A combination therapy for the treatment of non-small-cell lung cancer

Abstract

The present invention relates to a combination therapy for use in the treatment of cancer and a method for determining the response of cancer patients to a cancer treatment.

Description

[0001] A COMBINATION THERAPY FOR THE TREATMENT OF NON-SMALL-CELL LUNG CANCER

[0002] Technical Field

[0003] The present invention relates to a novel combination therapy for use in the treatment of cancer. A method for determining the treatment response of cancer patients is also presented.

[0004] Background of the Invention

[0005] Non-small-cell lung cancer (NSCLC), or non-small-cell lung carcinoma, is any type of epithelial lung cancer other than small-cell lung cancer (SCLC). NSCLC accounts for about 85% of all lung cancers. Patients with NSCLC are often diagnosed with aggressive / metastatic disease which has limited treatment options.

[0006] Chemotherapy is one of the first-line therapies in NSCLC patients. Cisplatin is a slightly more effective platinum-based treatment option. However, NSCLC patients may show de novo or acquired resistance to cisplatin. Moreover, cisplatin has been associated with wide range of side effects (Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. European journal of pharmacology 2014; 740: 364-378; Olaussen KA, et. al. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. The New England journal of medicine 2006; 355: 983-991). Molecular targeted therapies have significantly improved the prognosis of driver mutation positive NSCLC. However, in a vast majority of NSCLC cases, the carcinogenic driver is unknown (Haiyang Guo, et. al. Biomarker-Targeted Therapies in Non-Small Cell Lung Cancer: Current Status and Perspectives Cells. 2022; 11(20): 3200).

[0007] More recently, immunotherapy (IM) has had remarkable clinical success. The introduction of immune checkpoint inhibitors (ICIs), such as anti-PD-1, anti-PD-Ll and anti-CTLA-4, alone or in combination with chemotherapy, has had remarkable clinical benefit in lung cancer and melanoma patients (Liu, S.Y.M., et. al. Emerging Evidence and Treatment Paradigm of Non-Small Cell Lung Cancer. J. Hematol. Oncol. 2023, 16, 40; Otegui N, et. al. Cancer Cell-Intrinsic Alterations Associated with an Immunosuppressive Tumor Microenvironment and Resistance to Immunotherapy in Lung Cancer. Cancers (Basel). 2023 Jun 6;15(12):3076; Doroshow, D.B., et. al. Immunotherapy in Non-Small Cell Lung Cancer: Facts and Hopes. Clin. Cancer Res. 2019, 25, 4592-4602). In 2015, two ICIs targeting programmed cell death-1 (PD-1), namely nivolumab and pembrolizumab, were approved by Food and Drug Administration (FDA) for second-line therapy of NSCLC. Moreover, pembrolizumab also received approval in 2016 for first-line NSCLC treatment in patients with tumors expressing high PD-L1. However, nearly half of lung cancer patients will not respond to I Cis, and most responders will acquire resistance during the course of treatment, which deems it necessary to develop new treatment approaches (Topalian SL, et. al. Five-Year Survival and Correlates Among Patients With Advanced Melanoma, Renal Cell Carcinoma, or Non-Small Cell Lung Cancer Treated With Nivolumab.JAMA Oncol. 2019;5(10):1411-1420).

[0008] Radiation therapy (radiotherapy, RT) has emerged as a well-tolerated alternative to surgical resection in medically inoperable patients. In light of the historical challenges associated with radiation therapy, there is now compelling evidence to suggest that recent advances in delivery modalities have led to a surge in interest in this treatment approach. Nevertheless, the majority of large-scale, randomized trials have not demonstrated any benefit from the use of radiation therapy (Tannock IF. Combined modality treatment with radiotherapy and chemotherapy. Radiother Oncol. 1989 0ct;16(2):83-101). Consequently, it is necessary to combine different treatment methods with radiotherapy in order to achieve the desired results.

