlling tumor growth in a more clinically relevant model, OSCC PDX model. Tumor tissues from three patients with stage IV OSCC (designated patient #1, patient #2, patient #3, respectively) were used to establish the PDX model. Table 3 summarizes the clinical data for these three patients. The genetic profile of three patients was characterized by RNA-seq and whole exon sequencing (data not shown), with patient #2 having TP53 and CDKN2A mutations and patient #3 having TP53 and PIK3CA mutations (data not shown). Also, patient #2 and patient #3 were noted to have a significant increase in components of the PI3K/AKT/mTOR signaling pathway (data not shown). Gene expression of PIK3CA was also confirmed in three patients by qRT-PCR, with the ratio of PIK3CA expression in patient #2 and patient #3 being higher than 2.4, respectively, but not in patient #1 (data not shown). After establishing the OSCC PDX model, mice were divided into vehicle control group, cis-platinum group (5mg/kg), CHK1 inhibitor PF477736(20mg/kg), and cisplatin plus PF477736 group (fig. 5A). The results show that cisplatin or PF477736 monotherapy can partially (but not significantly) inhibit the growth of PDX tumors. In contrast, combination therapy showed synergistic inhibition of tumor growth in all PDX mouse models (fig. 5A, combination therapy vs. control, p 0.0024 in patient #1PDX, 0.006 in patient #2PDX, 0.03 in patient #3 PDX).
The results of IHC staining of Ki-67, an indicator of cell proliferation, on xenograft tumor sections are provided in FIG. 5B. Combination therapy alone showed a significant reduction in the number of Ki-67 positive cells (fig. 5B).
TABLE 3 clinical data of three OSCC patients related to the current PDX model
Patient numbering # 1 #2 #3
Age (year of old) 56 47 60
Sex M M M
Period T 4A 4A 2
Stage N 0 2B 2B
Pathology of disease Good effect Medium and high grade Medium and high grade
General points of view IV IV IV
Drinking wine Y Y Y
Chewing betel nut Y Y Y
Smoke extraction device Y N Y
Location of a body part Buccal mucosa Tongue with tongue-like portion Buccal mucosa
The effect of combined treatment with CHK1 and PI3K inhibitors on treatment of different PDX models was investigated as follows (fig. 5C). BYL719(50mg/kg) monotherapy and PF477736(20mg/kg) plus BYL719(50mg/kg) combination therapy failed to inhibit tumor growth in patient #1PDX model (fig. 5C, top panel; BYL719 monotherapy v.control where p is 0.66; combination therapy v.control where p is 0.844). At the same time, BYL719(50mg/kg) monotherapy slightly inhibited tumor growth (50mg/kg vs. control, p ═ 0.16), while combination therapy of BYL719(50mg/kg) plus PF477736(20mg/kg) synergistically slowed tumor growth in patient #2PDX (fig. 5C, bottom panel, combination treatment vs. control, p ═ 0.036). Patient #2 with specific genetic profiles (higher CHEK1 and PIK3CA expression) had a higher response of PDX to BYL719 plus PF477736 combination therapy. Furthermore, as shown in FIG. 5D, Ki-67 positive cells were significantly reduced in BYL719 plus PF477736 combination therapy, indicating reduced cancer cell proliferation under this treatment. In addition, no significant weight loss was observed in any of the treatment groups in patient #2PDX model (fig. 5E).
In summary, the data provided in this disclosure indicate that inhibitors of CHK1, alone or in combination with chemotherapy (e.g., cisplatin), may be more effective in treating cancer patients with higher expression of CHEK1 alone. In addition, the PI3K inhibitor and the CHK1 inhibitor can be used as a potential anti-cancer treatment method for treating cancer patients with high expression of CHEK1 and PIK3CA/PIK3 CD. Patients with specific genetic characteristics as described herein may particularly benefit from personalized anti-cancer treatments as described in the present disclosure.
It should be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those skilled in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the present invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.
