JP2024051970A - Small Cell Lung Cancer Treatment - Google Patents

Abstract

[Problem] The present invention is aimed at a therapy for treating and/or preventing small cell lung cancer. [Solution] The present invention relates to a pharmaceutical composition for preventing or treating small cell lung cancer, which contains an inhibitor of HPRT1, specifically 6-mercaptopurine, an inhibitor of HPRT1 and a folate synthesis inhibitor, or an inhibitor of HPRT1, a folate synthesis inhibitor and an inhibitor of glutamine synthetase. [Selected Figure] None

Description

The present invention belongs to the field of cancer, particularly the treatment of small cell lung cancer. In particular, the present invention relates to a pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of HPRT1, or an inhibitor of HPRT1 and a folate synthesis inhibitor, and a pharmaceutical composition for treating and/or preventing cancer, comprising an inhibitor of HPRT1, a folate synthesis inhibitor, and an inhibitor of glutamine synthetase.

Small cell lung cancer (SCLC) is one of the most aggressive cancers, characterized by rapid proliferation and high metastatic potential. Cisplatin has been used as the first-line drug for SCLC, but most patients eventually acquire resistance to this drug, and the cancer is defined as "untreatable" with a very low 5-year survival rate of about 7% (Non-Patent Documents 1, 2, 3). Cisplatin was approved as the first-line drug for SCLC in 1984, but until recently, nearly 40 years later, no specific and effective treatment has been developed. Despite various chemotherapy combinations implemented in clinical trials for SCLC, the survival time of SCLC patients has not been extended, which is a hopeless situation (Non-Patent Documents 3, 4).

Recently, immune checkpoint therapy has shown a certain effect on the overall survival of extensive-stage small cell lung cancer. However, it has become clear that small cell lung cancer is more resistant to treatment than non-small cell lung cancer, which is highly sensitive to immune checkpoint therapy, revealing the limitations of the effectiveness of immune checkpoint therapy for small cell lung cancer. In this context, the establishment of more effective and novel treatment methods for small cell lung cancer is eagerly awaited (Non-Patent Documents 5, 6, 7). In order to improve the prognosis of small cell lung cancer patients, it is necessary to clarify the root cause of the malignancy of small cell lung cancer through molecular biological analysis (Non-Patent Documents 3, 8). In recent years, comprehensive genetic profiles of small cell lung cancer have been revealed, but the details of its malignancy remain unclear.

It has become clear that many types of cancer are increasingly dependent on the de novo nucleic acid synthesis pathway, particularly in small cell lung cancer (Non-Patent Documents 9-15). De novo purine and pyrimidine synthesis, which synthesizes purines and pyrimidines anew rather than recycling materials, requires glutamine, which is mainly supplied from the host, as nitrogen (Non-Patent Document 16). Most of the orally ingested glutamine is catabolized in the intestinal endothelium, and only a small amount of glutamine enters the bloodstream directly (Non-Patent Documents 17, 18). Rather, glutamine in serum is supplied from organs such as the lungs and skeletal muscles, where glutamine synthetase (GS), which synthesizes glutamine from glutamate and ammonia, is highly expressed (Non-Patent Document 16). After being taken up into cells, glutamine is metabolized by phosphoribosyl pyrophosphate amidotransferase (PPAT), the rate-limiting enzyme in de novo purine synthesis, and the nitrogen derived from glutamine is transferred to purine precursors. In previous studies, the inventors have demonstrated that the expression level of PPAT is closely related to the prognosis of small cell lung cancer patients, and that inhibition of PPAT is effective in suppressing the proliferation of small cell lung cancer cells (Non-Patent Document 20). Purine nucleic acids are synthesized not only by the de novo pathway, but also by the salvage pathway, in which hypoxanthine phosphoribosyl diphosphate (PRPP) and hypoxanthine are metabolized by hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1). However, because the plasma concentration of hypoxanthine is low at around 1 μM, the contribution of the salvage pathway to the demand for nucleic acids that support cancer proliferation was unclear (Non-Patent Document 19).

George, J. et al. Comprehensive genomic profiles of small cell lung cancer. Nature 524, 47-53, 14664 (2015). Gazdar, A. F., Bunn, P. A. & Minna, J. D. Small-cell lung cancer: what we know, what we need to know and the path forward. Nat. Rev. Cancer 17, 725-737, 87 (2017). Zhang, W. et al. Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes. Transl Lung Cancer Res 7, 32-49, 02.02 (2018). Koinis, F., Kotsakis, A. & Georgoulias, V. Small cell lung cancer (SCLC): no treatment advances in recent years. Transl. Lung Cancer Res. 5, 39-50, (2016). Horn, L. et al. First-Line Atezolizumab plus Chemotherapy in Extensive-Stage Small-Cell Lung Cancer. N. Engl. J. Med. 379, 2220-2229, (2018). Sabari, J. K., Lok, B. H., Laird, J. H., Poirier, J. T. & Rudin, C. M. Unravelling the biology of SCLC: implications for therapy. Nat. Rev. Clin. Oncol. 14, 549-561, (2017). Remon, J. et al. Small cell lung cancer: a slightly less orphan disease after immunotherapy. Ann. Oncol. 32, 698-709, (2021). Stewart, C. A. et al. Single-cell analyses reveal increased intratumoral heterogeneity after the onset of therapy resistance in small-cell lung cancer. Nature Cancer 1, 423-436, (2020). Kodama, M. et al. A shift in glutamine nitrogen metabolism contributes to the malignant progression of cancer. Nature Communications 11, (2020). Kodama, M. & Nakayama, K. I. A second Warburg-like effect in cancer metabolism: The metabolic shift of glutamine-derived nitrogen: A shift in glutamine-derived nitrogen metabolism from glutaminolysis to de novo nucleotide biosynthesis contributes to malignant evolution of cancer. Bioessays, (2020). Huang, F. et al. Inosine Monophosphate Dehydrogenase Dependence in a Subset of Small Cell Lung Cancers. Cell Metab. 28, 369-382 e365, (2018). Augert, A. et al. MAX Functions as a Tumor Suppressor and Rewires Metabolism in Small Cell Lung Cancer. Cancer Cell 38, 97-114 e117, (2020). Li, L. et al. Identification of DHODH as a therapeutic target in small cell lung cancer. Sci. Transl. Med. 11, (2019). Morita, M. et al. PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth. Cancer Cell 33, 355-367 e357, (2018). Ruano-Ravina, A., Provencio-Pulla, M. & Perez-Rios, M. Small Cell Lung Cancer-A Neglected Disease With More Data Needed. JAMA Netw Open 5, e224837, (2022). Lee, J. S. et al. Urea Cycle Dysregulation Generates Clinically Relevant Genomic and Biochemical Signatures. Cell 174, 1559-1570 e1522, (2018). Windmueller, H. G. & Spaeth, A. E. Uptake and metabolism of plasma glutamine by the small intestine. J. Biol. Chem. 249, 5070-5079 (1974). Hensley, C. T., Wasti, A. T. & DeBerardinis, R. J. Glutamine and cancer: cell biology, physiology, and clinical opportunities. J. Clin. Invest. 123, 3678-3684, (2013). Luengo, A., Gui, D. Y. & Vander Heiden, M. G. Targeting Metabolism for Cancer Therapy. Cell Chem Biol 24, 1161-1180, (2017). Kodama, M. et al. A shift in glutamine nitrogen metabolism contributes to the malignant progression of cancer. Nat. Commun. 11, 1320 (2020).

Small cell lung cancer is one of the cancers with an extremely poor prognosis, with a 5-year survival rate of approximately 7%, and accounts for approximately 15% of all lung cancers. Due to its disseminated nature, by the time the cancer is discovered, there are often numerous metastatic lesions and surgery is no longer an option. As mentioned above, chemotherapy is currently being performed, mainly with cisplatin (cDDP), but the response rate is low, which has become a major medical problem, and there is a constant demand for drug therapies that can fundamentally treat small cell lung cancer.

The inventors analyzed patient samples with small cell lung cancer using the iMPAQT system, which they developed independently, and found that the proliferation of small cell lung cancer is strongly dependent on the nucleic acid synthesis pathway. In particular, they discovered that simultaneous inhibition of the main pathway (de novo pathway) and alternative pathway (salvage pathway) of the nucleic acid synthesis pathway using methotrexate (MTX) and 6-mercaptopurine (6-MP) can have a strong proliferation-suppressing effect on small cell lung cancer cell lines, and thus completed the present invention.

Therefore, the present invention includes the following aspects.
<Pharmaceutical composition containing an HPRT1 inhibitor>
[1]
A pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1).
[2]
The pharmaceutical composition according to [1], wherein the HPRT1 inhibitor is 6-mercaptopurine.
[3]
The pharmaceutical composition described in [1], wherein the HPRT1 inhibitor is an inhibitor of HPRT1 expression.
[4]
The pharmaceutical composition according to [3], wherein the inhibitor of HPRT1 expression is a substance that inhibits the expression of a nucleic acid sequence shown in either (a) or (b) below:
(a) the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing, or (b) a nucleic acid sequence containing a base sequence in which one or more bases have been deleted, substituted or added in the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing.
[5]
The pharmaceutical composition according to [4], wherein the inhibitor of HPRT1 expression is a nucleic acid sequence that hybridizes under stringent conditions with a base sequence complementary to the nucleic acid sequence of HPRT1 shown in SEQ ID NO: 1 of the sequence listing.
[6]
The pharmaceutical composition according to claim 4, wherein the inhibitor of HPRT1 expression is selected from the group consisting of siRNA, antisense and ribozyme.
[7]
The pharmaceutical composition according to any one of [1] to [6], wherein the small cell lung cancer is resistant to cisplatin.

<Pharmaceutical composition containing an HPRT1 inhibitor and a folic acid synthesis inhibitor>
[8]
A pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of HPRT1 and an inhibitor of folic acid synthesis.
[9]
The pharmaceutical composition according to [8], wherein the HPRT1 inhibitor is 6-mercaptopurine.
[10]
The pharmaceutical composition according to [9], wherein the folic acid synthesis inhibitor is selected from methotrexate, pralatrexate, pemetrexed, and raltitrexed.
[11]
The pharmaceutical composition according to any one of [8] to [11], wherein the small cell lung cancer is resistant to cisplatin.

