Cancer therapeutic targets and their applications

Abstract

This invention relates to the use of cytarabine, mitochondrial metabolism inhibitors, and agents that downregulate the expression or activity of CDA in the treatment of cancer.

Description

Technical Field

[0001] This invention belongs to the field of cancer treatment, and relates to cancer treatment targets and their uses, specifically a new method for treating the evolution of B-cell acute lymphoblastic leukemia and chemotherapy resistance. Background Technology

[0002] B-cell acute lymphoblastic leukemia (B-ALL) is a severe malignant hematopoietic disorder characterized by clonal expansion of hematopoietic stem / progenitor cells, commonly seen in children and adolescents. Several treatment regimens have proven effective in treating B-ALL, including chemotherapy, bone marrow transplantation, and CAR-T therapy. However, in the case of CAR-T therapy, the effectiveness of current strategies is hampered by drug resistance, lack of MHC-matched donor HSCs, patient unresponsiveness, or induced toxicity. Although chemotherapy is considered one of the most effective treatments for B-ALL, 20% of patients relapse after treatment with cytarabine (Ara-C), anthracyclines, or other chemotherapeutic agents. Growing evidence suggests that a small subset of leukemia cells, called leukemia initiating cells (LICs), may contribute to drug resistance or leukemia relapse. For example, the immunophenotype CD34+CD19+ LICs may be closely associated with leukemia progression and drug resistance in human B-ALL. However, the mechanisms leading to chemotherapy resistance remain unclear.

[0003] Recent studies have revealed that metabolism plays a crucial role in the development and progression of many different types of cancer, including hematologic malignancies and solid tumors. For example, mutations in isocitrate dehydrogenase 1 / 2 (IDH1 / 2) can effectively induce various cancers, such as acute myeloid leukemia, glioblastoma, and colon cancer. Acute / chronic myeloid leukemia primarily utilizes glycolysis as an energy source; fructose metabolism enhances the proliferation of Ph+-B-ALL cells; and fasting can effectively induce B-ALL differentiation in both early and late stages of the disease. However, whether cellular metabolism in B-ALL is related to drug resistance formation remains unclear.

[0004] Currently, drug resistance in B-ALL cells is believed to be caused by alterations in many endogenous factors (such as transcription factors or epigenetic modifications) and exogenous factors (such as the tissue microenvironment). For example, mutations in the transcription factor IKZF1 lead to a significant decrease in the sensitivity of B-ALL cells to tyrosine inhibitor therapy; downregulation of TWIST2 also results in resistance to the chemotherapeutic drugs etoposide, daunorubicin, and dexamethasone. Furthermore, the bone marrow (BM) microenvironment has been found to play a crucial role in leukemia development and chemotherapy resistance. The remodeled leukemia microenvironment may contain different types of cells (such as endothelial cells, osteoblasts, and mesenchymal stem cells) and their secreted proteins / cytokines / growth factors (such as SDF1, IL3, IL6, and hyaluronic acid), which may differ from those secreted under physiological conditions to enhance leukemia cell expansion or chemotherapy resistance. Although most recent research has focused on the microenvironmental components in the development and drug resistance process of acute myeloid leukemia (AML), some evidence suggests that microenvironmental factors also support the initiation and drug resistance of B-ALL. For example, GDF15 secreted by Nestin+ cells remodels the BM microenvironment and leads to chemotherapy resistance after Ara-C treatment. However, which specific cellular metabolic pathways lead to drug resistance in B-ALL cells requires further investigation.

[0005] The field still needs to find the mechanisms and therapeutic targets of Ara-C resistance, as well as methods for efficiently screening drugs targeting therapeutic targets. Summary of the Invention

[0006] The first aspect of this invention provides the use of mitochondrial metabolism inhibitors in the preparation of medicaments for treating cancer.

[0007] In one or more embodiments, the cancer is one in which the tricarboxylic acid cycle (TCA) or electron transport chain (ETC) is enhanced.

[0008] In one or more embodiments, the cancer is a cancer in which the expression or activity of PDHX and / or CREB is increased.

[0009] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, and mixed-cell leukemia.

[0010] In one or more embodiments, the cancer is B-cell acute lymphoblastic leukemia, preferably B-cell acute lymphoblastic leukemia in which the expression or activity of PDHX and / or CREB is increased.

[0011] In one or more embodiments, mitochondrial metabolic inhibitors include agents that weaken the tricarboxylic acid cycle (TCA) or electron transport chain (ETC).

[0012] In one or more embodiments, mitochondrial metabolism inhibitors include agents that downregulate the expression or activity of PDHX and / or CREB.

[0013] In one or more embodiments, the agents that downregulate the expression or activity of PDHX and / or CREB include:

[0014] Antibodies or ligands that specifically bind to PDHX and / or CREB; or

[0015] Repressive molecules that specifically interfere with the transcription and / or expression of PDHX and / or CREB genes.

[0016] In one or more embodiments, the antibody that specifically binds to PDHX and / or CREB is a polyclonal antibody or a monoclonal antibody.

[0017] In one or more embodiments, the repressor molecules target the PDHX and / or CREB genes or their transcripts. The sequences of the PDHX gene or its transcripts are shown as gene ID: 8050 or 27402; the sequences of the CREB gene or its transcripts are shown as gene ID: 1385 or 12912.

[0018] In one or more embodiments, the repressor molecule targets the 3'UTR or CDS region of the PDHX and / or CREB gene transcripts.

[0019] In one or more embodiments, the repressor molecule is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNAs, siRNAs, shRNAs, RNAi, dsRNAs, sgRNAs, or combinations thereof, and (2) nucleic acid constructs capable of expressing or forming (1). Preferably, the repressor molecule is an shRNA or construct that targets the PDHX and / or CREB genes or their transcripts.

[0020] In one or more embodiments, the inhibitory molecule is a reagent, such as sgRNA, used to knock down or knock out PDHX and / or CREB using a technology selected from ZFN, TALEN, and CRISPR. In one or more embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

[0021] In one or more embodiments, the mitochondrial metabolism inhibitor may also be selected from venetoclax, metformin, berberine, CREB inhibitor 666-15, or a pharmaceutically acceptable salt, isomer, racemate, solvate, or prodrug thereof.

[0022] The first aspect of the present invention also provides a method for treating cancer, the method comprising: downregulating the mitochondrial metabolic intensity of cancer cells.

[0023] In one or more embodiments, the method includes administering an agent to a patient in need that weakens the tricarboxylic acid cycle (TCA) or electron transport chain (ETC).

[0024] In one or more embodiments, the method includes administering to a patient in need an agent that downregulates the expression or activity of PDHX and / or CREB.

[0025] In one or more embodiments, the agent that downregulates the expression or activity of PDHX and / or CREB is: an antibody or ligand that specifically binds to PDHX and / or CREB; or an inhibitory molecule that specifically interferes with the transcription and / or expression of the PDHX and / or CREB genes.

[0026] In one or more embodiments, the mitochondrial metabolism inhibitor is selected from venetoclax, metformin, berberine, CREB inhibitor 666-15, or a pharmaceutically acceptable salt, isomer, racemate, solvate, or prodrug thereof.

[0027] In one or more embodiments, the cancer is a cancer in which the expression or activity of PDHX and / or CREB is increased.

[0028] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, and mixed-cell leukemia.

[0029] In one or more embodiments, the cancer is B-cell acute lymphoblastic leukemia, preferably B-cell acute lymphoblastic leukemia in which the expression or activity of PDHX and / or CREB is increased.

[0030] In one or more embodiments, other features of the method are as described in any of the embodiments of the first aspect herein.

[0031] The first aspect of the present invention also provides a substance that reduces the expression or activity of PDHX and / or CREB, said substance having a structure represented by Formula I:

[0032] Seq 正向 -X-Seq 反向 Formula I,

[0033] In Equation I, Seq 正向 To identify polynucleotides encoding PDHX and / or CREB sequences, Seq 反向 To be with Seq 正向Reverse complementary polynucleotides;

[0034] X is the interval sequence located between the forward and reverse directions of Seq, and the interval sequence is related to Seq. 正向 and Seq 反向 They are not complementary.

[0035] In one or more implementations, Seq 正向 The length is 5-20bp, preferably 8-15bp.

[0036] In one or more implementations, Seq 正向 It contains any of the sequences shown in SEQ ID NO:1-9.

[0037] In one or more embodiments, X includes SEQ ID NO:16.

[0038] The first aspect of the invention also provides a pharmaceutical composition comprising a mitochondrial metabolism inhibitor as described in any embodiment of the first aspect herein and a pharmaceutically acceptable excipient.

[0039] In one or more embodiments, the mitochondrial metabolism inhibitor is a substance that reduces the expression or activity of PDHX and / or CREB as described in any of the embodiments herein.

[0040] A second aspect of the present invention provides the use of reagents for detecting mitochondrial metabolic intensity in the preparation of cancer diagnostic kits or kits for identifying metabolic and functional subpopulations of cancer cells.

[0041] In one or more embodiments, the reagents for detecting mitochondrial metabolism include reagents for detecting the strength of the tricarboxylic acid cycle (TCA) or the electron transport chain (ETC).

[0042] In one or more embodiments, the reagents for detecting mitochondrial metabolism include reagents for detecting the expression or activity of PDHX and / or CREB. These include: (1) primers or probes targeting PDHX and / or CREB or their transcripts, or (2) antibodies or ligands that specifically bind to PDHX and / or CREB.

[0043] In one or more embodiments, the kit further comprises reagents required for RT-PCR, such as reverse transcriptase, RNA extraction reagents, nucleic acid polymerase, dNTPs, PCR buffer, etc.

[0044] In one or more embodiments, the kit further comprises reagents required for Northern blotting, such as RNA extraction reagents, ribonuclease inhibitors, Northern buffer, etc.

[0045] In one or more embodiments, the kit further comprises reagents required by Western blotting, such as protein extraction reagents, acrylamide, guanidine isothiocyanate, Tris, SDS, TEMED, etc.

[0046] In one or more embodiments, the sequence of the PDHX gene or its transcript is as shown in gene ID: 8050 or 27402.

[0047] In one or more embodiments, the sequence of the PDHX protein is as shown by protein ID: NP_003468 or AAH61231.

[0048] In one or more embodiments, the sequence of the CREB gene or its transcript is as shown in gene ID: 1385 or 12912.

[0049] In one or more embodiments, the sequence of the CREB protein is as shown in protein ID: AAQ24858.1 or AAB64015.1.

[0050] A third aspect of the present invention provides the use of cytarabine and reagents that inhibit mitochondrial metabolism and / or downregulate the expression or activity of CDA in the preparation of medicaments for treating cancer.