Sequence listing
<110> university of ChangG
Changgeng medical consortium legal person Linkou Changgeng Memorial Hospital
<120> method for treating oral cancer
<130> P4100-CN
<160> 8
<170> BiSSAP 1.3.6
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tcggtataat aatcgtgagc g 21
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<213> Artificial sequence
<220>
<223> synthetic sequence
<400> 2
acaggaccaa acatcaactg 20
<210> 3
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<213> Artificial sequence
<220>
<223> synthetic sequence
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acgatggaca actgtttca 19
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gtctttgtgc attcttggg 19
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gacatccagt atctcaagga c 21
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Claims (8)

1. A kit for determining the susceptibility of a subject suffering from oral cancer to a kinase inhibitor, comprising seq id no: 1 and 2, sequence number: 3 and 4, or sequence number: 5 and 6; the sequence numbers are as follows: 1 and 2 is used for detecting the expression level of the CHEK1 gene of the individual; the sequence numbers are as follows: 3 and 4 for detecting the expression level of the PIK3CA gene of the individual; the sequence numbers are as follows: 5 and 6 for detecting the expression level of the PIK3CD gene of the individual; and the kinase inhibitor is selected from the group consisting of: checkpoint kinase 1 inhibitors, and phosphatidylinositol 3-kinase inhibitors.
2. The kit of claim 1, further comprising the amino acid sequence of seq id no: 7 and 8, the sequence numbers: 7 and 8 is used for detecting the expression level of the TBP gene of the individual.
3. The kit of claim 1, wherein the oral cancer is oral squamous cell carcinoma.
4. The kit of claim 1, wherein the checkpoint kinase 1 inhibitor is selected from the group consisting of:
(S) -5- (3-fluorophenyl) -N- (piperidin-3-yl) -3-ureidothiophene-2-carboxamide;
4- (((3S) -1-azabicyclo (2.2.2) oct-3-yl) amino) -3- (1H-benzoimidazol-2-yl) -6-chloroquinolin-2 (1H) -one;
carrying out Labu color substitution;
porisin;
4- (2, 6-dichlorophenyl) -9-hydroxy-6- (3- (methylamino) propyl) pyrrolo [3,4-c ] carbazole-1, 3(2H,6H) -dione;
(R) -2-amino-2-cyclohexyl-N- (2- (1-methyl-1H-pyrazol-4-yl) -6-oxo-5, 6-dihydro-1H- [1,2] diazepino [4,5,6-cd ] indol-8-yl) acetamide;
(R) -6-bromo-3- (1-methyl-1H-pyrazol-4-yl) -5- (piperidin-3-yl) pyrazolo [1,5-a ] pyrimidin-7-amine;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) nicotinamide;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl isobutyramide;
(R) -N- (5-bromo-4- (3- (methylamino) piperidin-1-yl) -1H-pyrrolo [2,3-b ] pyridin-3-yl) nicotinamide;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-methylnicotinamide;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) cyclopropanecarboxamide;
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -3-methylbutanamide; and
(R) -N- (4- (3-aminopiperidin-1-yl) -5-bromo-1H-pyrrolo [2,3-b ] pyridin-3-yl) -2-cyclopropylacetamide.
5. The kit of claim 4, wherein the checkpoint kinase 1 inhibitor is:
(S) -5- (3-fluorophenyl) -N- (piperidin-3-yl) -3-ureidothiophene-2-carboxamide;
porisin; or
(R) -2-amino-2-cyclohexyl-N- (2- (1-methyl-1H-pyrazol-4-yl) -6-oxo-5, 6-dihydro-1H- [1,2] diazepino [4,5,6-cd ] indol-8-yl) acetamide.