Pharmaceutical Compositions Containing an HPRT1 Inhibitor, a Folic Acid Synthesis Inhibitor, and a Glutamine Synthetase Inhibitor
[12]
A pharmaceutical composition for treating and/or preventing cancer, comprising an inhibitor of HPRT1, an inhibitor of folate synthesis, and an inhibitor of glutamine synthetase (GS).
[13]
The pharmaceutical composition according to [12], wherein the GS inhibitor is methionine sulfoximine.
[14]
The pharmaceutical composition according to [12], wherein the GS inhibitor is an inhibitor of GS expression.
[15]
The pharmaceutical composition according to [14], wherein the inhibitor of GS expression is a substance that inhibits the expression of a nucleic acid sequence shown in either (a) or (b) below:
(a) the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing, or (b) a nucleic acid sequence containing a base sequence in which one or more bases have been deleted, substituted or added in the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing.
[16]
The pharmaceutical composition according to [15], wherein the inhibitor of GS expression is a nucleic acid sequence that hybridizes under stringent conditions with a base sequence complementary to the nucleic acid sequence of HPRT1 shown in SEQ ID NO: 2 of the sequence listing.
[17]
The pharmaceutical composition according to claim 15, wherein the inhibitor of GS expression is selected from the group consisting of siRNA, antisense and ribozyme.
[18]
The pharmaceutical composition according to any one of [12] to [17], wherein the small cell lung cancer is resistant to cisplatin.
[19]
The pharmaceutical composition according to [12], wherein the folic acid synthesis inhibitor is selected from methotrexate, pralatrexate, pemetrexed, and raltitrexed.
[20]
The pharmaceutical composition according to [19], wherein the cancer is small cell lung cancer, pancreatic adenocarcinoma, or glioblastoma.
[21]
The pharmaceutical composition according to [20], wherein the cancer is small cell lung cancer.
[22]
The pharmaceutical composition according to [21], wherein the small cell lung cancer is cisplatin-resistant.

<Prognosis of small cell lung cancer>
[23]
A method for determining prognosis in small cell lung cancer, comprising:
(a1) measuring the amount of HPRT1 (test biomarker amount) in a subject;
(b1) comparing the amount of a test biomarker with the amount of HPRT1 in a healthy specimen (a control biomarker amount); and (c1) determining that the specimen has a poor prognosis for small cell lung cancer if the amount of the test biomarker is greater than the amount of the control biomarker.
[24]
A biomarker, HPRT1, that can determine prognosis in small cell lung cancer.
[25]
Use of HPRT1 as a prognostic biomarker in small cell lung cancer.

The present invention enables therapies to treat and/or prevent cancer, particularly small cell carcinoma.

Overall, Figure 1a-d show that tumors from patients with small cell lung cancer are characterized by high expression of purine and pyrimidine nucleic acid synthetases. In the figures, SCLC stands for small cell lung cancer, NSCLC-Ad stands for lung adenocarcinoma, and NSCLC-Sq stands for lung squamous cell carcinoma. Figure 1a shows a heatmap for hierarchical clustering of metabolic enzymes measured by iMPAQT in small cell lung cancer (patients: 12), lung adenocarcinoma (patients: 12), and lung squamous cell carcinoma (patients: 12). Figure 1b shows a simplified graph of the expression patterns of metabolic enzymes in small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma, using principal component analysis. PC1 indicates the first principal component, and PC2 indicates the second principal component. The numbers in parentheses indicate the contribution rate of each principal component. FIG. 1c shows the results for expression of purine nucleotide and pyrimidine nucleotide synthesis enzymes plotted by GSEA against a list of genes that are up- or down-regulated in small cell lung cancer and lung adenocarcinoma tumors. FIG. 1d shows the results for expression of purine and pyrimidine nucleotide synthesis enzymes plotted by GSEA against a list of genes that are up- or down-regulated in small cell lung cancer and squamous cell carcinoma tumors.

Overall, Figures 2a-f show that the nucleic acid synthesis enzyme HPRT1 is a feature of small cell lung cancer. Figure 2a is a volcano plot depicting differential expression of nucleic acid synthesis enzymes in small cell lung cancer versus lung adenocarcinoma and in small cell lung cancer versus lung squamous cell carcinoma. FIG. 2b is a Venn diagram depicting nucleic acid synthesis enzymes whose expression levels are significantly increased in small cell lung cancer compared to lung adenocarcinoma and lung squamous cell carcinoma. FIG. 2c is a box plot showing the expression levels of HPRT1, DUT and POLR2F in small cell lung cancer, lung adenocarcinoma or squamous cell carcinoma. Figure 2d shows a comparison of the enzyme expression levels in tumors with sizes of 3 cm or less and those with sizes greater than 3 cm. Compared with DUT and POLF2F, it was revealed that the expression level of HPRT1 tends to increase with tumor size. Figure 2e shows the results of a comparative analysis of the expression levels of HPRT1, DUT, and POLR2F in normal tissues (n = 6) or cancer tissues (n = 6) from small cell lung cancer patients. Figure 2f shows the results of Kaplan-Meier survival analysis of patients with small cell lung cancer with high or low expression of HPRT1, DUT, and POLR2F genes in their tumor tissues. Patients were divided by median protein expression levels. Figure 3a shows images of HPRT1 by immunohistochemistry (IHC) in tumors from patients with small cell lung cancer, showing the prognostic impact of HPRT1 in this cancer. In the images, arrows indicate HPRT1-positive cells. Figure 3b shows the results of a Kaplan-Meier survival analysis of small cell lung cancer samples with high (solid line) or low (dashed line) HPRT1 expression levels. Patient diagnoses were based on the WHO lung tumor classification. Overall, Figure 4a-g show that genetic disruption of HPRT1 attenuates the tumorigenicity of small cell lung cancer. Figure 4a shows dose-response curves (n=4) of cell viability in small cell lung cancer cell lines (n=10) and a panel of other cancers (n=20) to 6-mercaptopurine (6-MP). Figure 4b shows the results of immunoblot analysis of HPRT1 and Hsp90 (loading control) in AAVS1 control, HPRT1-KO#1, HPRT1-KO#3, and human HPRT1-overexpressing HPRT1-OE small cell lung cancer cell lines (HPRT1-KO#1 and HPRT1-KO#3). FIG. 4c shows the results of overexpressing exogenous HPRT1 in HPRT1-deficient cells (HPRT1-KO#1 and HPRT1-KO#3) and examining the effect on cell viability. Figure 4d shows the results of the proliferation efficiency of small cell lung cancer cells in 2D culture using physiological medium in the AAVS1 control, HPRT1-KO#1, HPRT1-KO#3, and human HPRT1-overexpressing HPRT1-OE small cell lung cancer cell lines (HPRT1-KO#1 and HPRT1-KO#3). n=4. Figure 4e shows the results of proliferation efficiency of small cell lung cancer cells in 3D culture using physiological medium in AAVS1 control, HPRT1-KO#1, HPRT1-KO#3, and human HPRT1 overexpressing HPRT1-OE small cell lung cancer cell lines (HPRT1-KO#1 and HPRT1-KO#3). n=4. FIG. 4f is a schematic diagram of de novo purine metabolism and salvage metabolism. FIG. 4g shows the dose-response curves of cell viability of AAVS1 control cells and HPRT1-KO small cell lung cancer cell lines (SBC-3 and SBC-5) treated with MTX. Figure 4h shows the effect of HPRT1 deficiency on tumor formation in mouse tumor-bearing transplantation assays. Data are mean + s.d. *P<0.05, **P<0.01, ***P<0.001 (Student t-test); NS, not significant. Overall, Figures 5a-c show that nucleotide salvage metabolism promotes the growth of glutamine-starved small cell lung cancer cells. Figure 5a shows the effect of adding hypoxanthine, a substrate used in salvage purine nucleic acid synthesis, to glutamine-starved medium on the proliferation of SBC-3 and SBC-5 cells. FIG. 5b is a dose-response curve of cell viability of SBC-3 and SBC-5 cells treated with 6-MP for 96 hours during glutamine starvation (n=4). Figure 5c shows the effect of 6-MP treatment combined with glutamine starvation on the proliferation of small cell lung cancer cell lines. Data are mean + s.d. *P<0.05, **P<0.01, ***P<0.001 (Student t-test); NS, not significant. Collectively, Figures 6a-e show that perturbation of host glutamine metabolism evokes sensitivity to nucleotide rescue inhibition in small cell lung cancer. Figure 6a is a schematic diagram of the chemical reaction of glutamine synthetase (GS). Figure 6b shows the growth of small cell lung cancer cells SBC-3 and SBC-5 treated with MSO under various glutamine concentrations (0, 300, 600 μM). Plasma glutamine and glutamate were measured using Glutamine/Glutamate-Glo (Promega). The thick line in the figure indicates the mean value. FIG. 6b shows dose-response curves of cell viability of SBC-3 and SBC-5 cells treated with MSO for 96 hours under glutamine starvation conditions (n=4). Figure 6d shows the change in tumor weight of SBC-3 and SBC-5 cell lines in nude mice by administration of MSO alone, MSO in combination with 6-MP, and MSO in combination with 6-MP and MTX. Data are mean + s.d. *P<0.05, **P<0.01, ***P<0.001 (Student t-test); NS, not significant. Figure 6e shows the results of viability analysis of SBC-3 and SBC-5 cell lines in nude mice treated with MSO alone, MSO in combination with 6-MP, and MSO in combination with 6-MP and MTX. Data are mean + s.d. *P<0.05, **P<0.01, ***P<0.001 (Student t-test); NS, not significant. Overall, Figure 7a-h show that the combination of MTX and 6-MP can provide an effective therapeutic intervention against cisplatin-resistant small cell lung cancer. Figure 7a shows the dose-response curves of cell viability of cDDP-resistant MS-1-L and STC-1 cells treated with cisplatin (cDDP) for 96 hours (n=4). Small cell lung cancer cells were cultured with the Cmax concentration for 48 hours, and the cell population that survived within 30 days was designated resistant-1 (R1), and this cycle was repeated three times, resulting in a cisplatin-resistant population designated resistant-3 (R3). FIG. 7b shows the results confirming in vivo cisplatin resistance of cisplatin-resistant small cell lung cancer cells MS-1-L and MS-1-L R3. FIG. 7c shows the results of confirming in vivo cisplatin resistance of cisplatin-resistant small cell lung cancer cells STC-1 and STC-1 R3. FIG. 7d shows dose-response curves of cell viability of cDDP-resistant MS-1-L and STC-1 cells treated with 6-MP, MTX, or 6-MP+MTX (n=4). FIG. 7e shows the results of investigating the tumor volume in cisplatin-resistant small cell lung cancer MS-1-L R3 and STC-1 R3 treated with 6-MP, MTX, or 6-MP+MTX. FIG. 7f shows the results of Kaplan-Meier survival curves in cisplatin-resistant small cell lung cancer MS-1-L R3 and STC-1 R3 treated with 6-MP, MTX, or 6-MP+MTX.