[0051] In one or more embodiments, the cancer is one in which the tricarboxylic acid cycle (TCA) or electron transport chain (ETC) is enhanced.

[0052] In one or more embodiments, the cancer is a cancer in which the expression or activity of PDHX and / or CREB and / or CDA is increased.

[0053] In one or more embodiments, the cancer is a cancer in which the expression or activity of CDA is increased.

[0054] In one or more embodiments, the cancer is a cancer that is resistant to cytarabine.

[0055] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, and mixed-cell leukemia.

[0056] In one or more embodiments, the cancer is B-cell acute lymphoblastic leukemia, preferably B-cell acute lymphoblastic leukemia in which the expression or activity of PDHX and / or CREB and / or CDA is increased.

[0057] In one or more embodiments, mitochondrial metabolic inhibitors include agents that weaken the tricarboxylic acid cycle (TCA) or electron transport chain (ETC).

[0058] In one or more embodiments, mitochondrial metabolism inhibitors include agents that downregulate the expression or activity of PDHX and / or CREB.

[0059] In one or more embodiments, the agents that downregulate the expression or activity of PDHX and / or CREB include: antibodies or ligands that specifically bind to PDHX and / or CREB; or inhibitory molecules that specifically interfere with the transcription and / or expression of PDHX and / or CREB genes.

[0060] In one or more embodiments, the antibody that specifically binds to PDHX and / or CREB is a polyclonal antibody or a monoclonal antibody.

[0061] In one or more embodiments, the repressive molecule that specifically interferes with the transcription and / or expression of the PDHX and / or CREB genes targets the PDHX and / or CREB genes or their transcripts. The sequences of the PDHX gene or its transcripts are shown as gene ID: 8050 or 27402; the sequences of the CREB gene or its transcripts are shown as gene ID: 1385 or 12912.

[0062] In one or more embodiments, the repressor molecules that specifically interfere with the transcription and / or expression of PDHX and / or CREB genes target the 3'UTR or CDS region of the PDHX and / or CREB gene transcripts.

[0063] In one or more embodiments, the repressor molecule that specifically interferes with the transcription and / or expression of the PDHX and / or CREB genes is selected from the group consisting of (1) small molecule compounds, antisense nucleic acids, microRNAs, siRNAs, shRNAs, RNAi, dsRNAs, sgRNAs, or combinations thereof, and (2) nucleic acid constructs capable of expressing or forming (1). Preferably, the repressor molecule is an shRNA or construct that targets the PDHX and / or CREB genes or their transcripts.

[0064] In one or more embodiments, the repressor molecule that specifically interferes with the transcription and / or expression of the PDHX and / or CREB genes is an agent that knocks down or knocks out PDHX and / or CREB using a technology selected from ZFN, TALEN, and CRISPR, such as sgRNA.

[0065] In one or more embodiments, the mitochondrial metabolism inhibitor is selected from venetoclax, metformin, berberine, CREB inhibitor 666-15, or a pharmaceutically acceptable salt, isomer, racemate, solvate, or prodrug thereof.

[0066] In one or more embodiments, the agents that downregulate the expression or activity of CDA include: antibodies or ligands that specifically bind to CDA; or inhibitory molecules that specifically interfere with the transcription and / or expression of the CDA gene.

[0067] In one or more embodiments, the antibody that specifically binds to CDA is a polyclonal antibody or a monoclonal antibody.

[0068] In one or more embodiments, the repressive molecule that specifically interferes with CDA gene transcription and / or expression targets the CDA gene or its transcript. The sequence of the CDA gene or its transcript is shown as gene ID: 978 or 72269.

[0069] In one or more embodiments, the repressor molecule that specifically interferes with CDA gene transcription and / or expression targets the 3'UTR or CDS region of the CDA gene transcript.

[0070] In one or more embodiments, the repressor molecule that specifically interferes with CDA gene transcription and / or expression is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNAs, siRNAs, shRNAs, RNAi, dsRNAs, sgRNAs, or combinations thereof, and (2) nucleic acid constructs capable of expressing or forming (1). Preferably, the repressor molecule is an shRNA or construct that targets the CDA gene or its transcripts.

[0071] In one or more embodiments, the repressor molecule that specifically interferes with CDA gene transcription and / or expression is a reagent that knocks down or knocks out CDA using a technique selected from ZFN, TALEN, and CRISPR, such as sgRNA.

[0072] In one or more embodiments, the repressor molecule (including a repressor molecule that specifically interferes with the transcription and / or expression of the PDHX, CREB, or CDA gene) is shRNA or a nucleic acid construct thereof, the construct containing the structure shown in Formula I:

[0073] Seq 正向 -X-Seq 反向 Formula I,

[0074] In Equation I, Seq 正向 To identify polynucleotides in the PDHX, CREB, or CDA genes or their transcripts, Seq 反向 To be with Seq 正向 Reverse complementary polynucleotides;

[0075] X is the interval sequence located between the forward and reverse directions of Seq, and the interval sequence is related to Seq. 正向 and Seq反向 They are not complementary.

[0076] In one or more implementations, Seq 正向 The length is 5-20bp, preferably 8-15bp.

[0077] In one or more implementations, Seq 正向 It contains any of the sequences shown in SEQ ID NO:1-15.

[0078] In one or more embodiments, X includes SEQ ID NO:16.

[0079] A third aspect of the present invention also provides a method for treating cancer, the method comprising: administering cytarabine, and mitochondrial metabolism inhibitors and / or agents that downregulate the expression or activity of CDA to a patient in need.

[0080] In one or more embodiments, the mitochondrial metabolic inhibitor is an agent that weakens the tricarboxylic acid cycle (TCA) or electron transport chain (ETC).

[0081] In one or more embodiments, the mitochondrial metabolism inhibitor is an agent that downregulates the expression or activity of PDHX and / or CREB.

[0082] In one or more embodiments, the agent that downregulates the expression or activity of PDHX and / or CREB is: an antibody or ligand that specifically binds to PDHX and / or CREB; or an inhibitory molecule that specifically interferes with the transcription and / or expression of the PDHX and / or CREB genes.

[0083] In one or more embodiments, the mitochondrial metabolism inhibitor is selected from venetoclax, metformin, berberine, CREB inhibitor 666-15, or a pharmaceutically acceptable salt, isomer, racemate, solvate, or prodrug thereof.

[0084] In one or more embodiments, the agents that downregulate the expression or activity of CDA include: antibodies or ligands that specifically bind to CDA; or inhibitory molecules that specifically interfere with the transcription and / or expression of the CDA gene.

[0085] In one or more embodiments, the cancer is one in which the tricarboxylic acid cycle (TCA) or electron transport chain (ETC) is enhanced.

[0086] In one or more embodiments, the cancer is a cancer in which the expression or activity of PDHX and / or CREB and / or CDA is increased.

[0087] In one or more embodiments, the cancer is a cancer in which the expression or activity of CDA is increased.

[0088] In one or more embodiments, the cancer is a cancer that is resistant to cytarabine.

[0089] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, and mixed-cell leukemia.

[0090] In one or more embodiments, the cancer is B-cell acute lymphoblastic leukemia, preferably B-cell acute lymphoblastic leukemia in which the expression or activity of PDHX and / or CREB and / or CDA is increased.

[0091] In one or more embodiments, other features of the method are as described in any of the embodiments in the third aspect of this document.

[0092] In one or more embodiments, the repressor molecule (including a repressor molecule that specifically interferes with the transcription and / or expression of the PDHX, CREB, or CDA gene) is shRNA or a nucleic acid construct thereof, the construct containing the structure shown in Formula I:

[0093] Seq 正向 -X-Seq 反向 Formula I,

[0094] In Equation I, Seq 正向 To identify polynucleotides in the PDHX, CREB, or CDA genes or their transcripts, Seq 反向 To be with Seq 正向 Reverse complementary polynucleotides;

[0095] X is the interval sequence located between the forward and reverse directions of Seq, and the interval sequence is related to Seq. 正向 and Seq 反向 They are not complementary.

[0096] In one or more implementations, Seq 正向 The length is 5-20bp, preferably 8-15bp.

[0097] In one or more implementations, Seq 正向 It contains any of the sequences shown in SEQ ID NO:1-15.

[0098] In one or more embodiments, X includes SEQ ID NO:16.

[0099] A third aspect of the invention also provides a substance that reduces the expression or activity of CDA, said substance having the structure shown in Formula I:

[0100] Seq forward - X-Seq reverse I,

[0101] In Equation I, the forward direction of Seq represents the polynucleotide that recognizes the CDA coding sequence, and the reverse direction of Seq represents the polynucleotide that is complementary to the forward and reverse directions of Seq.

[0102] X is an interval sequence located between the forward and reverse sides of Seq, and the interval sequence is not complementary to the forward and reverse sides of Seq.

[0103] In one or more embodiments, the forward length of the Seq is 5-20 bp, preferably 8-15 bp.

[0104] In one or more embodiments, the Seq forward direction comprises any of the sequences shown in SEQ ID NO:10-15.

[0105] In one or more embodiments, X includes SEQ ID NO:16.

[0106] The third aspect of the present invention also provides a pharmaceutical composition comprising: (1) cytarabine, (2) a mitochondrial metabolism inhibitor and / or a reagent that downregulates the expression or activity of CDA, and (3) a pharmaceutically acceptable excipient.

[0107] In one or more embodiments, the pharmaceutical composition is used to treat cancer. The cancer is one in which the tricarboxylic acid cycle (TCA) or electron transport chain (ETC) is enhanced.

[0108] In one or more embodiments, the cancer is a cancer in which the expression or activity of PDHX and / or CREB and / or CDA is increased.

[0109] In one or more embodiments, the cancer is a cancer in which the expression or activity of CDA is increased.

[0110] In one or more embodiments, the cancer is a cancer that is resistant to cytarabine.

[0111] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, and mixed-cell leukemia.

[0112] In one or more embodiments, the cancer is B-cell acute lymphoblastic leukemia, preferably B-cell acute lymphoblastic leukemia in which the expression or activity of PDHX and / or CREB and / or CDA is increased.

[0113] In one or more embodiments, mitochondrial metabolic inhibitors include agents that weaken the tricarboxylic acid cycle (TCA) or electron transport chain (ETC).

[0114] In one or more embodiments, mitochondrial metabolism inhibitors include agents that downregulate the expression or activity of PDHX and / or CREB.

[0115] In one or more embodiments, the agents that downregulate the expression or activity of PDHX and / or CREB include: antibodies or ligands that specifically bind to PDHX and / or CREB; or inhibitory molecules that specifically interfere with the transcription and / or expression of PDHX and / or CREB genes.

[0116] In one or more embodiments, the antibody that specifically binds to PDHX and / or CREB is a polyclonal antibody or a monoclonal antibody.