[0009] Recently, radio-immunotherapy combination therapy has emerged as a promising treatment option. The results of the KEYNOTEOOl study revealed that patients with advanced NSCLC who previously had radiotherapy showed improved survival when treated with pembrolizumab, compared to those who did not have previous radiotherapy (Dovedi S], et. al. Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res. 2014 Oct l;74(19):5458-68; Gong X, et. al. Combined Radiotherapy and Anti-PD-Ll Antibody Synergistically Enhances Antitumor Effect in Non-Small Cell Lung Cancer. J Thorac Oncol. 2017 ]ul;12(7):1085- 1097). Further trials are needed to determine the best treatment option for advanced non-small cell lung cancer, considering the impact of prior radiotherapy on the efficacy of immune checkpoint inhibitors. A further recent phase 2 study (PEMBRO-RT) demonstrated that patients with advanced NSCLC exhibited significantly enhanced survival outcomes when they received pembrolizumab following stereotactic body radiation therapy (SBRT), particularly those with negative PD-L1 expression. However, the augmented response and survival benefits observed in the experimental group did not reach a statistically significant level in comparison to the control group (Meng L, et. al. (2021 ) The Combination of Radiotherapy With Immunotherapy and Potential Predictive Biomarkers for Treatment of Non-Small Cell Lung Cancer Patients. Front. Immunol. 12:723609; Theelen WSME, et. al. Effect of Pembrolizumab After Stereotactic Body Radiotherapy vs Pembrolizumab Alone on Tumor Response in Patients With Advanced Non-Small Cell Lung Cancer: Results of the PEMBRO-RT Phase 2 Randomized Clinical Trial. JAMA Oncol. 2019 Sep 1;5(9):1276- 1282). This means combining radiotherapy with immunotherapy shows potential benefits, but so far the results from randomized clinical trials have been inconclusive. The results of clinical trials on the effects of radiation on the immune system have been inconsistent due to the unclear biological mechanisms involved. Radiation can either stimulate or suppress the immune system, but the specific mechanisms involved are poorly understood. Although radiotherapy induces neoantigens, which trigger the host immune response, cytotoxic radiotherapy also kills host immune cells, which are required for an anti-tumour immune response. Furthermore, radiotherapy results in the generation of subclonal mutations that are associated with immune evasion and inadequate response to ICIs (Schreiber RD, Old LJ MJS. Cancer Immunoediting: Integrating Immunity's Roles in Cancer Suppression and Promotion. Science (2011) 331(6024):1565-70).

[0010] Several mechanisms within the tumor microenvironment have been proposed to explain the suppression of immune cells. These include low levels of oxygen, high levels of lactate, and high competition for glucose, which have the potential to contribute to T-cell dysfunction in the tumor microenvironment (Chelakkot C, et. al. Modulating Glycolysis to Improve Cancer Therapy. Int J Mol Sci. 2023 Jan 30;24(3):2606). Deregulation of glucose metabolism in tumors may lead to immune cell dysfunction, which may account for the failure of immunotherapy. As tumor cells have high rates of glycolysis, they are also likely to produce large amounts of the by-product lactate. Lactic acid has a suppressive effect on CD4+ and CD 8+ T cells by reducing proliferation and cytokine (Ruoshi Shi, et. al. Organoid Cultures as Preclinical Models ofNon-Small Cell Lung Cancer. Clin Cancer Res., 20201;26(5):1162-1174; Cunha BR, et. al. Cellular interactions in the tumor Microenvironment: The role of Secretome. J Cancer 2019; 10:4574-87; Kathleen A. Luckett and Karuna Ganesh. Engineering the Immune Microenvironment into Organoid Models. Anna. Rev. Cancer Biol. 2023. 7:171-187; Jin Yuan, et. al. Cancer organoid co-culture model system: Novel approach to guide precision medicine. Front Immunol. 2022; 13: 1061388). Furthermore, the competition for glucose between cancer cells and immune cells leads to the activation of glycolysis in cancer cells. This affects the function and survival of T-cells and is linked to higher expression of glycolysis-related genes in cancer cells, resulting in lower T-cell infiltration (Weng, C.-Y., et. al. Immuno-Metabolism: The Role of Cancer Niche in Immune Checkpoint Inhibitor Resistance. Int. J. Mol. Sci. 2021, 22, 1258).