In one embodiment, the present invention provides a pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), such as 6-mercaptopurine (6-MP).

HPRT1 is an enzyme involved in purine metabolism, which is a metabolic pathway for the synthesis and decomposition of purine bases contained in living organisms. In the present invention, it was found that the purine synthesis pathway is a promising target for the treatment of small cell lung cancer. It is also believed that cells with high HPRT1 expression and high metabolic activity have enhanced sensitivity to 6-MP, which has the effect of competitively inhibiting the metabolic activity of HPRT1. In the present invention, it was shown that small cell lung cancer cell lines are more sensitive to growth inhibition by 6-MP than other cancer cells, and from this, it was inferred that small cell lung cancer has high HPRT1 activity (Figure 4).

The nucleic acid or DNA sequence encoding HPRT1 is as follows:
atg gcgacccgca gccctggcgt cgtgattagtgatgatgaac caggttatga ccttgattta ttttgcatac ctaatcatta tgctgaggatttggaaaggg tgtttattcc tcatggacta attatggaca ggactgaacg tcttgctcgagatgtgatga aggagatggg aggccatcac attgtagccc tctgtgtgct caaggggggctataaattct ttgctgacct gctggattac atcaaagcac tgaatagaaa tagtgatagatccattccta tgactgtaga ttttatcaga ctgaagagct attgtaatga ccagtcaacaggggacataa aagtaattgg tggagatgat ctctcaactt taactggaaa gaatgtcttgattgtggaag atataattga cactggcaaa acaatgcaga ctttgctttc cttggtcaggcagtataatc caaagatggt caaggtcgca agcttgctgg tgaaaaggac cccacgaagtgttggatata agccagactt tgttggattt gaaattccag acaagtttgt tgtaggatatgcccttgact ataatgaata cttcagggat ttgaatcatg tttgtgtcat tagtgaaactggaaaagcaa aatacaaagc ctaa (SEQ ID NO: 1)

In the present invention, the term "inhibition" in "inhibitor" refers to the process of slowing down or stopping purine metabolism as HPRT1 activity by inhibiting or suppressing the expression or activity of the HPRT1 protein.

In one embodiment of the present invention, the inhibitor of HPRT1 is 6-mercaptopurine. The chemical name of 6-mercaptopurine is 1,7-dihydro-6H-purine-6-thione, and it has been shown to be highly effective not only in treating acute childhood leukemia, but also in treating acute adult leukemia. 6-MP is currently used in the treatment of human leukemia, so there are few safety concerns.

In another embodiment of the present invention, the inhibitor of HPRT1 is an inhibitor of HPRT1 expression. In this embodiment, the inhibitor of HPRT1 expression is a substance that inhibits the expression of a nucleic acid sequence shown in either (a) or (b) below:
(a) the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing, or (b) a nucleic acid sequence containing a base sequence in which one or more bases have been deleted, substituted or added in the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing.

In the present invention, specific examples of inhibitors of HPRT1 expression include nucleic acid sequences that hybridize under stringent conditions with a base sequence complementary to a nucleic acid containing the base sequence shown in SEQ ID NO:1 in the sequence listing.

In the present invention, the mRNA may be any mRNA encoded by a target nucleic acid or one that encodes a protein encoded by a target nucleic acid. Preferably, the mRNA is encoded by the nucleic acid sequence of HPRT1.

In one embodiment of the present invention, the inhibitor of HPRT1 expression is preferably selected from the group consisting of siRNA, antisense and ribozyme.

The ribozyme of the present invention refers to an RNA molecule that specifically cleaves other single-stranded RNA molecules by a mechanism similar to that of DNA restriction endonucleases. By appropriately modifying the nucleic acid sequence of RNA using known techniques, it is possible to prepare a ribozyme that recognizes and cleaves a specific base sequence in a single-stranded RNA (Science, 239, p. 1412-1416, 1988).

The siRNA of the present invention refers to double-stranded RNA that suppresses the expression of a nucleic acid sequence (target nucleic acid), and means "RNAi agent", "short interfering RNA", "short interfering nucleic acid", or "siNA", and is a nucleic acid molecule that can inhibit or down-regulate gene expression or viral replication through sequence-specific RNA interference (RNAi) or gene silencing. It may be composed of only RNA, or may be a fusion of DNA and RNA. The siRNA can be prepared according to a conventional method from the target nucleic acid and information obtained by searching the NCBI database. In designing siRNA oligonucleotides, a sequence with a GC content as close to 50% as possible is selected from the coding region of the mRNA encoded by the target nucleic acid. The GC content is ideally between 45% and 55%. The region of 50 to 100 nucleotides near the AUG start codon or 50 to 100 nucleotides near the termination codon is avoided, and 19 nucleotides following AA (adenine-adenine) are selected, and dTdT (deoxythymine-deoxythymine) is added to the 3' end of this sense strand 19 nucleotides as an overhang. The overhang can be selected from RNA oligos or RNA/DNA chimeric oligos. Thymine (TT) or uracil (UU) is usually used as a two-base overhang, but other overhangs can also be used. However, the RNAi effect may be affected by blunting the siRNA end, overhanging only the 5' end, or changing the length of the overhang. The base sequence of the nucleotide should avoid sequences with three or more consecutive guanosines or cytosines, and it is necessary to confirm that there is no homology with any other genes. The antisense strand is a complementary strand of the sense 19 nucleotides, and dTdT is added to the 3' end. Based on the base sequence designed in this way, the sense strand and the antisense strand are synthesized using a DNA/RNA synthesizer. The synthesized sense strand and antisense strand are purified using a NAP-10 column or the like, concentrated and dried, dissolved again in a buffer, heated, and annealed to form a double-stranded siRNA. In addition to the method of preparing siRNA using a DNA/RNA synthesizer, it is also possible to incorporate the siRNA target sequence of the present invention together with a loop sequence into an expression vector and express it in a cell to suppress the expression of the target nucleic acid. It is also possible to express the sense strand and antisense strand of the siRNA target sequence of the present invention separately in a cell, hybridize them in the cell to form siRNA, and suppress the expression of the target nucleic acid. Furthermore, it is also possible to introduce a long dsRNA containing the siRNA target sequence of the present invention into a cell, cleave it in the cell to form siRNA, and suppress the expression of the target nucleic acid.

The siRNA of the present invention inhibits the expression of the nucleic acid sequence of HPRT1, and therefore can be used as a substance that regulates the expression of a target nucleic acid, as a pharmaceutical composition for preventing or treating small cell lung cancer.

A nucleic acid sequence having an antisense sequence of the present invention (sometimes simply referred to as antisense) is a nucleic acid having a base sequence that suppresses the expression of a target nucleic acid, and is prepared according to conventional methods. First, a candidate target site of the mRNA encoded by the target nucleic acid is selected. As a selection method, an mRNA higher-order structure prediction method based on energy calculation (Methods in Enzymol., 180, p. 262, 1989; Ann. Rev. Biophys. Biophys. Chem., 17, p. 167, 1988), a random oligo/RNase H method (Nucleic Acid Res., 25, p. 5010, 1997), a reverse transcriptase method (Nature Biotech, 15, p. 537, 1997), a fluorescent nucleic acid probe method (Nucleic Acid Res., 27, p. 2387, 1999), a FRET method (Biochemistry, 31, p. 12055, 1992), etc. can be used. Next, the sequence of the antisense oligonucleotide is determined from the candidate region of the selected mRNA. In this case, the chain length of the antisense oligonucleotide is generally 15-30 mer, and the antisense oligonucleotide is designed so that it does not form a double strand or a self-stem-loop structure, and a sequence with four or more consecutive Gs is avoided because it is likely to interact with proteins, and the "CpG" sequence in the antisense oligonucleotide is avoided because it binds to the receptor of B cells. The structure of the antisense oligonucleotide can be selected from phosphate-linked types (natural type, phosphorothioate type, methylphosphonate type, phosphoramidate type, 2'-O-methyl type) and non-phosphate types (morpholidate type, polyamide nucleic acid). It is also possible to introduce peptides or cholesterol to increase cell permeability, or to give crosslinking ability by introducing alkylating agents or photocrosslinking agents. The antisense oligonucleotide designed in this way is prepared by a DNA synthesizer or the like, and purified by reverse phase HPLC, ion exchange HPLC, gel electrophoresis, ethanol precipitation, etc.

The nucleic acid having the antisense sequence of the present invention inhibits the expression of the target nucleic acid (the nucleic acid sequence of HPRT1), and can therefore be used as a substance that regulates the expression of the target nucleic acid, as a pharmaceutical composition for preventing or treating small cell lung cancer.

<Pharmaceutical composition containing an HPRT1 inhibitor and a folic acid synthesis inhibitor>
In another embodiment, the present invention relates to a pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of HPRT1, such as 6-mercaptopurine, and an inhibitor of folic acid synthesis, such as methotrexate, pralatrexate, pemetrexed, or raltitrexed. In the present invention, it has been shown that the combination of HPRT1 inhibition and MTX may be an effective therapy for small cell lung cancer.