[0117] In one or more embodiments, the repressive molecule that specifically interferes with the transcription and / or expression of the PDHX and / or CREB genes targets the PDHX and / or CREB genes or their transcripts. The sequences of the PDHX gene or its transcripts are shown as gene ID: 8050 or 27402; the sequences of the CREB gene or its transcripts are shown as gene ID: 1385 or 12912.

[0118] In one or more embodiments, the repressor molecules that specifically interfere with the transcription and / or expression of PDHX and / or CREB genes target the 3'UTR or CDS region of the PDHX and / or CREB gene transcripts.

[0119] In one or more embodiments, the inhibitory molecules that specifically interfere with the transcription and / or expression of the PDHX and / or CREB genes are selected from the group consisting of (1) small molecule compounds, antisense nucleic acids, microRNAs, siRNAs, shRNAs, RNAi, dsRNAs, sgRNAs, or combinations thereof, and (2) nucleic acid constructs that can express or form (1).

[0120] In one or more embodiments, the repressor molecule that specifically interferes with the transcription and / or expression of the PDHX and / or CREB genes is an agent, such as sgRNA, that knocks down or knocks out PDHX and / or CREB using a technology selected from ZFN, TALEN, and CRISPR. In one or more embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

[0121] In one or more embodiments, mitochondrial metabolism inhibitors venetoclax, metformin, and berberine, CREB inhibitor 666-15, or pharmaceutically acceptable salts, isomers, racemates, solvates, or prodrugs thereof.

[0122] In one or more embodiments, the agents that downregulate the expression or activity of CDA include: antibodies or ligands that specifically bind to CDA; or inhibitory molecules that specifically interfere with the transcription and / or expression of the CDA gene.

[0123] In one or more embodiments, the antibody that specifically binds to CDA is a polyclonal antibody or a monoclonal antibody.

[0124] In one or more embodiments, the repressive molecule that specifically interferes with CDA gene transcription and / or expression targets the CDA gene or its transcript. The sequence of the CDA gene or its transcript is shown as gene ID: 978 or 72269.

[0125] In one or more embodiments, the repressor molecule that specifically interferes with CDA gene transcription and / or expression targets the 3'UTR or CDS region of the CDA gene transcript.

[0126] In one or more embodiments, the repressor molecule that specifically interferes with CDA gene transcription and / or expression is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNAs, siRNAs, shRNAs, RNAi, dsRNAs, sgRNAs, or combinations thereof, and (2) nucleic acid constructs capable of expressing or forming (1). Preferably, the repressor molecule is an shRNA or construct that targets the CDA gene or its transcripts.

[0127] In one or more embodiments, the repressor molecule that specifically interferes with CDA gene transcription and / or expression is a reagent, such as sgRNA, that knocks down or knocks out CDA using a technology selected from ZFN, TALEN, and CRISPR. In one or more embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

[0128] In one or more embodiments, the repressor molecule (including a repressor molecule that specifically interferes with the transcription and / or expression of the PDHX, CREB, or CDA gene) is shRNA or a nucleic acid construct thereof, the construct containing the structure shown in Formula I:

[0129] Seq 正向 -X-Seq 反向 Formula I,

[0130] In Equation I, Seq 正向 To identify polynucleotides in the PDHX, CREB, or CDA genes or their transcripts, Seq 反向 To be with Seq 正向 Reverse complementary polynucleotides;

[0131] X is the interval sequence located between the forward and reverse directions of Seq, and the interval sequence is related to Seq. 正向 and Seq 反向 They are not complementary.

[0132] In one or more implementations, Seq 正向 The length is 5-20bp, preferably 8-15bp.

[0133] In one or more implementations, Seq 正向 It contains any of the sequences shown in SEQ ID NO:1-15.

[0134] In one or more embodiments, X includes SEQ ID NO:16.

[0135] The fourth aspect of the present invention provides the use of the probe shown in any one of SEQ ID NO:23-28 in the preparation of a kit for distinguishing metabolic and functional subsets of cancer cells.

[0136] In one or more embodiments, the probe is used to introduce cancer cells or express them therein, and the fluorescence intensity or fluorescence intensity ratio of the probe is detected, particularly the ratio of fluorescence intensity generated by excitation at 405nm-420nm to fluorescence intensity generated by excitation at 485nm-488nm.

[0137] In one or more embodiments, the fluorescence generated by excitation at 405nm-420nm is the fluorescence generated at 528nm by excitation at 405nm-420nm.

[0138] In one or more embodiments, the fluorescence generated by excitation at 485nm-488nm is the fluorescence generated at 528nm by excitation at 405nm-420nm.

[0139] In one or more embodiments, the probe is preferably as shown in SEQ ID NO:27.

[0140] In one or more embodiments, the smaller the ratio of fluorescence intensity, the stronger the metabolism of the tricarboxylic acid cycle and / or oxidative phosphorylation pathway in the cancer cells, the stronger their tumorigenic capacity, and the shorter the patient survival.

[0141] In one or more embodiments, when the fluorescence intensity ratio is less than or equal to about 1.0 (e.g., less than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), the cancer cells exhibit strong metabolism of the tricarboxylic acid cycle and / or oxidative phosphorylation pathway, strong tumorigenicity, and short patient survival; when the fluorescence intensity ratio is greater than or equal to about 1.0 (e.g., greater than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), the cancer cells exhibit weak metabolism of the tricarboxylic acid cycle and / or oxidative phosphorylation pathway, weak tumorigenicity, and long patient survival.

[0142] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, preferably B-cell acute lymphoblastic leukemia.

[0143] The fourth aspect of the present invention also provides a method for distinguishing metabolic and functional subpopulations of cancer cells, comprising introducing or expressing a probe shown in any one of SEQ ID NO:23-28 in cancer cells and detecting the fluorescence intensity or fluorescence intensity ratio of the probe, particularly the ratio of fluorescence intensity generated by excitation at 405nm-420nm to fluorescence intensity generated by excitation at 485nm-488nm.

[0144] In one or more embodiments, the fluorescence generated by excitation at 405nm-420nm is the fluorescence generated at 528nm by excitation at 405nm-420nm.

[0145] In one or more embodiments, the fluorescence generated by excitation at 485nm-488nm is the fluorescence generated at 528nm by excitation at 405nm-420nm.

[0146] In one or more embodiments, the probe is preferably as shown in SEQ ID NO:27.

[0147] In one or more embodiments, the smaller the ratio of fluorescence intensity, the stronger the metabolism of the tricarboxylic acid cycle and / or oxidative phosphorylation pathway in the cancer cells, the stronger their tumorigenic capacity, and the shorter the patient survival.

[0148] In one or more embodiments, when the fluorescence intensity ratio is less than or equal to about 1.0 (e.g., less than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), the cancer cells exhibit strong metabolism of the tricarboxylic acid cycle and / or oxidative phosphorylation pathway, strong tumorigenicity, and short patient survival; when the fluorescence intensity ratio is greater than or equal to about 1.0 (e.g., greater than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), the cancer cells exhibit weak metabolism of the tricarboxylic acid cycle and / or oxidative phosphorylation pathway, weak tumorigenicity, and long patient survival.

[0149] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, preferably B-cell acute lymphoblastic leukemia.

[0150] The fourth aspect of the present invention also provides a method for selecting a treatment plan, comprising:

[0151] (1) Introduce or express the probe shown in any one of SEQ ID NO:23-28 in cancer cells and detect the fluorescence intensity or fluorescence intensity ratio of the probe, especially the ratio of the fluorescence intensity generated by excitation at 405nm-420nm to the fluorescence intensity generated by excitation at 485nm-488nm.

[0152] (2) When the ratio of fluorescence intensity is less than or equal to about 1.0 (e.g., less than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), a pharmaceutical composition comprising: (1) cytarabine, (2) a mitochondrial metabolism inhibitor and / or a reagent that downregulates the expression or activity of CDA, and (3) a pharmaceutically acceptable excipient is selected.

[0153] In one or more embodiments, the fluorescence generated by excitation at 405nm-420nm is the fluorescence generated at 528nm by excitation at 405nm-420nm.

[0154] In one or more embodiments, the fluorescence generated by excitation at 485nm-488nm is the fluorescence generated at 528nm by excitation at 405nm-420nm.

[0155] In one or more embodiments, the probe is preferably as shown in SEQ ID NO:27.

[0156] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, preferably B-cell acute lymphoblastic leukemia.

[0157] In one or more embodiments, the pharmaceutical composition is as described in any embodiment of the third aspect herein.

[0158] A fourth aspect of the present invention also provides an apparatus for selecting a treatment plan, the apparatus comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that the processor, when executing the program, performs the following steps:

[0159] (1) Introduce or express the probe shown in any one of SEQ ID NO:23-28 in cancer cells and detect the fluorescence intensity or fluorescence intensity ratio of the probe, especially the ratio of the fluorescence intensity generated by excitation at 405nm-420nm to the fluorescence intensity generated by excitation at 485nm-488nm.

[0160] (2) When the ratio of fluorescence intensity is less than or equal to about 1.0 (e.g., less than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), the pharmaceutical composition described herein is selected, comprising: (1) cytarabine, (2) a mitochondrial metabolism inhibitor and / or a reagent that downregulates the expression or activity of CDA, and (3) a pharmaceutically acceptable excipient.

[0161] In one or more embodiments, the probe is preferably as shown in SEQ ID NO:27.

[0162] In one or more embodiments, the fluorescence generated by excitation at 405nm-420nm is the fluorescence generated at 528nm by excitation at 405nm-420nm.

[0163] In one or more embodiments, the fluorescence generated by excitation at 485nm-488nm is the fluorescence generated at 528nm by excitation at 405nm-420nm.

[0164] In one or more embodiments, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, preferably B-cell acute lymphoblastic leukemia.