[0011] As with immune-resistance, numerous studies have demonstrated that the Warburg effect, or aerobic glycolysis, contributes to the radio-resistance of various malignant tumors. Ionising radiation induces DNA damage, which, if left unrepaired, results in programmed cell death or apoptosis. The induction of glycolysis is linked to radio-resistance, as it facilitates the DNA repair process (Bhattacharya S, Asaithamby A. Repurposing DNA repair factors to eradicate tumor cells upon radiotherapy. TransI Cancer Res. 2017 JuI;6(SuppI 5):S822-S839; Front. Cell Dev. Biol., 2021. Cell Metabolism and DNA Repair Pathways: Implications for Cancer Therapy. Front. Cell Dev. Biol. 9:633305). It has been demonstrated that DNA repair deficiency regulates the immune response in cancers, since the development of effective immune responses depends on the generation of genetically diverse antigen receptors in T and B cells, which is achieved through the processes of DNA rearrangement and repair (Xu et al., Cancers (Basel). 2023 Mar; 15(5): 1619. DNA Repair Deficiency Regulates Immunity Response in Cancers: Molecular Mechanism and Approaches for Combining Immunotherapy). Concurrently, the objective of radiotherapy is to inflict sufficient DNA damage upon cancer cells to exceed their capacity for repair, thereby leading to cell death. Recent studies suggest a link between DNA repair and glycolysis. In particular, new functions for glycolytic proteins in DNA repair pathways have been suggested, mainly based on the observation that several glycolytic proteins, including Hexokinase II, Fumarase, and ATP-citrate lyase (ACLY), relocate to the nucleus after being exposed to genomic stress (Ohba S, Johannessen TA, Chatla K, Yang X, Pieper RO, Mukherjee J. Phosphoglycerate Mutase 1 Activates DNA Damage Repair via Regulation ofWIPl Activity. Cell reports 2020; 31: 107518, Hitosugi T., Zhou L., ElfS., Fan J., Kang H. B., Seo J. H., et al. (2012). Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth. Cancer Cell 22 585-600; Van Vugt M. A. T. M. (2017). Shutting down the power supply for DNA repair in cancer cells. J. Cell Biol. 216295-297; Yuan W., Wu S., Guo ]., Chen Z., Ge ]., Yang P., et al. (2010). Silencing ofTKTLl by siRNA inhibits proliferation of human gastric cancer cells in vitro and in vivo. Cancer Biol. Ther. 9 710-716).

[0012] Phosphorylation of H2AXat Ser 139 (y-H2AX) is crucial for the DNA damage response (DDR). It is considered to be the most sensitive marker for assessing DNA damage and subsequent repair (Podhorecka et al. J Nucleic Acids. 2010; 2010: 920161. H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy). In a recent study, the link between glycolysis and DNA repair was established by demonstrating the role of both hexokinase 1 (HK1) and HKlb isoforms in the up-regulation of DNA repair pathways. The disruption of cancer metabolism by depleting both HK1 and HKlb using CRISPR-Cas9 technology revealed the role of glycolysis in promoting the DNA repair response and indicated that both HK1 and HKlb are critical mediators between DNA repair and glycolysis pathways. The findings also indicate that both HKland HKlb play a role in the induction of drug resistance pathways (Yasemin Yozgat, Emre Karakoc, Ozgur Sahin, Seyma Cimen, Wael M Rabeh, Mehmet Serif Aydin, Adil Mardinoglu, Ihsan Gursel, et al. Hexokinase lb is a novel target for Non-small-cell lung cancerbioRxiv, 2022.06. 27.497447). However, there is no study in the literature investigating the link between HKland HKlb isoforms and the resistance to immunotherapy and / or radiotherapy.

[0013] Patient-derived organoids (PDOs) are three-dimensional models derived from patients, offering a more accurate representation of cancer than traditional models. They are of significant value in the development of cancer treatment strategies. Radiotherapy represents a pivotal component of cancer treatment, and can be integrated with chemotherapy, immunotherapy, or radiodensities drugs to enhance its efficacy. Nevertheless, the absence of reliable preclinical models has impeded the approval of these combinations. PDOs are advantageous in thatthey allow for a reliable model. PDO technology can also replicate the variability of individual patient tumors and predict treatment response. Given the necessity for combined regimens and the regulatory requirements of clinical trials, the PDO platform may provide a timely and effective solution. Therefore, the PDO platform is well-suited for the testing of novel combinations of IM, RT, as well as other targeted therapies, such as inhibitors of glycolysis. PDO models are promising in terms of facilitating a better understanding of cancer biology and for evaluating drug efficacy in vitro. However, complex organotypic models using organoids in co-culture with stromal and immune cellular components of the tumor have yet to be established (Liu, Y. et. al. Palliative treatment efficacy of glucose inhibition combined with chemotherapy for non-small cell lung cancer with widespread bone and brain metastases: A case report. Biomed. Rep. 2017, 7, 553-557).