Methotrexate (MTX) is an immunosuppressant antirheumatic drug that is most commonly used worldwide to treat rheumatoid arthritis. MTX inhibits the activity of dihydrofolate reductase (DHFR), which reduces folic acid to active folate, which is necessary for nucleic acid synthesis, and depletes tetrahydrofolate (THF), thereby inhibiting the thymidylate synthesis pathway and suppressing cell proliferation. As a result, MTX also inhibits purine metabolism (DNA synthesis) and amino acid metabolism, and shows antitumor effects against a wide range of malignant tumors, including leukemia and breast cancer. Since MTX is currently used to treat human leukemia, it is thought that there are few safety issues. Although both 6-MP and MTX are used to treat leukemia, neither has been used to treat small cell lung cancer, and there is no evidence that a combination of the two is useful for treating small cell lung cancer.

The first-line treatment for small cell lung cancer is treatment with cisplatin-based platinum preparations, but the therapeutic effect is often transient, and most cases acquire cisplatin resistance. Cisplatin-resistant non-small cell lung cancer has recently been shown to undergo glutamine metabolic remodeling and to be more sensitive to glutamine starvation (30-32). It has also been shown that nucleic acid synthesis is promoted in cisplatin-resistant and various other chemotherapy-resistant cancers, and sensitivity to nucleic acid synthesis inhibitors is increased (30-32). In this invention, we investigated whether simultaneous inhibition of the de novo purine synthesis pathway (HPRT1 inhibitor) and the salvage purine synthesis pathway (folate synthesis inhibitor) is effective against cisplatin-resistant small cell lung cancer, and found that it shows a definitive therapeutic effect against not only cisplatin-sensitive small cell lung cancer but also cisplatin-resistant small cell lung cancer.

In yet another embodiment, the present invention relates to a pharmaceutical composition for treating and/or preventing cancer, comprising an inhibitor of HPRT1, an inhibitor of folate synthesis, and an inhibitor of glutamine synthetase, such as methionine sulfoximine.
In the present invention, glutamine depletion was shown to increase sensitivity to 6-MP. That is, glutamine is an essential amino acid for cancer growth, being both an essential nitrogen donor in de novo nucleotide biosynthesis and a supplementary substrate for the glutamate oxidation pathway of the TCA cycle (22, 23). It is known that cancer cells growing in a host organism are exposed to glutamine starvation. The glutamine concentration in human plasma is ∼600 μM, whereas the glutamine concentration in the outer layer of solid tumors is ∼400 μM and even ∼100 μM in the deeper layers of tumors (24-26). These findings suggest that cancer cells may rely on salvage pathways to maintain their proliferation and viability when glutamine supply is limited. To test this hypothesis, we investigated the effect of adding hypoxanthine to glutamine-starved medium on the growth of small cell lung cancer cells (Example 3). Hypoxanthine is a substrate used in salvage purine nucleic acid synthesis, and its presence allows nucleic acid precursors to be synthesized even in the absence of glutamine. Therefore, when cancer cells are exposed to glutamine starvation, HPRT1 may metabolize hypoxanthine and function as an escape route for the de novo nucleic acid synthesis pathway. In the present invention, we found that the addition of hypoxanthine in a glutamine starvation environment promotes the proliferation of small cell lung cancer cells. We also found that the combination of 6-MP treatment and glutamine starvation is more effective at inhibiting the proliferation of small cell lung cancer cells than either treatment alone, and that the additional treatment with MTX further enhances this effect.

The nucleic acid or DNA sequence encoding glutamine synthetase (GS) is as follows:
atgac cacctcagca agttcccact taaataaagg catcaagcag gtgtacatgtccctgcctca gggtgagaaa gtccaggcca tgtatatctg gatcgatggt actggagaaggactgcgctg caagacccgg accctggaca gtgagcccaa gtgtgtggaa gagttgcctgagtggaattt cgatggctct agtactttac agtctgaggg ttccaacagt gacatgtatctcgtgcctgc tgccatgttt cgggacccct tccgtaagga ccctaacaag ctggtgttatgtgaagtttt caagtacaat cgaaggcctg cagagaccaa tttgaggcac acctgtaaacggataatgga catggtgagc aaccagcacc cctggtttgg catggagcag gagtataccctcatggggac agatgggcac ccctttggtt ggccttccaa cggcttccca gggccccagggtccatatta ctgtggtgtg ggagcagaca gagcctatgg cagggacatc gtggaggcccattaccgggc ctgcttgtat gctggagtca agattgcggg gactaatgcc gaggtcatgcctgcccagtg ggaatttcag attggacctt gtgaaggaat cagcatggga gatcatctctgggtggcccg tttcatcttg catcgtgtgt gtgaagactt tggagtgata gcaacctttgatcctaagcc cattcctggg aactggaatg gtgcaggctg ccataccaac ttcagcaccaaggccatgcg ggaggagaat ggtctgaagt acatcgagga ggccattgag aaactaagcaagcggcacca gtaccacatc cgtgcctatg atcccaaggg aggcctggac aatgcccgacgtctaactgg attccatgaa acctccaaca tcaacgactt ttctgctggt gtagccaatcgtagcgccag catacgcatt ccccggactg ttggccagga gaagaagggt tactttgaagatcgtcgccc ctctgccaac tgcgacccct tttcggtgac agaagccctc atccgcacgtgtcttctcaa tgaaaccggc gatgagccct tccagtacaa aaattaa (SEQ ID NO: 2)

In the present invention, the term "inhibition" in "inhibitor" refers to the process of slowing down or stopping the production of glutamine by inhibiting or suppressing the expression or activity of GS protein.

In one embodiment of the present invention, the GS inhibitor is methionine sulfoximine. Methionine sulfoximine (MSO) inhibits the enzymatic activity of glutamine synthetase (GS) in synthesizing glutamine from glutamate and ammonia (Figure 6a).

In another embodiment of the present invention, the GS inhibitor is an inhibitor of GS expression. In this embodiment, the GS expression inhibitor is an inhibitor of the expression of a nucleic acid sequence shown in either (a) or (b) below:
(a) the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing, or (b) a nucleic acid sequence containing a base sequence in which one or more bases have been deleted, substituted or added in the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing.

In the present invention, specific examples of inhibitors of GS expression include nucleic acid sequences that hybridize under stringent conditions with a base sequence complementary to a nucleic acid containing the base sequence shown in SEQ ID NO:2 in the sequence listing.

In the present invention, the mRNA may be any mRNA encoded by a target nucleic acid or one that encodes a protein encoded by a target nucleic acid. Preferably, the mRNA is encoded by a nucleic acid sequence of GS.

In one embodiment of the present invention, the inhibitor of GS expression is preferably selected from the group consisting of siRNA, antisense and ribozyme.

The ribozyme of the present invention refers to an RNA molecule that specifically cleaves other single-stranded RNA molecules by a mechanism similar to that of DNA restriction endonucleases. By appropriately modifying the nucleic acid sequence of RNA using known techniques, it is possible to prepare a ribozyme that recognizes and cleaves a specific base sequence in a single-stranded RNA (Science, 239, p. 1412-1416, 1988).

The siRNA of the present invention refers to double-stranded RNA that suppresses the expression of a nucleic acid sequence (target nucleic acid), and means "RNAi agent", "short interfering RNA", "short interfering nucleic acid", or "siNA", and is a nucleic acid molecule that can inhibit or down-regulate gene expression or viral replication through sequence-specific RNA interference (RNAi) or gene silencing. It may be composed of only RNA, or may be a fusion of DNA and RNA. The siRNA can be prepared according to a conventional method from the target nucleic acid and information obtained by searching the NCBI database. In designing siRNA oligonucleotides, a sequence with a GC content as close to 50% as possible is selected from the coding region of the mRNA encoded by the target nucleic acid. The GC content is ideally between 45% and 55%. The region of 50 to 100 nucleotides near the AUG start codon or 50 to 100 nucleotides near the termination codon is avoided, and 19 nucleotides following AA (adenine-adenine) are selected, and dTdT (deoxythymine-deoxythymine) is added to the 3' end of this sense strand 19 nucleotides as an overhang. The overhang can be selected from RNA oligos or RNA/DNA chimeric oligos. Thymine (TT) or uracil (UU) is usually used as a two-base overhang, but other overhangs can also be used. However, the RNAi effect may be affected by blunting the siRNA end, overhanging only the 5' end, or changing the length of the overhang. The base sequence of the nucleotide should avoid sequences with three or more consecutive guanosines or cytosines, and it is necessary to confirm that there is no homology with any other genes. The antisense strand is a complementary strand of the sense 19 nucleotides, and dTdT is added to the 3' end. Based on the base sequence designed in this way, the sense strand and the antisense strand are synthesized using a DNA/RNA synthesizer. The synthesized sense strand and antisense strand are purified using a NAP-10 column or the like, concentrated and dried, dissolved again in a buffer, heated, and annealed to form a double-stranded siRNA. In addition to the method of preparing siRNA using a DNA/RNA synthesizer, it is also possible to incorporate the siRNA target sequence of the present invention together with a loop sequence into an expression vector and express it in a cell to suppress the expression of the target nucleic acid. It is also possible to express the sense strand and antisense strand of the siRNA target sequence of the present invention separately in a cell, hybridize them in the cell to form siRNA, and suppress the expression of the target nucleic acid. Furthermore, it is also possible to introduce a long dsRNA containing the siRNA target sequence of the present invention into a cell, cleave it in the cell to form siRNA, and suppress the expression of the target nucleic acid.

The siRNA of the present invention inhibits the expression of the nucleic acid sequence of GS, and therefore can be used as a substance that regulates the expression of target nucleic acids and as a pharmaceutical composition for preventing or treating small cell lung cancer.