[0165] In one or more embodiments, the pharmaceutical composition is as described in any embodiment of the third aspect herein. Attached Figure Description

[0166] Figure 1 SoNar probes differentiate metabolic and functional subsets of acute B-lymphoblastic leukemia cells. (A) Statistical analysis of the SoNar ratio (F405 / F488 nm) of 204 mouse SoNar B-ALL cells. (B) Evaluation of the SoNar ratio of high-SoNar and low-SoNar B-ALL cells by flow cytometry. (C) ATP levels in high-SoNar and low-SoNar B-ALL cells measured by bioluminescence. (D) Quantification of mean fluorescence intensity (MFI) in high-SoNar and low-SoNar B-ALL cells. (E) OCRs of high-SoNar and low-SoNar B-ALL cells. (F) OCR of high-SoNar and low-SoNar B-ALL cells. bas OCR ATPBasal respiration, ATP turnover, and maximal respiration in (and max resp). (G) Detection of mitochondrial DNA (mtDNA) copy number in SoNar-high and SoNar-low B-ALL cells. (H) Extracellular acidification rate (ECAR) in SoNar-high and SoNar-low B-ALL cells. (I) Glycolysis and glycolytic capacity of SoNar-high and SoNar-low B-ALL cells in ECAR. (J) Extracellular lactate levels in SoNar-high and SoNar-low B-ALL cells. (K) In vitro... 13 After C-labeling glucose, intermediate metabolites from glycolysis and the tricarboxylic acid cycle in B-ALL cells with high and low SoNar- were measured. (L) 13 C-labeled glucose was used to label SoNar-B-ALL cells in vivo, and intermediate metabolites from glycolysis and the tricarboxylic acid cycle were measured in SoNar-high and SoNar-low leukemia cells. (M) Overall survival of recipient mice transplanted with SoNar-high, SoNar-medium, and SoNar-low cells was determined. (N) Functional LIC frequency of primary B-ALL cells was determined using the L-Calc software limiting dilution method. (OQ) Overall survival of non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice (AML#1 to AML#3) transplanted with human primary SoNar-high / SoNar-low B-ALL cells was measured. Data are expressed as mean ± SEM. Statistical significance was assessed using a two-tailed unpaired t-test; *p<0.05; **p<0.01; ***p<0.001.

[0167] Figure 2Upregulated expression of genes such as Pdhx in SoNar-low cell subsets promotes mitochondrial metabolism. (A) Using microarray data, KEGG analysis revealed upregulated pathways in SoNar-low B-ALL cells, with candidate pathways of oxidative phosphorylation highlighted in boxes. (B) Quantitative RT-PCR analysis was used to analyze the expression levels of genes related to glycolysis, oxidative phosphorylation, fatty acid oxidation, and glutamine breakdown in SoNar-high and SoNar-low B-ALL cells. (C) Relative mRNA expression in Pdhx-mixed and Pdhx-knockout (shPdhx#2) B-ALL cells. (D) In ​​L1210 cells (mouse B-ALL cell line), the relative mRNA expression levels of Pdhx after shNRAs gene knockout (shPdhx#1-#3) were detected. (E) PDHX protein levels in the mixed (#1-#3) and PDHX-knockdown mouse primary B-ALL cells (#1-#3) from (C) were detected using Western blotting. The PDHX / actin ratio was quantified and normalized to that of the mixed #1 cells. (FG) Oxygen consumption rate (OCR, F) and extracellular acidification rate (ECAR, G) of the mixed and pdhx-knockout B-ALL cells were measured using a Seahorse XF96 analyzer. (H) Overall survival of recipient mice receiving mixed or Pdhx-knockout (shPdhx#2) B-ALL cells. (I) Overall survival of recipient mice transplanted with mixed and pdhx-knockout Nalm6 cells was measured. Data are expressed as mean ± SEM. Statistical significance was assessed using a two-tailed unpaired t-test; *p<0.05; **p<0.01; ***p<0.001.

[0168] Figure 3The Cda gene in the SoNar-low cell subset mediates resistance to cytarabine Ara-C, a therapeutic agent for acute B-lymphoblastic leukemia. (A) SoNar ratios of 50 B-ALL cells were counted. (B) Cell numbers were assessed in mouse SoNar-high and SoNar-low B-ALL cells after Ara-C treatment at specified time points. (C) Quantitative RT-PCR analysis was performed to determine the expression levels of Ara-C transporter (Slc29a1) and catabolites (Cda, DcK, Dctd, and Cmpk1) in SoNar-high and low B-ALL cells. (D) Overall survival was determined in recipient mice transplanted with TIB205 cells with / without Ara-C scrambling and Cda suppression. (E) The relative expression levels of Cda in scrambled and Cda-knockdown TIB205 cells were determined by quantitative RT-PCR. (F) The knockdown efficiency of shRNA (shPdhx#1-#3) in Cda of TIB205 cells was measured by quantitative RT-PCR. (G) The knockdown efficiency of shRNA (shCDA#1-#3) in CDA of Nalm6 cells was determined by Western blotting. Data are expressed as mean ± SEM. Statistical significance was assessed using a two-tailed unpaired t-test; **p<0.01; ***p<0.001.

[0169] Figure 4 CREB phosphorylation enhances Pdhx transcription in the SoNar-low cell subset. (A) CREB and p-CREB levels in SoNar-high and low B-ALL cells were measured by Western blotting. (B) CREB protein levels in scrambled (#1-#3) and Creb-knockdown B-ALL cells (#1-#3) in (D) were measured by Western blotting. (C) The percentage of mCherry+GFP+ and Creb-knockdown B-ALL cells in mouse peripheral blood was determined by flow cytometry 3 weeks post-transplantation. (D) Survival rate of recipient mice transplanted with scrambled Cda-knockdown B-ALL cells. Data are expressed as mean ± SEM. Statistical significance was determined using a two-tailed unpaired t-test, ***p<0.001.

[0170] Figure 5: Combination therapy of mitochondrial metabolism inhibitors or CREB inhibitors with cytarabine Ara-C enhances the therapeutic effect of acute B-lymphoblastic leukemia. (AB) Evaluation of the proliferation rates of Nalm6 cells (A) and Ara-C-resistant Nalm6 cells (B) after treatment with Ara-C, CREB inhibitor (666-15), and Ara-C combined with CREB inhibitor. (C) Evaluation of the proliferation rate of anti-Ara-C Nalm6 cells after treatment with the oxidative phosphorylation inhibitor Venetoclax. (D) Quantitative data on the survival rate of peripheral blood B-ALL cells in leukemia mice after treatment with PBS, Ara-C, Venetoclax, and Venetoclax combined with Ara-C (3 weeks post-transplantation). (EF) Evaluation of the proliferation rate of anti-Ara-C Nalm6 cells after treatment with mitochondrial metabolism-targeting drugs (Metformin and Berberine). (G) Quantitative data on the survival rate of peripheral blood B-ALL cells in leukemia mice treated with PBS, Ara-C, Metformin, Berberine, Metformin+Ara-C, and Berberine+Ara-C, respectively (3 weeks post-transplantation). (H) Quantitative data on the survival rate of peripheral blood B-ALL cells in leukemia mice treated with PBS, Ara-C, Venetoclax, and Venetoclax combined with Ara-C, respectively (3 weeks post-transplantation). (I) Quantitative data on the survival rate of peripheral blood B-ALL cells in leukemia mice treated with PBS, Ara-C, Metformin, Berberine, Metformin+Ara-C, and Berberine+Ara-C, respectively (3 weeks post-transplantation). The overall survival rate of recipient mice transplanted with B-ALL cells was determined in (G) (n = 5 mice per group, log-rank test). Detailed Implementation

[0171] The inventors used SoNar fluorescent probes to specifically monitor intracellular NADH / NAD+ levels and discovered that tumor cells of B-cell acute lymphoblastic leukemia exhibit different mitochondrial metabolic activities. In cells with higher mitochondrial metabolic activity, the pyruvate dehydrogenase complex member gene Pdhx was significantly upregulated. Simultaneously, the inventors discovered for the first time that resistance to cytarabine (CAS#147-94-4) is related to the mitochondrial metabolic activity of tumor cells; the stronger the mitochondrial metabolic activity, the stronger the resistance to cytarabine. By knocking down the expression of the Cda gene, the inventors confirmed that the Cda gene mediates resistance to cytarabine in cells with higher mitochondrial metabolic activity. Immunoblotting and expression knockdown experiments showed that phosphorylation of the transcription enhancer CREB enhances the transcription of both the Pdhx and Cda genes. Based on this, the inventors screened mitochondrial metabolic inhibitors and found that these inhibitors can significantly reduce tumor cell resistance to cytarabine.

[0172] The "SoNar fluorescent probe" described herein is the fused fluorescent probe described in patent CN104403003A, which is incorporated herein by reference in its entirety. This probe is an inherent ratiometric sensor with two excitation wavelengths (405-420 nm and 485-488 nm, with slight variations depending on the instrument), and its response to NADH and NAD+ is inversely proportional to the fluorescence ratio. Specifically, SEQ ID NO:23-28 described herein corresponds to SEQ ID NO:144-149 in that patent.

[0173] Genes and their inhibitors

[0174] As used in this article, the sequences of the Pdhx gene or its transcripts are shown as gene ID (Entrez ID): 8050 or 27402; the sequences of the PDHX protein are shown as protein ID (Genbank ID): NP_003468 or AAH61231.1; the sequences of the Creb gene or its transcripts are shown as gene ID (Entrez ID): 1385 or 12912; the sequences of the CREB protein are shown as protein ID (Genbank ID): AAQ24858.1 or AAB64015.1; the sequences of the Cda gene or its transcripts are shown as gene ID (Entrez ID): 978 or 72269; and the sequences of the CDA protein are shown as protein ID (Genbank ID): NP_001776.1 or NP_082452.1.

[0175] In this invention, the terms "PDHX," "CREB," and "CDA" also include variations of the sequences shown that have the same function as the proteins shown. These variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, 1-20, 1-10, 1-8, or 1-5), and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. In the art, amino acids of similar properties often refer to families of amino acids with similar side chains, which are well-defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not alter the function of the polypeptide or protein. Conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conserved substitutions for other non-coding amino acids can be determined based on a comparison of their physical properties with those of their genetically encoded amino acids.

[0176] The variant forms of peptides include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants. Any peptide that shares high homology with the PDHX, CREB, or CDA (e.g., 70% or higher homology with the illustrated sequence; preferably, 80% or higher; more preferably, 90% or higher homology, such as 95%, 98%, or 99% homology) and has similar or identical functions is also included in this invention. The peptide fragments, derivatives, or analogs of this invention can also be: (i) peptides formed by fusing a mature peptide with another compound (e.g., a compound that extends the half-life of the peptide, such as polyethylene glycol); or (ii) peptides formed by fusing an additional amino acid sequence to this peptide sequence (e.g., a leader sequence, secreted sequence, or sequence used to purify the peptide, or a proteogenic sequence, or a fusion protein). These fragments, derivatives, and analogs, as defined herein, are within the scope well known to those skilled in the art.

[0177] This invention also relates to inhibitors of PDHX, CREB, or CDA and their uses. Any substance that can reduce the activity, stability, expression, duration of action, interaction with associated proteins, or transcription and translation of PDHX, CREB, or CDA may be used in this invention as a downregulator, antagonist, or inhibitor of PDHX, CREB, or CDA. Exemplary inhibitors include antibodies or ligands that specifically bind to PDHX, CREB, or CDA; repressive molecules that specifically interfere with the transcription and / or expression of PDHX, CREB, or CDA genes (such as interfering molecules that can form shRNA); or small molecule compounds that inhibit the activity of PDHX, CREB, or CDA.