6. The kit of claim 1, wherein the phosphatidylinositol 3-kinase inhibitor is selected from the group consisting of:
(2S) -N1- [5- (2-tert-butyl-4-thiazolyl) -4-methyl-2-thiazolyl ] pyrrolidine-1, 2-dicarboxamide;
(Z) -5- ((5- (4-fluoro-2-hydroxyphenyl) furan-2-yl) methylene) thiazolidine-2, 4-dione;
5- (2,2-difluoro-benzo [1,3] dioxolan-5-ylmethylene) -thiazolidine-2, 4-dione;
(R) -2- (1- (7-methyl-2-morpholinyl-4-oxo-4H-pyrido [1,2-a ] pyrimidin-9-yl) ethylamino) benzoic acid;
2-amino-N- [7-methoxy-8- (3-morpholinylpropoxy) -2,3-dihydroimidazo [1,2-c ] quinazoline;
crompernese;
8- (6-methoxypyridin-3-yl) -3-methyl-1- (4- (piperazin-1-yl) -3- (trifluoromethyl) phenyl) -1H-imidazo [4,5-c ] quinolin-2(3H) -one maleate;
buparville sodium salt;
abaciside;
(5E) -5- { [5- (4-fluorophenyl) furan-2-yl ] methylene } -1,3-thiazolidine-2, 4-dione;
5- (2-amino-8-fluoro- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) -N-tert-butylpyridin-3-sulfonamide;
vickers;
2- ((6-amino-9H-purin-9-yl) methyl) -5-methyl-3-o-tolylquinazolin-4(3H) -one;
esdalafil plug;
cerilide plug;
taselisaint;
a Pituity plug;
apixaglicen;
(Z) -5- ((4- (pyridin-4-yl) quinolin-6-yl) methylene) thiazolidine-2, 4-dione;
(ii) Omibacil;
2-methyl-1- (2-methyl-3- (trifluoromethyl) benzyl) -6-morpholinyl-1H-benzo [ d ] imidazole-4-carboxylic acid;
6- [5- [ (benzenesulfonyl) amino ] -3-pyridyl ] -imidazo [1,2-a ] pyridine-3-carboxylic acid, ethyl ester;
2-amino-N, N-dimethyl-5- (3- (2-methylpyridin-4-yl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) pyridine-3-sulfonamide;
2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one;
dartorii plug;
pirifoxine;
2-amino-8- [4- (2-hydroxyethoxy) cyclohexyl ] -6- (6-methoxypyridin-3-yl) -4-methylpyrido [2,3-d ] pyrimidin-7-one;
a Jedalie plug;
3- (4-morpholinopyrido [ 3', 2': 4,5] furo [3,2-d ] pyrimidin-2-yl) phenol;
2-methyl-5-nitro-2- [ (6-bromoimidazo [1,2-a ] pyridin-3-yl) methylene ] -1-methylhydrazide-benzenesulfonic acid;
n- (2,3-dihydro-7,8-dimethoxyimidazo [1,2-c ] quinazolin-5-yl) -3-pyridinecarboxamide;
2- [ (4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl ] -5-methyl-3- (2-methylphenyl) -4(3H) -quinazolinone;
2- [ [4-amino-3- (3-hydroxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl ] methyl ] -5-methyl-3- (2-methylphenyl) -4(3H) -quinazolinone;
1- (4- (3-ethyl-7-morpholinyl-3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-yl) phenyl) -3- (4- (4-methylpiperazine-1-carbonyl) phenyl) urea;
sorbierite;
(S) -2- (2- (2-methylindolin-1-yl) -2-oxoethyl) -6-morpholinylpyrimidin-4 (3H) -one;
a pita plug;
(2S) -2- [ [ (2S) -3-carboxy-2- [ [2- [ [ (2S) -5- (diaminomethyleneamino) -2- [ [4-oxo-4- [ [4- (4-oxo-8-phenylchromen-2-yl) morpholin-4-ium-4-yl ] methoxy ] butanoyl ] amino ] pentanoyl ] amino ] acetyl ] amino ] propanoyl ] amino ] -3-hydroxypropanoate;
3- (2,4-diaminopteridin-6-yl) phenol;
lapariella plug;
5- (9-isopropyl-8-methyl-2-morpholinyl-9H-purin-6-yl) pyrimidin-2-amine;
vodarglia; and
4, 4' - (6- (2- (difluoromethyl) -1H-benzo [ d ] imidazol-1-yl) -1,3,5-triazine-2,4-diyl) dimorpholine.
7. The kit of claim 6, wherein the phosphatidylinositol 3-kinase inhibitor is:
abaciside;
a Pituity plug; or
(Z) -5- ((4- (pyridin-4-yl) quinolin-6-yl) methylene) thiazolidine-2, 4-dione.
8. The kit of claim 1, wherein the subject is a human.
CN202010409087.3A 2020-05-14 2020-05-14 Method for treating oral cancer Pending CN113667747A (en)

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US20160317517A1 (en) * 2013-12-20 2016-11-03 Biomed Valley Discoveries, Inc. Cancer treatments using combinations of pi3k/akt pathway and erk inhibitors
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