A nucleic acid sequence having an antisense sequence of the present invention (sometimes simply referred to as antisense) is a nucleic acid having a base sequence that suppresses the expression of a target nucleic acid, and is prepared according to conventional methods. First, a candidate target site of the mRNA encoded by the target nucleic acid is selected. As the selection method, the mRNA higher-order structure prediction method based on energy calculation (Methods in Enzymol., 180, p. 262, 1989; Ann. Rev. Biophys. Biophys. Chem., 17, p. 167, 1988), the random oligo/RNase H method (Nucleic Acid Res., 25, p. 5010, 1997), the reverse transcriptase method (Nature Biotech, 15, p. 537, 1997), the fluorescent nucleic acid probe method (Nucleic Acid Res., 27, p. 2387, 1999), the FRET method (Biochemistry, 31, p. 12055, 1992), etc. can be used. Next, the sequence of the antisense oligonucleotide is determined from the candidate region of the selected mRNA. In this case, the chain length of the antisense oligonucleotide is generally 15-30 mer, and the antisense oligonucleotide is designed so that it does not form a double strand or a self-stem-loop structure, and a sequence with four or more consecutive Gs is avoided because it is likely to interact with proteins, and the "CpG" sequence in the antisense oligonucleotide is avoided because it binds to the receptor of B cells. The structure of the antisense oligonucleotide can be selected from phosphate-linked types (natural type, phosphorothioate type, methylphosphonate type, phosphoramidate type, 2'-O-methyl type) and non-phosphate types (morpholidate type, polyamide nucleic acid). It is also possible to introduce peptides or cholesterol to increase cell permeability, or to give crosslinking ability by introducing alkylating agents or photocrosslinking agents. The antisense oligonucleotide designed in this way is prepared by a DNA synthesizer or the like, and purified by reverse phase HPLC, ion exchange HPLC, gel electrophoresis, ethanol precipitation, etc.

The nucleic acid having the antisense sequence of the present invention inhibits the expression of the target nucleic acid (GS nucleic acid sequence), and can therefore be used as a substance that regulates the expression of the target nucleic acid, as a pharmaceutical composition for preventing or treating small cell lung cancer.

In this embodiment, "cancer" includes small cell lung cancer, pancreatic adenocarcinoma, glioblastoma, and the like.

In the present invention, "treatment" means a method or process intended to (1) delay the onset of cancer, such as small cell lung cancer; (2) slow or stop the progression, worsening or deterioration of symptoms of cancer, such as small cell lung cancer; (3) bring about remission of symptoms of cancer, such as small cell lung cancer; or (4) cure cancer, such as small cell lung cancer. Treatment may be administered prior to the onset of a disease or condition as a preventative measure, or treatment may be administered after the onset of a disease.

In the present invention, "prevention" means preventing the onset of cancer, such as small cell lung cancer, in advance.

The pharmaceutical composition of the present invention can be formulated by a method known to those skilled in the art. The pharmaceutical composition of the present invention can be administered either parenterally or orally. In the case of parenteral administration, the composition can be, for example, an injection type, a nasal administration type, a pulmonary administration type, or a transdermal administration type. For example, the composition can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. In the case of oral administration, the composition can be in the form of tablets, capsules, pills, granules, powders, syrups, or the like.

The administration method can be selected appropriately depending on the patient's age and symptoms. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can determine an appropriate dosage and administration method taking these conditions into account.

<Prognosis of small cell lung cancer>
In another aspect, the present invention provides a method for determining prognosis in small cell lung cancer, comprising the steps of:
(a1) measuring the amount of HPRT1 (test biomarker amount) in a subject;
(b1) comparing the amount of the test biomarker with the amount of HPRT1 in a healthy specimen (a control biomarker amount); and (c1) determining that the specimen has a poor prognosis for small cell lung cancer if the amount of the test biomarker is greater than the amount of the control biomarker.
In this regard, in yet another aspect, the present invention provides a biomarker that is HPRT1, which can determine the prognosis in small cell lung cancer. Furthermore, the present invention provides the use of HPRT1 as a biomarker that can determine the prognosis in small cell lung cancer.

The present inventors performed a Kaplan-Meier survival analysis comparing high and low expression levels of the HPRT1, DUT, and POLR2F genes in cancer tissues, and found that HPRT1, which controls the rate-limiting step of the salvage nucleic acid synthesis pathway, is a definitive prognostic marker for small cell lung cancer, and completed the determination method, biomarker, and use of blood HPRT1 of the present invention. If the prognosis of a subject can be determined in advance using the determination method, biomarker, and blood HPRT1 of the present invention, this can be of great help in subsequent cancer treatment and is also extremely beneficial for the prevention of small cell lung cancer.

To measure HPRT1 in a subject, cancer tissue is collected from a human patient, and the amount of HPRT1 therein can usually be measured by an immunological method. For example, it can be measured by an immunological method well known to those skilled in the art, such as Western blotting or immunostaining.

The present invention will be described in detail below with reference to examples and examples. However, it should be noted that these do not limit the scope of the present invention and are merely illustrative.

The materials and methods used in the examples are as follows.
Materials and Methods Patient and tissue samples
For the iMPQAT analysis, frozen tumor specimens were retrospectively collected from 36 patients with lung adenocarcinoma (n = 12), squamous cell carcinoma (n = 12), and limited-stage small cell lung cancer (n = 12) who underwent surgical resection at Kyushu University Hospital between January 2012 and July 2018. Frozen normal lung tissue specimens were available for 6 of the 12 patients with small cell lung cancer. In addition, formalin-fixed paraffin-embedded tumor samples were collected from 36 patients with untreated extensive-stage small cell lung cancer who underwent bronchoscopic biopsy at Kyushu University Hospital between July 2000 and December 2017 and used for immunohistochemical analysis. Patient characteristics, including age, sex, smoking history including Brinkman index (BI), histological type, TNM classification, driver oncogenes for 12 patients with adenocarcinoma, and type of epidermal growth factor receptor (EGFR) mutation, were extracted from medical records. This study complied with the Kyushu University "Ethical Guidelines for Medical Research Involving Human Subjects". Patient diagnoses were based on the WHO classification of lung tumors (42). This study adhered to the "Ethical Guidelines for Medical Research Involving Human Subjects" of the National Hospital Organization Kyushu Medical Center and Kyushu University. This study was conducted with ethical approval from the appropriate Institutional Review Boards (IRBs). The IRB number for Kyushu Medical Center is 20C076. The IRB numbers for Kyushu University Hospital are 2020-446 and 2021-102.

iMPAQT sample preparation: Frozen tissues were disrupted at 2500 rpm in a bead shocker (Yasuikikai MB1200), lysed in 100 μl of a solution containing 2% SDS, 7 M urea, and 100 mM Tris-HCl (pH 8.8), sonicated five times with 30 s intervals using a Bioruptor (Sonicbio), diluted with an equal volume of water, sonicated again using the same protocol, and protein concentration was measured by bicinchoninic acid (BCA) assay. To block cysteine/cysteine residues, 200 μg of protein was treated with 10 mM tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher) for 45 min at 37°C, followed by alkylation with 20 mM 2-iodoacetamide (Sigma) for 30 min at room temperature. These samples were vigorously vortexed with 5 volumes of acetone and left at -30°C for 1 h, followed by successive centrifugations in a swing rotor (Tomy AX-521) at 2000 g for 5 min and a fixed-angle rotor (KUBOTA 3520) at 12000 g for 2 min to obtain protein pellets. The protein pellets were washed twice with ice-cold 90% acetone, and then 100 μl of digestion buffer (50 mM triethylammonium bicarbonate) was added and sonicated under the same conditions as for the lysate treatment. Each sample was digested with lysyl endopeptidase (2 μg, Wako) at 37°C for 3 h, followed by further digestion with trypsin (4 μg, Thermo Fisher) at 37°C for 14 h. The resulting tissue digests were lyophilized and labeled with mTRAQ0 reagent (1U, SCIEX) at room temperature for 2 h.

MRM data analysis Peptides of metabolic enzymes were selected from the iMPAQT database and chemically synthesized. The peptides were labeled with mTRAQ4 reagents and added to the mTRAQ0-compatible sample digest. The corresponding MRM (multiple reaction monitoring) transitions with expected retention times were obtained from the iMPAQT database. MRM was performed using a triple quadrupole mass spectrometer (QTRAP6500, SCIEX) coupled to a liquid chromatography system (ACQUITY UPLC H-Class, Waters). The abundance of endogenous peptides was determined by multiplying the ratio of the summed light to heavy intensities for each transition by the known amount of synthetic peptide.

Immunohistochemical analysis of PPAT and HPRT-1 expression in small cell lung cancer. Immunohistochemical staining was performed on all 36 cases. Formalin-fixed, paraffin-embedded tissues were sectioned at 3 μm thickness. The primary antibody was HPRT-1 (dilution 1:150; Abcam, Cambridge, UK). Immune complexes were detected with the DAKO EnVision Detection System. Cancer cells showing cytoplasmic staining of HPRT-1 were scored using the Allred score. The cutoff values for HPRT-1 in the Allred score were set at 4 and 6. All immunohistochemical images were assessed by an investigator blinded to the results.

Physiological medium: DMEM (catalog no. 04833575, Wako) containing high glucose and sodium pyruvate and lacking amino acids, was supplemented with 10% dialyzed FBS and amino acids and hypoxanthine (1 μM) at concentrations similar to those present in human plasma (24).

Cell culture Cancer cells were cultured in physiological medium at 37°C under a 5% _CO2 atmosphere. Human cancer cell lines were confirmed to be free of mycoplasma contamination. For retroviral infection, amphotropic viruses produced in HEK293T cells transfected with both pCX4-EcoVR and pGP/pE-ampho (Takara Bio) were used to introduce retroviruses into cells via the mouse ecotropic virus receptor. Complementary DNA for HPRT1 in pCX4 was introduced into HEK293T cells together with pGP/pE-eco (Takara Bio) to produce recombinant retroviruses. Cancer cells expressing the mouse ecotropic retrovirus receptor were then infected with the retrovirus encoding HPRT1.

antibody
Antibody against HPRT1 (1:1000, ab32072) was obtained from Abcam, and antibody against Hsp90 (1:5000, 610419) was obtained from BD Transduction Laboratories.

Anticancer drug reagents
MTX was obtained from Fujifilm Wako (1391571). 6-MP was obtained from Sigma-Aldrich (852678). MSO was obtained from Sigma (M5379). GLS1 inhibitor CB-839 was obtained from Kaiman Chemical (22038). cDDP was obtained from Fujifilm Wako (033-20091).