[0178] Any antibody or ligand known in the art that specifically binds to PDHX, CREB, or CDA can be used in this invention. The antibody can be a monoclonal or polyclonal antibody. Polyclonal antibodies can be produced by immunizing animals, such as rabbits, mice, rats, and camels, with the FBXW2 protein; various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Similarly, cells expressing FBXW2 or its antigenic fragments can be used to immunize animals to produce antibodies. The antibody can also be a monoclonal antibody, which can be prepared using hybridoma technology or single-cell screening. Furthermore, after knowing the antibody sequence, the antibody coding sequence can be loaded into an expression vector, thereby operatively linking the antibody gene with promoters, terminators, etc., and expressing it in host cells to produce antibodies. Other components required for the expression vector, such as promoters, terminators, and the expression vector itself, can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid and vector can be used as long as it can replicate and stabilize in the host. Those skilled in the art know how to select appropriate vectors, promoters, enhancers, and host cells.

[0179] Once the target sequence is known, methods for preparing interfering molecules that interfere with the expression of specific genes are well known to those skilled in the art. As a preferred embodiment of the present invention, the PDHX, CREB, or CDA inhibitor is a PDHX, CREB, or CDA-specific shRNA or a construct thereof, wherein the shRNA specifically recognizes the PDHX, CREB, or CDA gene or its transcript. In this document, "transcript" includes a UTR region (e.g., 3'UTR) and a CDS region. Exemplary shRNAs targeting PDHX have the following structure: (any of SEQ ID NO: 1-6)-(SEQ ID NO: 16)-(reverse complement of any of SEQ ID NO: 1-6); Exemplary shRNAs targeting CREB have the following structure: (any of SEQ ID NO: 7-9)-(SEQ ID NO: 16)-(reverse complement of any of SEQ ID NO: 7-9); Exemplary shRNAs targeting CDA have the following structure: (any of SEQ ID NO: 10-15)-(SEQ ID NO: 16)-(reverse complement of any of SEQ ID NO: 10-15).

[0180] Furthermore, to downregulate the expression or activity of PDHX, CREB, or CDA genes, gene knockout vectors can be introduced into cells, and / or gene editing technologies such as ZFN, TALEN, or CRISPR / Cas9 can be used to edit the gene. The ZFN, TALEN, and CRISPR / Cas9 technologies applicable to this invention are well known in the art. Each technology achieves target gene knockout through the combined action of a DNA recognition domain and an endonuclease.

[0181] Downregulators or inhibitors of PDHX, CREB, or CDA can also be small molecule compounds that inhibit the activity of PDHX, CREB, or CDA, such as the CREB inhibitor 666-15 (CAS#1433286-70-4).

[0182] Mitochondrial metabolism inhibitors

[0183] Mitochondrial metabolism inhibitors include agents that weaken the tricarboxylic acid cycle (TCA) or electron transport chain (ETC), such as venetoclax (CAS#1257044-40-8), metformin (CAS#657-24-9), berberine (CAS#633-65-8), or their pharmaceutically acceptable salts, isomers, racemates, solvates, or prodrugs.

[0184] In this invention, the “compound” (including cytarabine, venetoclax, metformin, berberine, CREB inhibitor 666-15 or a pharmaceutically acceptable salt, isomer, racemate, solvate or prodrug thereof) may be a compound in pure form or a compound with a purity greater than 85% (preferably greater than 90%, for example 95%, 98%, 99%).

[0185] Those skilled in the art will understand that, upon learning the structure of the compounds of the present invention, they can be obtained using various methods well-known in the art and using known raw materials, such as chemical synthesis or extraction from biological sources (e.g., microorganisms), all of which are included in this invention. Furthermore, the compounds can also be commercially available pharmaceutical products, and therefore their finished products are readily available to those skilled in the art.

[0186] This invention also includes pharmaceutically acceptable salts of the compounds, which retain the chemical activity of the respective compounds. In this invention, a "pharmaceutically acceptable" component is a substance suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The "pharmaceutically acceptable salt" can be an acidic or basic salt of the compound in question.

[0187] "Pharmaceutically acceptable acid salts" refer to salts that retain the biological activity and properties of the free base without exhibiting undesirable changes in biological activity or other aspects. These salts can be composed of inorganic acids, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and similar acids. They can also be composed of organic acids, such as, but not limited to, acetic acid, dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, camphoric acid, camphorsulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, formic acid, and fumaric acid. (acid), galactoic acid, gentian acid, glucoheponic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, viscous acid, naphthalene-1,5-disulfonic acid, 2-naphthalenesulfonic acid, 1-naphthol-2-carboxylic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, undecenoic acid and similar acids.

[0188] "Pharmaceutically acceptable basic salts" refer to salts that retain the biological activity and properties of the free acid without exhibiting undesirable changes in biological activity or other aspects. These salts are produced by adding an inorganic or organic base to the free acid. Salts obtained by using inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and similar salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts obtained from organic bases include, but are not limited to, primary, secondary, and tertiary ammonium salts. Substituted amines include naturally substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, tannin, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, halamine, choline, betaine, phenethylbenzylamine, N,N'-bisbenzylethylenediamine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, thiazoline, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resins, and similar structures. Preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0189] Crystallization typically produces solvated products of the disclosed compounds. When used herein, the term "solvent" refers to a polymer comprising one or more molecules of the compounds disclosed herein and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may also be an organic solvent. Therefore, the compounds disclosed herein can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and similar structures, and can also exist as corresponding solvated products. The compounds disclosed herein can be true solvates, while in other cases, the compounds disclosed herein may also be containing only a portion of water, or a mixture of water and some solvent.

[0190] This invention also includes prodrugs of compounds (including cytarabine, venetoclax, metformin, berberine, and CREB inhibitor 666-15), wherein a "prodrug" refers to a compound that, when taken in an appropriate manner, undergoes metabolism or chemical reaction in the patient's body to transform into the compound.

[0191] The compounds described herein (cytarabine, venetoc, metformin, berberine, CREB inhibitor 666-15) also include analogs known in the art that have at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 99%) potency of the compound. Methods for determining the potency of compounds and analogs are well known in the art, such as experiments based on solution systems, subcellular systems, cellular systems, or in vivo systems.

[0192] Mitochondrial metabolism inhibitors also include antibodies, nucleic acids, and small molecule compounds that inhibit the expression or activity of related genes. Therefore, mitochondrial metabolism inhibitors include any of the above-mentioned agents that downregulate the expression or activity of PDHX and / or CREB.

[0193] Pharmaceutical Composition

[0194] The present invention also provides a composition comprising an effective amount (e.g., 0.000001-50 wt%; preferably 0.00001-20 wt%; more preferably 0.0001-10 wt%) of cytarabine and the aforementioned mitochondrial metabolism inhibitor and / or agent for downregulating CDA expression or activity (CDA inhibitor), as well as pharmaceutically acceptable excipients. The composition may be used for the prevention and treatment of cancer. Any of the aforementioned mitochondrial metabolism inhibitors may be used in the preparation of the composition.

[0195] The cancers described herein include any cancer in which the tricarboxylic acid cycle or electron transport chain is enhanced, such as cancers in which the expression or activity of PDHX and / or CREB is increased. In a preferred embodiment, the cancer is leukemia, including but not limited to lymphocytic leukemia, myeloid leukemia, and mixed-cell leukemia, such as B-cell acute lymphoblastic leukemia. In one or more embodiments, the cancer is a cancer resistant to cytarabine.

[0196] As used herein, "effective amount" means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. "Pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc. The carriers may also contain cell transfection reagents.

[0197] Having learned of the uses of cytarabine and the aforementioned inhibitors (mitochondrial metabolism inhibitors and / or CDA inhibitors), a variety of methods well known in the art can be used to administer cytarabine and the aforementioned inhibitors, or their encoding genes, or pharmaceutical compositions thereof, to mammals. These include, but are not limited to, subcutaneous injection, intramuscular injection, transdermal administration, local administration, implantation, and sustained-release administration; preferably, the administration method is off-enteric.

[0198] Preferably, gene therapy can be used. For example, mitochondrial metabolism inhibitors and / or CDA inhibitors, along with cytarabine, can be directly administered to the subject via methods such as injection; alternatively, expression units (such as antibody expression vectors, viruses, or shRNA constructs) carrying mitochondrial metabolism inhibitors and / or CDA inhibitors and cytarabine can be delivered to the target site (e.g., tumor cells) through a specific route, thereby inducing the expression of the inhibitors, depending on the type of inhibitors mentioned. These methods are well known to those skilled in the art. Cytarabine and the inhibitors can be administered simultaneously or sequentially, for example, at intervals of at least 1 hour, at least half a day, at least 3 days, or at least 1 week.

[0199] The effective dose of cytarabine and its inhibitor described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective dose can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: the pharmacokinetic parameters of the cytarabine and its inhibitor, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the cytarabine or its inhibitor of this invention is administered daily at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (e.g., 0.0001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0200] Drug screening

[0201] Knowing the close correlation between mitochondrial metabolism and CDA with cytarabine, substances that inhibit cytarabine resistance can be screened based on this characteristic. Drugs for use in combination with cytarabine to prevent or treat cancer (cancers resistant to cytarabine) can be found among these substances.

[0202] Therefore, this invention provides a method for screening potential substances for preventing or treating cancers resistant to cytarabine. The method includes: treating a system for detecting mitochondrial metabolism with a candidate substance; and detecting the intensity of mitochondrial metabolism in the system. If the candidate substance inhibits high mitochondrial metabolism, it indicates that the candidate substance is a potential substance for reducing cytarabine resistance. For example, the system for detecting mitochondrial metabolism can be a system expressing a SoNar probe, such as cells or cell cultures. The cells can be endogenously expressing a SoNar probe or recombinantly expressing a SoNar probe. The SoNar-expressing system can also be a subcellular system, a solution system, a tissue system, an organ system, or an animal system (such as an animal model, preferably a non-human mammalian animal model, such as a mouse, rabbit, sheep, monkey, etc.). The detection method for the SoNar-expressing probe system is as described in patent CN104403003A (which is incorporated herein by reference in its entirety), for example, detecting the ratio of the fluorescence intensity generated by the probe when excited at 405nm-420nm to the fluorescence intensity generated when excited at 485nm-488nm.

[0203] In a preferred embodiment of the present invention, a control group may be set up during screening to facilitate observation. The control group may be a system expressing the SoNar probe without adding the candidate substance.