HPRT1 knockout cells
To generate HPRT1-deficient SBC-3 or SBC-5 cells using the CRISPR-Cas9 system, complementary oligonucleotides corresponding to the target sequence of the HPRT1 single guide RNA (#1: 5'-CACCGAGCTGCTCACCACGACGCCA-3' (SEQ ID NO: 3) and 5'-AAACTGGCGTCGTGGTGAGCAGCTC-3' (SEQ ID NO: 4);
#3: 5'-CACCGCTCATGGACTAATTATGGAC-3' (SEQ ID NO:5) and 5'-AAACGTCCATAATTAGTCCATGAGC-3' (SEQ ID NO:6) or AAVS1 (5'-CACCGGTCCCCTCCACCCCACAGTG-3' (SEQ ID NO:7) and 5'-AAACCACTGTGGGGTGGAGGGGACC-3' (SEQ ID NO:8)) were annealed and subcloned into the BbsI site of pX330 (Addgene). Cells were transfected with 1 μg of plasmid using FuGENE HD Transfection Reagent (Promega). For selection of HPRT1-deficient cells, 6-thioguanine was added to the medium (2 μg/ml) and cells were grown for 10 days.

Cell viability assay For cell viability assay, cells were plated at a density of 2500 cells per well in 96-well assay plates (Corning). Cell proliferation and viability were determined from cell counts at the indicated times using the PrestoBlue assay. Dose-response curves were generated from quadruplicate biological measurements using a four-parameter logistic model using JMP pro 15 software (version 15.1.0).

Establishment of cDDP-resistant SCLC cells We treated SCLC cells with a chemotherapy regimen mimicking that used in clinical trials. SCLC cells were cultured for 48 hours with the maximum plasma concentration (Cmax) of cDDP in human patients, and the cell population that survived within 30 days was designated Resistant-1 (R1). This cycle was repeated three times to establish a cisplatin-resistant population designated Resistant-3 (R3).

Mice Female nude (Balb/c-nu/nu) mice were obtained from CLEA Japan. All mice were housed under pathogen-free conditions with a 12-h dark-light cycle and automatically provided food and water. All mouse experiments were approved by the Kyushu University Animal Ethics Committee.

Tumorigenicity assay
SBC-3 or SBC-5 cells (2.5×10 ⁶ ), MS-1-L or MS-1-L R3 or STC-1 or STC-1 R3 cells (1×10 ⁷ ) were subcutaneously injected into 8-10 week old female nude mice, and tumor size was measured every week thereafter. Tumor volume (mm ³ ) was calculated as follows: (longest diameter×2)/2. In Fig. 4h, mice were treated with MTX (25 mg/kg, once daily for 5 days) or vehicle (saline) via IP injection. In Fig. 6d, mice were treated with 6-MP (50 mg/kg, once daily for 5 days), MTX (12.5 mg/kg, once daily for 5 days), MSO (25 mg/kg, once daily for 5 days) or administration vehicle (saline) delivered via IP injection. In Figure 7e, mice were treated with 6-MP (50 mg/kg, once daily for 5 days), MTX (12.5 mg/kg, once daily for 5 days), MSO (25 mg/kg, once daily for 5 days), cDDP (2.5 mg/kg, once daily for 5 days) or treatment vehicle (saline) administered by IP injection. Mice were euthanized when tumors reached the size limit of 1.4 cm or when other symptoms indicative of declining health appeared. These included lethargy, weight loss (20% from the start of the experiment), decreased appetite, lack of exercise, hunched posture, and hair loss (43).

Statistical analysis Categorical variables were expressed as numbers, and continuous variables (age) were expressed as median (range). For categorical variables, statistical differences between HPRT1 expression and clinical and immunohistochemical characteristics were tested using Fisher's exact test. Overall survival (OS) was defined as the time from the date of initial surgery or initial diagnosis to the date of last follow-up or death. OS was estimated using the Kaplan-Meier method and log-rank test. A p-value < 0.05 was considered statistically significant. Quantitative data are presented as mean ± sd unless otherwise noted, and between-group comparisons were performed with paired two-tailed Student's t-test. Principal component analysis was performed on heat maps obtained from hierarchical clustering of measured metabolic enzymes. A P-value < 0.05 was considered statistically significant. All statistical analyses were performed using JMP v15 (SAS Institute, Cary, NC, USA).

Example 1
To clarify the metabolic phenotype of small cell lung cancer, tumor samples were obtained from 12 patients with small cell lung cancer, 12 patients with lung adenocarcinoma, and 12 patients with lung squamous cell carcinoma, and a comparative analysis of metabolic enzyme expression levels at the protein level was performed using iMPAQT analysis (Non-Patent Document 9) (21). The results are shown in Figure 1a.

Principal component analysis of the metabolic enzyme expression patterns revealed that small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma each had an independent metabolic enzyme expression signature (Figure 1b). Gene Set Enrichment Analysis (GSEA) was performed between these samples. GSEA analysis can examine which "gene sets" prepared in advance contain a large number of genes whose gene expression has increased or decreased. As a result, it was revealed that the expression of purine nucleotide and pyrimidine nucleotide synthetase groups was significantly increased in small cell lung cancer compared to lung adenocarcinoma and lung squamous cell carcinoma (Figure 1c, d). A comparative analysis of the metabolic enzyme expression levels of these small cell lung cancers, lung squamous cell carcinomas, and lung adenocarcinomas revealed that the increased expression of purine pyrimidine nucleotide synthetase is a metabolic enzyme expression pattern that characterizes small cell lung cancer.

Next, to identify the enzyme with the most significant increase in expression among the purine pyrimidine nucleotide synthetases that characterize small cell lung cancer, we used a volcano plot to compare and analyze the changes in expression of purine pyrimidine nucleotide synthetases in small cell lung cancer versus squamous cell carcinoma, and in small cell lung cancer versus adenocarcinoma. The results are shown in Figure 2a. Enzymes with increased expression in both small cell lung cancer versus squamous cell carcinoma, and in small cell lung cancer versus adenocarcinoma, were extracted (Figures 2a and b). As a result, it was revealed that the expression of the purine salvage pathway enzymes hypoxanthine-guanine phosphoribosyltransferase (HPRT1), RNA polymerase II, I and III subunit F (POLR2F), and the pyrimidine metabolic enzyme deoxyuridine triphosphatase (DUT) was significantly increased in small cell lung cancer compared to squamous cell carcinoma and adenocarcinoma (Fig. 2b). Among the factors HPRT1, POLR2F, and DUT extracted as characteristics of small cell lung cancer, it was revealed that the expression level of HPRT1 was most significantly increased when small cell lung cancer was compared to squamous cell carcinoma and adenocarcinoma (Fig. 2a, c). In addition, we confirmed the changes in the expression levels of HPRT1, POLR2F, and DUT in tumors larger than 3 cm and smaller than 3 cm, which require surgical intervention, and found that the expression level of HPRT1 increased with increasing tumor size (Fig. 2d). For HPRT1, POLR2F, and DUT, which were extracted as factors characterizing small cell lung cancer, we performed a comparative analysis of the expression levels using iMPAQT in normal tissues and cancer tissues derived from small cell lung cancer patients (Fig. 2e). As a result, among HPRT1, POLR2F, and DUT, the difference in expression level of HPRT1 between normal tissues and cancer tissues was the largest, suggesting that HPRT1 is involved in the cause of malignant progression of small cell lung cancer (Fig. 2e).

Kaplan-Meier analysis was performed to evaluate the influence of these HPRT1, POLR2F, and DUT factors on the prognosis of patients with small cell lung cancer. The results showed that increased expression of HPRT1 was the factor that most increased the risk of death in patients with small cell lung cancer and was a definitive prognostic marker (Figure 2f). Thus, a multifaceted analysis using metabolic enzyme quantification values by iMPAQT in patients with small cell lung cancer revealed that HPRT1 is a malignant factor that contributes to a significant increase in expression in small cell lung cancer and an increased risk of death.

Next, to reaffirm that HPRT1 is a malignant factor that contributes to an increased risk of death, immunohistochemical staining (IHC) was performed on tumor tissue from patients with extensive-stage small cell lung cancer, and Kaplan-Meier survival analysis was also performed (Fig. 3a, b). The results of the HPRT1 immunostaining analysis also demonstrated that HPRT1 is a malignant factor that contributes to an increased risk of death (Fig. 3b). A multifaceted analysis of the protein amounts of these metabolic enzymes quantified by iMPAQT suggested that increased HPRT1 expression is strongly associated with the malignancy of small cell lung cancer tumors.

Example 2
Deletion of the HPRT1 nucleic acid sequence suppresses tumorigenesis in small cell lung cancer
To investigate whether HPRT1 is a functional factor that contributes to the malignant progression of small cell lung cancer, we treated 10 small cell lung cancer cell lines and 20 other cancer cell lines with 6-mercaptopurine (6-MP) for 96 h in physiological medium and compared the sensitivity of small cell lung cancer cells to 6-MP with other cancer cells. The results showed that small cell lung cancer cells were more sensitive to 6-MP than other cancer cells (Fig. 4a). In addition to pharmacological experiments, we used the CRISPR-Cas9 system to genetically disrupt the HPRT1 nucleic acid sequence in two independent small cell lung cancer cell lines, SBC-3 (NIBIOHN) and SBC-5 (JCRB). Specifically, for SBC-3 and SBC-5, we performed immunoblot analysis of HPRT1 and Hsp90 (loading control) in AAVS1 control cells (Kanarek, N., Petrova, B. & Sabatini, DM Dietary modifications for enhanced cancer therapy. Nature 579, 507-517, (2020).), HPRT1-KO#1, HPRT1-KO#3, and human HPRT1-overexpressing HPRT1-KO small cell lung cancer cell lines (HPRT1-KO#1 and HPRT1-KO#3 in +HPRT1-OE) infected with human HPRT1 retrovirus. Immunoblot analysis was performed in a single run. Immunoblot analysis showed that HPRT1 protein expression was not detected in these cells (Figure 4b). For rescue experiments, HPRT1-deficient SBC-3 and SBC-5 cells (HPRT1-KO#1 and HPRT1-KO#3) were exogenously overexpressed with HPRT1. Specifically, small cell lung cancer cells (2.5×10 ³ ) were plated and cultured in physiological medium in the presence of DMSO control (DMSO) or 100 μM 6-MP. Cell numbers were determined by PrestoBlue on days 3 or 6 (n=4). These HPRT1-deficient small cell lung cancer cells were resistant to 6-MP, which becomes cytotoxic when metabolized by HPRT1, whereas AAVS1 control cells were sensitive to this drug (Fig. 4c). In 2D culture in physiological medium prepared with nutrient levels of human plasma, the growth efficiency of HPRT1-deficient small cell lung cancer cells was significantly reduced compared to the control, but this was restored by forced expression of exogenous HPRT1 in HPRT1-deficient small cell lung cancer cells (Fig. 4d). Furthermore, HPRT1-deficient SCLC cells also exhibited reduced anchorage-independent growth in 3D culture compared with AAVS1 control cells (Fig. 4d), indicating that cancer-specific proliferation in suspension was suppressed.