[0204] Diagnostics and kits

[0205] Based on the relationship between the expression or activity of mitochondrial metabolism-related genes (e.g., PDHX and / or CREB) and cancer, cancer cell metabolism, this invention also provides a method for diagnosing cancer or identifying metabolic and functional subpopulations of cancer cells, the method comprising detecting the expression of mitochondrial metabolism-related genes (e.g., PDHX and / or CREB) in a subject. The method comprises: (1) obtaining a sample of the subject; (2) detecting the intensity of the tricarboxylic acid cycle (TCA) or electron transport chain (ETC) in the subject sample; and (3) comparing the intensity of the subject's TCA or ETC with that of a healthy control. If the intensity of the subject's TCA or ETC is increased, the subject is diagnosed with cancer, particularly cancer in which the TCA or ETC is enhanced. Reagents for detecting the intensity of the TCA or ETC are well known in the art. For example, detecting the expression or activity of genes related to the TCA or ETC.

[0206] Specifically, the method includes: (1) obtaining a sample of the subject, (2) detecting the expression or activity of mitochondrial metabolism-related genes in the sample of the subject, and (3) comparing the expression or activity of mitochondrial metabolism-related genes of the subject with that of mitochondrial metabolism-related genes of healthy controls. If the expression or activity of mitochondrial metabolism-related genes of the subject is increased, the subject is diagnosed with cancer, especially cancer in which the tricarboxylic acid cycle or electron transport chain is enhanced.

[0207] Typically, reagents used to detect the expression or activity of mitochondrial metabolism-related genes (e.g., PDHX and / or CREB) include: (1) primers or probes targeting the gene or its transcripts, or (2) antibodies or ligands that specifically bind to the gene. Primers, probes, antibodies, and ligands are described elsewhere herein.

[0208] This invention provides a kit for performing the above-mentioned diagnoses or identifications, comprising reagents for detecting the expression or activity of metabolism-related genes (e.g., PDHX and / or CREB). Furthermore, the kit may also contain reagents required for RT-PCR, such as reverse transcriptase, RNA extraction reagents, nucleic acid polymerases, dNTPs, PCR buffer, etc.; or, the kit may also contain reagents required for Northern PCR, such as RNA extraction reagents, ribonuclease inhibitors, Northern PCR buffer, etc.; or, the kit may further contain reagents required for Western PCR, such as protein extraction reagents, acrylamide, guanidine isothiocyanate, Tris, SDS, TEMED, etc.

[0209] Furthermore, this invention discloses a computer-readable storage medium storing a computer program, which, when executed, performs the methods described herein for identifying dairy product intake, detecting cardiovascular disease risk, identifying cardiovascular risk factors, or changes thereof. The steps of the methods or algorithms described in conjunction with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of both. If implemented as a computer program product in software, the functions may be stored as one or more instructions or codes on or transmitted via a computer-readable medium. A computer-readable medium includes both computer storage media and communication media, including any medium that facilitates the transfer of a computer program from one location to another. The storage medium may be any available medium accessible to a computer. By way of example and not limitation, such a computer-readable medium may include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to carry or store desired program code in the form of instructions or data structures and is accessible to a computer. An exemplary storage medium is coupled to a processor so that the processor can read and write information from / to the storage medium. In an alternative, the storage medium may be integrated into the processor. The processor and storage media can reside in the ASIC. The ASIC can reside in the user terminal. In an alternative, the processor and storage media can reside as discrete components in the user terminal.

[0210] Methods for identifying cancer cell metabolism

[0211] This invention develops a method for identifying metabolic and functional subpopulations of cancer cells using SoNar probes. The method includes introducing or expressing a probe shown in any of SEQ ID NO:23-28 (e.g., SEQ ID NO:27) in cancer cells and detecting the fluorescence intensity or fluorescence intensity ratio of the probe, particularly the ratio of fluorescence intensity generated by excitation at 405nm-420nm to fluorescence intensity generated by excitation at 485nm-488nm. A smaller fluorescence intensity ratio indicates stronger metabolism via the tricarboxylic acid cycle and / or oxidative phosphorylation pathways in the cancer cells, stronger tumorigenic capacity, and shorter patient survival. For example, when the fluorescence intensity ratio is less than or equal to about 1.0 (e.g., less than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), the cancer cells exhibit strong metabolism of the tricarboxylic acid cycle and / or oxidative phosphorylation pathway, strong tumorigenicity, and short patient survival; when the fluorescence intensity ratio is greater than or equal to about 1.0 (e.g., greater than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), the cancer cells exhibit weak metabolism of the tricarboxylic acid cycle and / or oxidative phosphorylation pathway, weak tumorigenicity, and long patient survival.

[0212] Methods and devices for selecting treatment options

[0213] The present invention also provides a method for selecting a treatment regimen, comprising: (1) introducing or expressing a probe shown in any one of SEQ ID NO: 23-28 in cancer cells and detecting the fluorescence intensity or fluorescence intensity ratio of the probe, particularly the ratio of fluorescence intensity generated by excitation at 405 nm-420 nm to fluorescence intensity generated by excitation at 485 nm-488 nm; and (2) determining a treatment regimen based on the fluorescence intensity or fluorescence intensity ratio. For example, when the fluorescence intensity ratio is less than or equal to about 1.0 (e.g., less than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), a pharmaceutical composition comprising: (1) cytarabine, (2) a mitochondrial metabolism inhibitor and / or a reagent that downregulates the expression or activity of CDA, and (3) a pharmaceutically acceptable excipient, is selected.

[0214] Furthermore, this invention discloses a computer-readable storage medium storing a computer program, which, upon execution, performs the methods described herein for identifying metabolic and functional subpopulations of cancer cells or for selecting treatment regimens. The steps of the methods or algorithms described in conjunction with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of both. If implemented in software as a computer program product, the functions may be stored as one or more instructions or codes on or transmitted via a computer-readable medium. A computer-readable medium includes both computer storage media and communication media, including any medium that facilitates the transfer of a computer program from one location to another. The storage medium may be any available medium accessible to a computer. By way of example and not limitation, such a computer-readable medium may include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to carry or store desired program code in the form of instructions or data structures and is accessible to a computer. An exemplary storage medium is coupled to a processor so that the processor can read and write information from / to the storage medium. In an alternative, the storage medium may be integrated into the processor. The processor and storage media can reside in the ASIC. The ASIC can reside in the user terminal. In an alternative, the processor and storage media can reside as discrete components in the user terminal.

[0215] Therefore, the above-described method for selecting a treatment regimen can be implemented by a device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that the processor executes the program to perform the following steps: (1) introducing or expressing a probe shown in any one of SEQ ID NO:23-28 in cancer cells and detecting the fluorescence intensity or fluorescence intensity ratio of the probe, particularly the ratio of fluorescence intensity generated by excitation at 405nm-420nm to fluorescence intensity generated by excitation at 485nm-488nm, (2) when the fluorescence intensity ratio is less than or equal to about 1.0 (e.g., less than or equal to about 0.9, 0.8, 0.7, 0.6, 0.5), selecting the pharmaceutical composition described herein comprising: (1) cytarabine, (2) a mitochondrial metabolism inhibitor and / or an agent that downregulates the expression or activity of CDA, and (3) pharmaceutically acceptable excipients.

[0216] Using transgenic mice encoding the NADH / NAD+ probe SoNar, the inventors discovered that acute lymphoblastic leukemia (B-ALL) cells primarily utilize oxidative phosphorylation as their energy source. B-ALL cells with low SoNar exhibit enhanced mitochondrial respiration, tend to reside in blood vessel walls, and are enriched in more functional leukemia-initiating cells (B-ALL stem cells) than cells with high SoNar. Cells with low SoNar are more resistant to Ara-C, a first-line drug for acute leukemia. Further studies showed that human primary B-ALL cells with low SoNar also favor oxidative phosphorylation and are resistant to Ara-C treatment. Inhibiting oxidative phosphorylation with drugs effectively attenuates Ara-C-induced resistance, both in vivo and in vitro. This invention provides an effective tool for understanding the potential link between metabolic characteristics and B-ALL cell fate determination, and for developing therapeutic strategies against cancer drug resistance.

[0217] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 2002), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0218] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0219] Example

[0220] I. Experimental Materials and Reagents

[0221] The embodiments mainly employ conventional genetic engineering molecular biology cloning methods, cell culture, and imaging methods, which are well known to those skilled in the art. Examples include: Jane Rothcams et al.'s "Molecular Biology Laboratory Reference Manual," J. Sambrook and DW Russell's "Molecular Cloning: A Laboratory Manual" (3rd edition, August 2002, Science Press, Beijing); Fereschney et al.'s "Animal Cell Culture: A Basic Technical Guide" (5th edition), translated by Zhang Jingbo and Xu Cunshuan et al.; and J.S. Bonnie Fesnon, M. Dassault et al.'s "A Concise Laboratory Manual of Cell Biology," translated by Zhang Jingbo et al.

[0222] All primers used for PCR were synthesized, purified, and identified correctly by mass spectrometry by Shanghai Jierui Biotechnology Co., Ltd. The expression plasmids constructed in the examples were all sequenced by BGI Genomics and J. Lee Sequencing. Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, Pfu DNA polymerase from Tiangen Biotech (Beijing) Co., Ltd., and primeSTAR DNA polymerase from TaKaRa. Corresponding polymerase buffers and dNTPs were included with each purchase. Restriction endonucleases, T4 ligases, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, with corresponding buffers included. The Lip2000 transfection kit was purchased from Invitrogen. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Ameresco. 96-well and 384-well fluorescence detection blackboards were purchased from Grenier. The DNA purification kits used in these examples were purchased from BBI (Canada), and the general plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. Nickel affinity chromatography columns and desalting column packing materials were both from GE Healthcare.

[0223] The main instruments used in the examples include: BD Aria2 flow cytometer, Biotek Synergy 2 multi-functional microplate reader (Bio-Tek, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd.), and nucleic acid electrophoresis apparatus (Shenneng Bocai Co., Ltd.).

[0224] Antibodies: Anti-CREB phosphate antibody (Abway), anti-CREB antibody (Abway), anti-CDA antibody (Abclonal), anti-PDHX antibody (Abclonal), anti-β-actin antibody (MBL).

[0225] Primers and shRNA: Primers and shRNA were designed using conventional methods based on the gene sequences of each protein.

[0226] Exemplary primers include SoNar-F: SEQ ID NO:17, SoNar-R: SEQ ID NO:18, Pdhx-F: SEQ ID NO:19, Pdhx-R: SEQ ID NO:20, CDA-F: SEQ ID NO:21, and CDA-R: SEQ ID NO:22.

[0227] The target sequences of shRNA are shown in the table below:

[0228] shRNA target sequence Mouse shPdhx#1 SEQ ID NO:1 Mouse shPdhx#2 SEQ ID NO:2 Mouse shPdhx#3 SEQ ID NO:3 Human shPDHX#1 SEQ ID NO:4 Human shPDHX#2 SEQ ID NO:5 Human shPDHX#3 SEQ ID NO:6 Mouse shCreb#1 SEQ ID NO:7 Mouse shCreb#2 SEQ ID NO:8 Mouse shCreb#3 SEQ ID NO:9 Mouse shCda#1 SEQ ID NO:10 Mouse shCda#2 SEQ ID NO:11 Mouse shCda#3 SEQ ID NO:12 Human shCDA#1 SEQ ID NO:13 Human shCDA#2 SEQ ID NO:14 Human shCDA#3 SEQ ID NO:15

[0229] II. Molecular Biology Methods and Cellular Experimental Methods

[0230] Routine procedures such as polymerase chain reaction (PCR), endonuclease digestion, and ligation of the target fragment and vector were performed according to *Molecular Cloning: A Laboratory Manual* (3rd edition, August 2002, Science Press, Beijing). DNA extraction was conducted according to the kit instructions, and RNA extraction used Trizol reagent. Reverse transcription used 6-nucleotide random primers, and qPCR used SYBR Green dye for 40 cycles, followed by melting curve analysis to test product uniformity. Immunoblotting was performed using 6-15% polyacrylamide gel electrophoresis based on protein molecular weight, transferring proteins to PVDF membranes using electrophoresis. The membranes were blocked with 5% skim milk powder, and antibody was diluted to a specific ratio before development.

[0231] All leukemia cells were cultured in Gibco IMDM medium with 2% fetal bovine serum in a 37°C, 5% CO2 cell culture incubator. Cells were centrifuged at 800g for 1 hour after lentiviral or retroviral infection, followed by incubation at 37°C. Cell proliferation assays were performed using a Thermo-Fisher cell counter. Flow cytometry analysis of antibodies was performed by incubation at 4°C for 15 to 60 minutes.

[0232] III. Animal Experimentation Methods

[0233] SoNar transgenic animals

[0234] The DNA coding sequence of the SoNar probe (SEQ ID NO:27) was amplified by PCR and inserted into the pCAG vector. The linearized targeting vector was purified and microinjected into FVB blastocysts. SoNar DNA was randomly integrated into the genome, and genotypes were identified by PCR. SoNar protein expression in various tissues was detected by fluorescence microscopy or flow cytometry. FVB mice were backcrossed with C57BL / 6 mice, and heterozygous mice were used in this experiment. Genotyping was performed using peripheral blood cell DNA, with primers SEQ ID NO:17-18. Immunodeficient mice and C57BL / 6 mice were purchased from Slack Animal Center. All animal experiments followed the laboratory animal ethics requirements of East China University of Science and Technology and Shanghai Jiao Tong University.

[0235] Establishment and analysis of a mouse model of acute B-lymphoblastic leukemia

[0236] Retroviral vectors MSCV–N-myc–IRES-mCherry or MSCV–BCR-ABL–p190-IRES-mCherry were used to package retroviruses, which were then used to infect SoNar-positive cells from mouse fetal livers. 200,000 infected cells were injected into C57BL / 6 recipient mice whose lymphocytes had been irradiated and killed. Leukemia progression was monitored by detecting the number of peripheral blood mCherry-positive cells. SoNar-low and SoNar-high leukemia cells, or B220 and CD43-positive but IgM and IgD-negative leukemia initiating cells, were used for metabolic and transplantation analyses. The frequency of leukemia initiating cells was determined using a limiting dilution method, in which bone marrow-derived SoNar-low and SoNar-high leukemia cells were co-transplanted with 200,000 competing cells into lymphocyte-irradiated mice. In the drug resistance assay, transplanted cells were treated with 1 μmol of Ara-C for 24 hours. After transplantation into recipient mice, the overall survival rate of the leukemia-inducing mice was recorded, followed by the use of STEMCELL's L-Calc software to measure the leukemia-inducing cell frequency. Lentiviral-mediated expression of double-stranded interfering RNA was used to knock down the Pdhx, Cda, or Creb genes. Target cells for infection included mouse N-myc–mCherry-positive leukemia cells, the mouse acute B-lymphoblastic leukemia cell line TIB205, human acute B-lymphoblastic leukemia cell lines Nalm6 cells / Sup-B15 cells / REH cells, or human primary acute B-lymphoblastic leukemia cells. The transplantation experiment used 10,000 mouse bone marrow-derived leukemia cells, 5 million TIB205 cells, 2 million Nalm6 cells, or 5 million human primary acute B-lymphoblastic leukemia cells.

[0237] Example 1: SoNar probes differentiate metabolic and functional subsets of acute B-lymphoblastic leukemia cells

[0238] After SoNar probe expression in mouse acute B-lymphoblastic leukemia, cells were divided into three subsets—SoNar-high, SoNar-medium, and SoNar-low—based on the probe fluorescence ratio F405 / F488. Figure 1 A). Compared to cells with high SoNar-SoNar- ( Figure 1 B), SoNar-low cell subsets have higher ATP levels ( Figure 1 C) Mitochondrial membrane potential ( Figure 1 D) Oxygen consumption rate ( Figure 1 (E, F) Higher mitochondrial DNA copy number ( Figure 1 (G), while there was no statistically significant difference between glycolysis and lactate secretion levels (G). Figure 1 (H,I,J). Use 13 Cell subpopulations of SoNar-high and SoNar-low were cultured with 13C-labeled glucose, and cellular metabolites were determined by high-performance liquid chromatography-mass spectrometry. The results showed that in SoNar-low cells, the levels of 13C-labeled tricarboxylic acid cycle intermediates, including citric acid, ketoglutarate, succinic acid, and malic acid, were tens to hundreds of times higher than in SoNar-high cells, while there was no difference in glycolytic intermediates, including glucose-6-phosphate, pyruvate, and lactate. Figure 1 The results (K,L) indicate that SoNar-low cells' metabolites of exogenous glucose uptake enter the mitochondria, significantly enhancing the metabolism of the tricarboxylic acid cycle and oxidative phosphorylation pathway.

[0239] Compared to cells with high SoNar- levels, cells with low SoNar- levels exhibited significantly different tumorigenic capabilities. When these two cell subsets were inoculated into immunodeficient mice, the survival time of mice inoculated with low SoNar- levels was significantly shortened. Figure 1 After inoculating each experimental mouse with 100-1000 cells, the mice inoculated with SoNar-low cell subsets showed a higher proportion of tumor cell clonal proliferation, indicating that leukemia-initiating cells accounted for 1 / 306 in the SoNar-low cell subset, while they accounted for only 1 / 1393 in the SoNar-high cell subset. Figure 1 Leukemia cell samples were obtained from patients with acute B-lymphoblastic leukemia, and SoNar probes were expressed during in vitro culture. Immunodeficient mice were then inoculated with these cells, and it was found that cells with low SoNar levels had shorter survival times in tumor-bearing mice. Figure 1 (O, P, Q). Therefore, SoNar probes can be used to indicate the metabolic status and pathological function of acute B-lymphoblastic leukemia.

[0240] Example 2: Upregulation of genes such as Pdhx in SoNar-low cell subsets promotes mitochondrial metabolism

[0241] Microarray analysis of the transcriptome of acute B-lymphoblastic leukemia revealed that genes upregulated in low SoNar-positive cell subsets were enriched in oxidative phosphorylation metabolic pathways. Figure 2 A). Quantitative PCR was used to detect the expression of genes related to pyruvate decarboxylation, the tricarboxylic acid cycle, and oxidative phosphorylation metabolism in mitochondria. The results showed that genes involved in all three metabolic pathways were upregulated, with the most significant increase observed in the expression of Pdhx, a member of the pyruvate dehydrogenase complex. Figure 2 , B). Using shRNA, the Pdhx gene can be silenced and knocked down in acute B-lymphoblastic leukemia cells. Figure 2 (C, D, E) Knocking down the Pdhx gene reduces mitochondrial oxygen consumption. Figure 2 F), increases lactic acid secretion ( Figure 2 (G). After inoculating immunodeficient mice with B-ALL cells or control cells with knocked-down Pdhx gene, the survival time of the Pdhx knockdown mouse group was significantly longer than that of the control group. Figure 2 H, I).

[0242] Example 3: Cda gene in SoNar-low cell subset mediates resistance to cytarabine Ara-C, a therapeutic agent for acute B-lymphoblastic leukemia.

[0243] Treatment of SoNar-low tumor cells with cytarabine Ara-C, a drug used to treat acute B-lymphoblastic leukemia, resulted in a decrease in the SoNar fluorescence ratio. Figure 3 A). SoNar-low acute B-lymphoblastic leukemia cell subsets are more resistant to Ara-C than SoNar-high cell subsets. Figure 3 B). Analysis of the expression of genes involved in drug transport and catabolism of Ara-C showed that the Cda gene was expressed at higher levels in the SoNar-low cell subset than in the SoNar-high subset. Figure 3 C). Using shRNA to knock down Cda gene expression, and then inoculating the cells into immunodeficient mice, it was found that for Ara-C treatment, the Cda knockdown group had a significantly longer survival time than the control group. Figure 3 (DG).

[0244] Example 4: CREB phosphorylation enhances Pdhx transcription in SoNar-low cell subsets

[0245] Immunoblotting experiments showed that the phosphorylation of the transcription enhancer CREB was higher in the SoNar-low acute B-lymphoblastic leukemia cell subset than in the SoNar-high subset. Figure 4 A). Knocking down Creb expression with shRNA significantly reduced the expression of Pdhx and Cda genes. Figure 4(B, C), while the survival time of tumor-bearing mice with Creb knockdown of leukemia cells was significantly longer than that of control mice. Figure 4 D).

[0246] Example 5: Combination therapy of mitochondrial metabolism inhibitors or CREB inhibitors with cytarabine Ara-C enhances the therapeutic effect of acute B-lymphoblastic leukemia.