Because purine synthesis is carried out by both the de novo and salvage pathways (Fig. 4e), it is expected that when the salvage pathway is no longer functional due to HPRT1 deficiency, purine synthesis becomes more dependent on the de novo pathway to meet the cell growth requirements. Therefore, we performed dose-response curves (n=4) of cell viability in AAVS1 control cells and HPRT1-KO small cell lung cancer cell lines (SBC-3 and SBC-5) treated for 96 hours with methotrexate (MTX), an inhibitor of folate metabolism that indirectly inhibits the de novo pathway. As expected, these HPRT1-deficient small cell lung cancer cells were more sensitive to MTX than AAVS1 control cells, and tumor size and tumor weight were suppressed (Fig. 4e).

Furthermore, we investigated the effect of HPRT1 deficiency on tumor formation in mouse tumor-bearing transplantation assays. That is, the tumorigenicity of AAVS1 control cells and HPRT1-KO small cell lung cancer cell lines in nude mice was examined for tumor volume (left panel) and tumor weight (right panel) in subcutaneous transplants treated with the indicated MTX (25 mg/kg, 5 days). MTX administration alone moderately suppressed tumor formation of AAVS1 control SBC-3 and SBC-5 cells (Fig. 4h). Transplantation of HPRT1-deficient small cell lung cancer cells resulted in much more tumor suppression than AAVS1 controls, and this reduction in tumor size was even more pronounced with MTX treatment. These results suggest that HPRT1 is essential for small cell lung cancer cell proliferation in vitro and tumor formation in vivo, and that the combination of HPRT1 inhibition and MTX may be an effective therapy for small cell lung cancer.

Example 3
To test the possibility that cancer cells rely on salvage pathways to maintain proliferation and viability when glutamine supply is limited, we examined the effect of adding hypoxanthine, a substrate used in salvage purine nucleic acid synthesis, to glutamine-starved medium on the proliferation of SBC-3 and SBC-5 cells. Small cell lung cancer cells (2.5 × 10 ³ ) were plated and then cultured in physiological medium in the presence of vehicle (saline) alone or physiological levels of hypoxanthine (1 μM or 10 μM). Cell numbers were determined by PrestoBlue at 96 h (n=4). The results showed that the addition of hypoxanthine improved the inhibition of proliferation of SBC-3 or SBC-5 under glutamine-starved conditions (Figure 5a). We next examined the sensitivity of SBC-3 and SBC-5 cells to 6-MP at glutamine concentrations of 0, 300, and 600 μM (Figure 5b). The results showed that the sensitivity of these small cell lung cancer cells to 6-MP was significantly increased under glutamine starvation conditions. Furthermore, the effect of the combination of 6-MP treatment and glutamine starvation on the proliferation of small cell lung cancer cell lines was examined. That is, equal numbers of cells (2.5 × 10 ³ ) of 10 independent small cell lung cancer cell lines were plated and then cultured under various cell culture conditions. 6-MP, MTX and CB-839 were added at 1 μM. Cell numbers were determined after 96 h by PrestoBlue (n = 4). The results showed that the combination of 6-MP treatment and glutamine starvation was more effective at inhibiting the proliferation of 10 independent small cell lung cancer cell lines than each treatment alone, and additional treatment with MTX further enhanced this effect (Figure 5c). Glutamine is largely metabolized via the nucleic acid synthesis and glutaminolysis pathways. To demonstrate that the glutaminolysis pathway was not being interfered with under the above conditions, we investigated whether inhibition of the rate-limiting enzyme GLS1 in the glutaminolysis pathway with CB-839 did not inhibit the proliferation of small cell lung cancer cells. As a result, most small cell lung cancer cell lines were resistant to CB-839, but some cells showed increased proliferation in the presence of CB-839 at physiological glutamine levels (600 μM). This verified that the perturbation of glutamine starvation in small cell lung cancer cells did not affect the glutaminolysis pathway, but rather the nucleic acid synthesis pathway. These results suggest that the de novo and salvage pathways are mutually dependent, and that the dependency on HPRT1 activity increases under glutamine-limited conditions.

Next, the effect of a decrease in glutamine supply on the growth of small cell lung cancer was examined in mice using a pharmacological approach. Methionine sulfoximine (MSO) inhibits the enzyme activity of glutamine synthetase (GS), which synthesizes glutamine from glutamate and ammonia (Fig. 6a). Administration of MSO to mice indeed reduced blood glutamine levels (Fig. 6b). This was consistent with previous studies in chickens (27). Small cell lung cancer cells SBC-3 and SBC-5 were treated with MSO under various glutamine concentrations (0, 300, and 600 μM) and the growth of these small cell lung cancer cells was examined. As in previous studies (28, 29), MSO treatment only moderately inhibited the growth of small cell lung cancer cells under conditions where glutamine was completely depleted, but did not inhibit the growth of small cell lung cancer cells under conditions where glutamine was supplied from the culture medium (Fig. 6c).

Next, we investigated the changes in tumor weight and survival rate of small cell lung cancer cells in nude mice by administration of MSO alone, the combination of MSO and 6-MP, and the additional combination of MSO, 6-MP, and MTX. That is, 5 days after transplantation of small cell lung cancer SBC-3 or SBC-5, nude mice were treated with MSO (25 mg/kg), MSO (25 mg/kg)/6-MP (50 mg/kg), and MSO (20 mg/kg)/6-MP (50 mg/kg)/MTX (12.5 mg/kg) for 5 days. As a result, in the mouse tumor-bearing model of SBC-3 or SBC-5, administration of MSO alone had no therapeutic effect, but the combination of MSO and 6-MP was significantly more effective in suppressing the growth of small cell lung cancer cells than administration of 6-MP alone (Figure 6d). This therapeutic effect was further enhanced by additional treatment with MTX. Indeed, mice treated with MSO + 6-MP or MSO + 6-MP + MTX did not die within the observation period of ~3 months after small cell lung cancer cells were transplanted into the mice (Fig. 6e). These results suggest that small cell lung cancer is more sensitive to 6-MP under conditions of limited glutamine availability, and that disruption of host glutamine metabolism by MSO enhances the efficacy of anticancer therapy by inhibiting the salvage pathway of small cell lung cancer in vivo.

Based on these results, if we can disrupt the host's glutamine metabolism and reduce the amount of glutamine in the host, it may be possible to enhance the 6-MP sensitivity of small cell lung cancer in vivo. To test this hypothesis, we performed an intervention method to manipulate the host's plasma glutamine level. To do this, we need to inhibit glutamine synthesis in the host to disrupt the host's glutamine metabolism in vivo and limit the supply of glutamine to cancer cells. Methionine sulfoximine (MSO) is known as a glutamine synthetase (GS) specific inhibitor (Fig. 6a), and it can suppress the amount of glutamine in plasma and organs such as the brain and liver in rodents and other animals. We administered MSO IP at 25 mg/kg to BALBc/Ajcl nu/nu nude mice and measured the plasma glutamine concentration, and found that the decrease in plasma glutamine concentration was sustained for up to 24 hours after administration (Fig. 6b). To confirm the direct growth-inhibitory effect of GS on small cell lung cancer, we varied the glutamine concentration in an in vitro culture environment and found that GS did not inhibit the growth of small cell lung cancer cells when glutamine was supplied from outside the cells (Fig. 6c). GS only slightly inhibited the growth of small cell lung cancer cells under the condition of complete glutamine depletion in an in vitro culture environment (22) (Fig. 6c). To confirm whether the perturbation of host plasma glutamine metabolism by MSO enhances sensitivity to 6-MP, we performed a xenograft assay. MSO alone did not show a significant tumor suppressive effect compared to the vehicle control of SBC-3 and SBC-5 (Fig. 6d, e). 6-MP alone showed a significant tumor suppressive effect compared to the vehicle control of SBC-3 and SBC-5 (Fig. 6d, f). Co-administration of MSO and 6-MP to SBC-3 and SBC-5 showed a significant and marked tumor suppression effect compared to vehicle control and extended the survival time of mice (Fig. 6d, e). Co-administration of MSO, 6-MP, and MTX to SBC-3 and SBC-5 also showed a significant and marked tumor suppression effect compared to vehicle control and further extended the survival time of mice compared to MSO and 6-MP (Fig. 6d, e). These results demonstrate that small cell lung cancer cells show increased sensitivity to 6-MP under conditions of limited glutamine availability in vitro and in vivo, and that perturbation of host glutamine metabolism using MSO enhances the efficacy of anticancer therapies via the salvage pathway.

Example 5
Cisplatin-resistant small cell lung cancer is sensitive to glutamine starvation and inhibition of nucleic acid synthesis
We investigated whether simultaneous inhibition of the de novo and salvage purine synthesis pathways is effective against cisplatin-resistant small cell lung cancer. To this end, we exposed small cell lung cancer cell lines MS-1-L and STC-1 to cisplatin to induce metabolic adaptation that occurs in cisplatin resistance. Exposure to 10 μM cisplatin, which corresponds to the maximum therapeutic concentration of cisplatin in human plasma (33), was repeated for 48 h in one cycle, and the exposure was continued until the third cycle, at which point cisplatin resistance appeared in the small cell lung cancer lines (Fig. 7a).

As a result, in vivo cisplatin resistance was confirmed in mouse tumor-bearing transplantation assays using these cisplatin-resistant small cell lung cancer cells (Fig. 7b, c). These cisplatin-resistant (cDDP-resistant) MS-1-L and STC-1 cells were treated with either 6-MP or MTX, or 6-MP + MTX, and cell viability of the cDDP-resistant small cell lung cancer cells was examined. The results showed that either 6-MP or MTX, or both, suppressed the growth of cisplatin-resistant small cell lung cancer lines more effectively than the control parental small cell lung cancer lines in some cases (Fig. 7d). Next, cisplatin-resistant small cell lung cancer MS-1-L R3 and STC-1 R3 were treated with 6-MP (50 mg/kg) + MTX (12.5 mg/kg) and MSO (25 mg/kg) + 6-MP (50 mg/kg) + MTX (12.5 mg/kg). As a result, MTX and 6-MP showed significant tumor growth inhibition compared to the control group in both cisplatin-sensitive and cisplatin-resistant small cell lung cancer (Fig. 7e, f). Combination therapy of MSO, 6-MP and MTX showed significant tumor inhibition compared to the control group in both cisplatin-sensitive and cisplatin-resistant small cell lung cancer, and further improved mouse survival rate compared to MSO and 6-MP combination therapy (Fig. 7e, f). These results suggest that the combination of manipulating plasma glutamine levels by MSO and inhibiting nucleic acid synthesis showed a decisive therapeutic effect not only against cisplatin-sensitive but also against cisplatin-resistant small cell lung cancer, and may be a novel treatment for small cell lung cancer.

The present invention can include the following embodiments.
<Pharmaceutical composition containing an HPRT1 inhibitor>
[101]
A pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1).
[102]
The pharmaceutical composition according to [101], wherein the HPRT1 inhibitor is 6-mercaptopurine.
[103]
The pharmaceutical composition according to [101], wherein the HPRT1 inhibitor is an inhibitor of HPRT1 expression.
[104]
The pharmaceutical composition according to [103], wherein the inhibitor of HPRT1 expression is a substance that inhibits the expression of a nucleic acid sequence shown in either (a) or (b) below:
(a) the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing, or (b) a nucleic acid sequence containing a base sequence in which one or more bases have been deleted, substituted or added in the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing.
[105]
The pharmaceutical composition according to [103] or [104], wherein the inhibitor of HPRT1 expression is a nucleic acid sequence that hybridizes under stringent conditions with a base sequence complementary to the nucleic acid sequence of HPRT1 shown in sequence number 1 of the sequence listing.
[106]
The pharmaceutical composition according to [103] or [104], wherein the inhibitor of HPRT1 expression is selected from the group consisting of siRNA, antisense and ribozyme.
[107]
The pharmaceutical composition according to any one of [101] to [106], wherein the small cell lung cancer is resistant to cisplatin.

<Pharmaceutical composition containing an HPRT1 inhibitor and a folic acid synthesis inhibitor>
[108]
A pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of HPRT1 and an inhibitor of folic acid synthesis.
[109]
The pharmaceutical composition according to [108], wherein the HPRT1 inhibitor is 6-mercaptopurine.
[110]
The pharmaceutical composition according to [108] or [109], wherein the folic acid synthesis inhibitor is selected from methotrexate, pralatrexate, pemetrexed, and raltitrexed.
[111]
The pharmaceutical composition according to any one of [108] to [110], wherein the small cell lung cancer is resistant to cisplatin.

Pharmaceutical Compositions Containing an HPRT1 Inhibitor, a Folic Acid Synthesis Inhibitor, and a Glutamine Synthetase Inhibitor
[112]
A pharmaceutical composition for treating and/or preventing cancer, comprising an inhibitor of HPRT1, an inhibitor of folate synthesis, and an inhibitor of glutamine synthetase (GS).
[113]
The pharmaceutical composition according to [112], wherein the GS inhibitor is methionine sulfoximine.
[114]
The pharmaceutical composition according to [112], wherein the GS inhibitor is an inhibitor of GS expression.
[115]
The pharmaceutical composition according to [114], wherein the inhibitor of GS expression is a substance that inhibits the expression of a nucleic acid sequence shown in either (a) or (b) below:
(a) the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing, or (b) a nucleic acid sequence containing a base sequence in which one or more bases have been deleted, substituted or added in the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing.
[116]
The pharmaceutical composition according to [114] or [115], wherein the inhibitor of GS expression is a nucleic acid sequence that hybridizes under stringent conditions with a base sequence complementary to the nucleic acid sequence of HPRT1 shown in sequence number 2 of the sequence listing.
[117]
The pharmaceutical composition according to [114] or [115], wherein the inhibitor of GS expression is selected from the group consisting of siRNA, antisense and ribozyme.
[118]
The pharmaceutical composition according to any one of [112] to [117], wherein the small cell lung cancer is resistant to cisplatin.
[119]
The pharmaceutical composition according to any one of [112] to [118], wherein the folic acid synthesis inhibitor is selected from methotrexate, pralatrexate, pemetrexed, and raltitrexed.
[120]
The pharmaceutical composition according to any one of [112] to [119], wherein the cancer is small cell lung cancer, pancreatic adenocarcinoma, or glioblastoma.
[121]
The pharmaceutical composition according to [120], wherein the cancer is small cell lung cancer.
[122]
The pharmaceutical composition according to [121], wherein the small cell lung cancer is cisplatin-resistant.

<Prognosis of small cell lung cancer>
[123]
A method for determining prognosis in small cell lung cancer, comprising:
(a1) measuring the amount of HPRT1 (test biomarker amount) in a subject;
(b1) comparing the amount of a test biomarker with the amount of HPRT1 in a healthy specimen (a control biomarker amount); and (c1) determining that the specimen has a poor prognosis for small cell lung cancer if the amount of the test biomarker is greater than the amount of the control biomarker.
[124]
A biomarker, HPRT1, that can determine prognosis in small cell lung cancer.
[125]
Use of HPRT1 as a prognostic biomarker in small cell lung cancer.

The references cited herein by number are as follows:
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Claims (25)

A pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1). The pharmaceutical composition according to claim 1, wherein the HPRT1 inhibitor is 6-mercaptopurine. The pharmaceutical composition according to claim 1, wherein the HPRT1 inhibitor is an inhibitor of HPRT1 expression. The pharmaceutical composition according to claim 3, wherein the inhibitor of HPRT1 expression is a substance that inhibits the expression of a nucleic acid sequence shown in either (a) or (b) below:
(a) the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing, or (b) a nucleic acid sequence containing a base sequence in which one or more bases have been deleted, substituted or added in the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing. The pharmaceutical composition according to claim 4, wherein the inhibitor of HPRT1 expression is a nucleic acid sequence that hybridizes under stringent conditions with a base sequence complementary to the nucleic acid sequence of HPRT1 shown in SEQ ID NO:1 in the sequence listing. The pharmaceutical composition according to claim 4, wherein the inhibitor of HPRT1 expression is selected from the group consisting of siRNA, antisense and ribozyme. The pharmaceutical composition according to any one of claims 1 to 6, wherein the small cell lung cancer is cisplatin-resistant. A pharmaceutical composition for treating and/or preventing small cell lung cancer, comprising an inhibitor of HPRT1 and an inhibitor of folic acid synthesis. The pharmaceutical composition according to claim 8, wherein the HPRT1 inhibitor is 6-mercaptopurine. The pharmaceutical composition according to claim 9, wherein the folic acid synthesis inhibitor is selected from methotrexate, pralatrexate, pemetrexed, and raltitrexed. The pharmaceutical composition according to any one of claims 8 to 10, wherein the small cell lung cancer is cisplatin-resistant. A pharmaceutical composition for treating and/or preventing cancer, comprising an inhibitor of HPRT1, an inhibitor of folate synthesis, and an inhibitor of glutamine synthetase (GS). The pharmaceutical composition according to claim 12, wherein the GS inhibitor is methionine sulfoximine. The pharmaceutical composition according to claim 12, wherein the GS inhibitor is an inhibitor of GS expression. The pharmaceutical composition according to claim 14, wherein the inhibitor of GS expression is a substance that inhibits the expression of a nucleic acid sequence shown in either (a) or (b) below:
(a) the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing, or (b) a nucleic acid sequence containing a base sequence in which one or more bases have been deleted, substituted or added in the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing. The pharmaceutical composition according to claim 15, wherein the inhibitor of GS expression is a nucleic acid sequence that hybridizes under stringent conditions with a base sequence complementary to the nucleic acid sequence of HPRT1 shown in SEQ ID NO:2 in the sequence listing. The pharmaceutical composition according to claim 15, wherein the inhibitor of GS expression is selected from the group consisting of siRNA, antisense and ribozyme. The pharmaceutical composition according to any one of claims 12 to 17, wherein the small cell lung cancer is cisplatin-resistant. The pharmaceutical composition according to claim 12, wherein the folic acid synthesis inhibitor is selected from methotrexate, pralatrexate, pemetrexed, and raltitrexed. The pharmaceutical composition according to claim 19, wherein the cancer is small cell lung cancer, pancreatic adenocarcinoma, or glioblastoma. The pharmaceutical composition according to claim 20, wherein the cancer is small cell lung cancer. The pharmaceutical composition according to claim 21, wherein the small cell lung cancer is cisplatin-resistant. A method for determining prognosis in small cell lung cancer, comprising:
(a1) measuring the amount of HPRT1 (test biomarker amount) in a subject;
(b1) comparing the amount of a test biomarker with the amount of HPRT1 in a healthy specimen (a control biomarker amount); and (c1) determining that the specimen has a poor prognosis for small cell lung cancer if the amount of the test biomarker is greater than the amount of the control biomarker. HPRT1 is a biomarker that can determine prognosis in small cell lung cancer. The use of HPRT1 as a biomarker for determining prognosis in small cell lung cancer.