[0247] The inhibitor 666-15 of the transcription enhancer CREB can enhance the inhibitory effect of cytarabine Ara-C on the proliferation of human acute B-lymphoblastic leukemia cells. Figure 5 Even in human acute B-lymphoblastic leukemia cell lines resistant to cytarabine Ara-C, 666-15 can still enhance tumor cell suppression. Figure 5 (B) The combination of cytarabine Ara-C and the leukemia treatment drug venetoclax can dose-dependently enhance the cell proliferation inhibitory effect of Ara-C. Figure 5 (C, D). Furthermore, the diabetes treatment drugs metformin or berberine can also dose-dependently enhance the cell proliferation inhibition effect of Ara-C (C). Figure 5 E, F, G). Any of the three drugs—venetoclax, metformin, or berberine—used in combination with Ara-C was more effective and prolonged in treating acute B-lymphoblastic leukemia tumor-bearing mice than Ara-C alone. Figure 5 H, I). sequence list <110> East China University of Science and Technology <120> Cancer therapeutic targets and their applications <130> 211834 <141> 2021-03-10 <160> 28 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 accaggtttc ttgaaacttt 20 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 ccagcttata acggtcacaa t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 gcgggtacat tcaccgaaat t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <400> 4 gcttacttac tccaatcata a 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 gcttacttac tccaatcata a 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <400> 6 ccaagagatt aactgaatct a 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <400> 7 cagcagctca tgcaacatca t 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <400> 8 actgatggac agcagattct em 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <400> 9 gcctgaaagc 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<211> 21 <212> DNA <213> Artificial Sequence <400> 21 aagtcagcct actgccccta c 21 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <400> 22 gatagcggtc cgttcagcac 20 <210> 23 <211> 386 <212> PRT <213> Artificial sequence <220> <223> Synthetic fluorescent sensor <400> 23 Met Asn Arg Lys Trp Gly Leu Cys Ile Val Gly Met Gly Arg Leu Gly 1 5 10 15 Ser Ala Leu Ala Asp Tyr Pro Gly Phe Gly Glu Ser Phe Glu Leu Arg 20 25 30 Gly Phe Phe Asp Val Asp Pro Glu Lys Val Gly Arg Pro Val Arg Gly 35 40 45 Gly Val Ile Glu His Val Asp Leu Leu Pro Gln Arg Val Pro Gly Arg 50 55 60 Ile Glu Ile Ala Leu Leu Thr Val Pro Arg Glu Ala Ala Gln Lys Ala 65 70 75 80 Ala Asp Leu Leu Val Ala Ala Gly Ile Lys Gly Ile Leu Asn Phe Ala 85 90 95 Pro Val Val Leu Glu Val Pro Lys Glu Val Ala Val Glu Asn Val Asp 100 105 110 Phe Ala Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln 115 120 125 Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp 130 135 140 Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly 145 150 155 160 Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser 165 170 175 Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu 180 185 190 Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr 195 200 205 Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu 210 215 220 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 225 230 235 240 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 245 250 255 Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val 260 265 270 Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe 275 280 285 Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 290 295 300 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 305 310 315 320 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 325 330 335 Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn 340 345 350 Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Gly Leu Ala Gly Leu 355 360 365 Thr Arg Leu Ser Phe Ala Ile Leu Asn Pro Lys Trp Arg Glu Glu Met 370 375 380 Met Gly 385 <210> 24 <211> 385 <212> PRT <213> Artificial sequence <220> <223> Synthetic fluorescent sensor <400> 24 Met Asn Arg Lys Trp Gly Leu Cys Ile Val Gly Met Gly Arg Leu Gly 1 5 10 15 Ser Ala Leu Ala Asp Tyr Pro Gly Phe Gly Glu Ser Phe Glu Leu Arg 20 25 30 Gly Phe Phe Asp Val Asp Pro Glu Lys Val Gly Arg Pro Val Arg Gly 35 40 45 Gly Val Ile Glu His Val Asp Leu Leu Pro Gln Arg Val Pro Gly Arg 50 55 60 Ile Glu Ile Ala Leu Leu Thr Val Pro Arg Glu Ala Ala Gln Lys Ala 65 70 75 80 Ala Asp Leu Leu Val Ala Ala Gly Ile Lys Gly Ile Leu Asn Phe Ala 85 90 95 Pro Val Val Leu Glu Val Pro Lys Glu Val Ala Val Glu Asn Val Asp 100 105 110 Phe Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys 115 120 125 Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly 130 135 140 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 145 150 155 160 Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val 165 170 175 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 180 185 190 Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn 195 200 205 Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe 210 215 220 Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 225 230 235 240 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly 245 250 255 Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro 260 265 270 Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala 275 280 285 Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met 290 295 300 Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly 305 310 315 320 Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val 325 330 335 Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile 340 345 350 Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Gly Leu Ala Gly Leu Thr 355 360 365 Arg Leu Ser Phe Ala Ile Leu Asn Pro Lys Trp Arg Glu Glu Met Met 370 375 380 Gly 385 <210> 25 <211> 384 <212> PRT <213> Artificial sequence <220> <223> Synthetic fluorescent sensor <400> 25 Met Asn Arg Lys Trp Gly Leu Cys Ile Val Gly Met Gly Arg Leu Gly 1 5 10 15 Ser Ala Leu Ala Asp Tyr Pro Gly Phe Gly Glu Ser Phe Glu Leu Arg 20 25 30 Gly Phe Phe Asp Val Asp Pro Glu Lys Val Gly Arg Pro Val Arg Gly 35 40 45 Gly Val Ile Glu His Val Asp Leu Leu Pro Gln Arg Val Pro Gly Arg 50 55 60 Ile Glu Ile Ala Leu Leu Thr Val Pro Arg Glu Ala Ala Gln Lys Ala 65 70 75 80 Ala Asp Leu Leu Val Ala Ala Gly Ile Lys Gly Ile Leu Asn Phe Ala 85 90 95 Pro Val Val Leu Glu Val Pro Lys Glu Val Ala Val Glu Asn Val Asp 100 105 110 Phe Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 115 120 125 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser 130 135 140 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 145 150 155 160 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu 165 170 175 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 180 185 190 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val 195 200 205 Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr 210 215 220 Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His 225 230 235 240 Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys 245 250 255 Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp 260 265 270 Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg 275 280 285 Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro 290 295 300 Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn 305 310 315 320 Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn 325 330 335 Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile Leu 340 345 350 Gly His Lys Leu Glu Tyr Asn Gly Thr Gly Leu Ala Gly Leu Thr Arg 355 360 365 Leu Ser Phe Ala Ile Leu Asn Pro Lys Trp Arg Glu Glu Met Met Gly 370 375 380 <210> 26 <211> 386 <212> PRT <213> Artificial sequence <220> <223> Synthetic fluorescent sensor <400> 26 Met Asn Arg Lys Trp Gly Leu Cys Ile Val Gly Met Gly Arg Leu Gly 1 5 10 15 Ser Ala Leu Ala Asp Tyr Pro Gly Phe Gly Glu Ser Phe Glu Leu Arg 20 25 30 Gly Phe Phe Asp Val Asp Pro Glu Lys Val Gly Arg Pro Val Arg Gly 35 40 45 Gly Val Ile Glu His Val Asp Leu Leu Pro Gln Arg Val Pro Gly Arg 50 55 60 Ile Glu Ile Ala Leu Leu Thr Val Pro Arg Glu Ala Ala Gln Lys Ala 65 70 75 80 Ala Asp Leu Leu Val Ala Ala Gly Ile Lys Gly Ile Leu Asn Phe Ala 85 90 95 Pro Val Val Leu Glu Val Pro Lys Glu Val Ala Val Glu Asn Val Asp 100 105 110 Phe Ser Ala Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys 115 120 125 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu 130 135 140 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 145 150 155 160 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln 165 170 175 Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 180 185 190 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 195 200 205 Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu 210 215 220 Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val 225 230 235 240 Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr 245 250 255 Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro 260 265 270 Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys 275 280 285 Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser 290 295 300 Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp 305 310 315 320 Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr 325 330 335 Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly 340 345 350 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Leu Ala Gly Leu 355 360 365 Thr Arg Leu Ser Phe Ala Ile Leu Asn Pro Lys Trp Arg Glu Glu Met 370 375 380 Met Gly 385 <210> 27 <211> 385 <212> PRT <213> Artificial sequence <220> <223> Synthetic fluorescent sensor <400> 27 Met Asn Arg Lys Trp Gly Leu Cys Ile Val Gly Met Gly Arg Leu Gly 1 5 10 15 Ser Ala Leu Ala Asp Tyr Pro Gly Phe Gly Glu Ser Phe Glu Leu Arg 20 25 30 Gly Phe Phe Asp Val Asp Pro Glu Lys Val Gly Arg Pro Val Arg Gly 35 40 45 Gly Val Ile Glu His Val Asp Leu Leu Pro Gln Arg Val Pro Gly Arg 50 55 60 Ile Glu Ile Ala Leu Leu Thr Val Pro Arg Glu Ala Ala Gln Lys Ala 65 70 75 80 Ala Asp Leu Leu Val Ala Ala Gly Ile Lys Gly Ile Leu Asn Phe Ala 85 90 95 Pro Val Val Leu Glu Val Pro Lys Glu Val Ala Val Glu Asn Val Asp 100 105 110 Phe Ser Ala Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys 115 120 125 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu 130 135 140 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 145 150 155 160 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln 165 170 175 Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 180 185 190 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 195 200 205 Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu 210 215 220 Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val 225 230 235 240 Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr 245 250 255 Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro 260 265 270 Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys 275 280 285 Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser 290 295 300 Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp 305 310 315 320 Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr 325 330 335 Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly 340 345 350 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Leu Ala Gly Leu Thr 355 360 365 Arg Leu Ser Phe Ala Ile Leu Asn Pro Lys Trp Arg Glu Glu Met Met 370 375 380 Gly 385 <210> 28 <211> 384 <212> PRT <213> Artificial sequence <220> <223> Synthetic fluorescent sensor <400> 28 Met Asn Arg Lys Trp Gly Leu Cys Ile Val Gly Met Gly Arg Leu Gly 1 5 10 15 Ser Ala Leu Ala Asp Tyr Pro Gly Phe Gly Glu Ser Phe Glu Leu Arg 20 25 30 Gly Phe Phe Asp Val Asp Pro Glu Lys Val Gly Arg Pro Val Arg Gly 35 40 45 Gly Val Ile Glu His Val Asp Leu Leu Pro Gln Arg Val Pro Gly Arg 50 55 60 Ile Glu Ile Ala Leu Leu Thr Val Pro Arg Glu Ala Ala Gln Lys Ala 65 70 75 80 Ala Asp Leu Leu Val Ala Ala Gly Ile Lys Gly Ile Leu Asn Phe Ala 85 90 95 Pro Val Val Leu Glu Val Pro Lys Glu Val Ala Val Glu Asn Val Asp 100 105 110 Phe Ser Ala Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys 115 120 125 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu 130 135 140 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 145 150 155 160 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln 165 170 175 Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 180 185 190 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 195 200 205 Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu 210 215 220 Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val 225 230 235 240 Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr 245 250 255 Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro 260 265 270 Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys 275 280 285 Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser 290 295 300 Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp 305 310 315 320 Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr 325 330 335 Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly 340 345 350 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Leu Ala Gly Leu Thr Arg 355 360 365 Leu Ser Phe Ala Ile Leu Asn Pro Lys Trp Arg Glu Glu Met Met Gly 370 375 380

Claims

1. Use of cytarabine and venetoclax in the preparation of drugs for the treatment of B-cell acute lymphoblastic leukemia.

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