[0014] In light of the above, it can be seen that there is still a need for the new treatment approaches towards cancer, especially towards NSCLC, in the relevant technical field. Combination therapies and the resistance to the same need to be revealed in order to provide efficient treatment approaches that overcome poor response to treatment. Moreover, methods for determining treatment responses need to be provided, so that treatment of cancer can be achieved without increasing treatment complexity and costs. In order to meet these needs in the relevant technical field, the present invention provides a combination therapy for use in the treatment of cancer, and a method for determining the treatment response of a subject diagnosed with cancer.

[0015] Brief Description of the Invention

[0016] In an aspect of the present invention, a combination therapy comprising at least one immune checkpoint inhibitor, at least one further co-therapeutic agent, and optionally metformin, for use in treatment of a subject diagnosed with cancer is provided. Herein;

[0017] - the subject is diagnosed with a glycolytic activity of a tumor tissue, which is equal to or lower compared to a control sample, as measured based on a level of HK1 and / or HKlb isoform (s) in the tumor tissue, whereby the combination therapy does not comprise administration of metformin, or

[0018] - the subject is diagnosed with a glycolytic activity of a tumor tissue, which is higher compared to a control sample, as measured based on a level of HK1 and / or HKlb isoform (s) in the tumor tissue, whereby the combination therapy comprises administration of metformin. At least one immune checkpoint inhibitor according to the invention is preferably pembrolizumab.

[0019] At least one further co-therapeutic agent is preferably radiotherapy, chemotherapy or targeted therapy. More preferably, at least one further co-therapeutic agent is radiotherapy.

[0020] In preferred embodiments, the cancer is selected from a group comprising non-small-cell lung cancer, breast cancer, high grade sereous over carcinoma, colorectal cancer and glioblastoma. More preferably, the cancer is non-small-cell lung cancer.

[0021] Preferably, the level of HK1 and / or HKlb isoform(s) in the tumor tissue is determined by FDG- PET.

[0022] In another aspect, present invention provides a method for determining the treatment response of a subject diagnosed with cancer to a cancer treatment, wherein a change in the level of HK1 and / or HKlb isoform(s) of a tumor tissue compared to a control sample is a predictive of the subject's response to treatment.

[0023] Said cancer treatment is preferably a combination therapy comprising immunotherapy and at least one further co-therapeutic agent Said at least one further co-therapeutic agent may be selected as radiotherapy, chemotherapy or targeted therapy.

[0024] In preferred embodiments, immunotherapy comprises at least one immune checkpoint inhibitor, which may be selected as pembrolizumab.

[0025] In another aspect, present invention provides an inhibitor of HK-lb for use as an inhibitor of glycolysis.

[0026] In a further aspect, a method of screening comprising [18F]FDG-PET, wherein the method identifies the existence and / or level of HKlb isoform is provided.

[0027] In a further aspect, a method for immune cell distribution profiling in NSCLC patient-derived organoids (PDOs) is provided. Herein the method comprises the identification at least one marker selected from a group comprising CD4+, CD8+, immune checkpoint receptor PD-1 and immune checkpoint receptor PD-L1. Detailed Description of the Invention

[0028] In this detailed description, aspects and embodiments of the present invention are further described for better understanding of the subject matter.

[0029] With the present invention, it is aimed to present a novel and efficient combination therapy for the treatment of cancer. Another aim of the present invention is enhancing response to a cancer combination therapy comprising immunotherapy, through interaction of the DNA damage response and the glycolytic pathway in cancers, especially in non-small-cell lung cancer. Also provided is a new strategy for overcoming resistance and / or poor response to treatment observed in patients diagnosed with cancer, for example NSCLC.

[0030] Studies in the relevant technical field offer inconsistent results with regards to whether the combination of immunotherapy with chemotherapy, radiotherapy or targeted therapy shows desired clinical benefits. The underlying cellular mechanisms that govern resistance to cancer therapies remain yet to be elucidated, and approaches aimed at overcoming them are necessary. Currently, Warburg effect is thought to be the most likely cause of resistance to radioimmunotherapy treatment, by contributing to T-cell dysfunction in the tumor microenvironment and to radio-resistance of various malignanttumors. Metabolic reprogramming of cancer cells and within tumor microenvironment results in resistance to cancer therapies, especially to immunotherapy and radiotherapy. However, metabolic inhibitors tested in pre-clinical and clinical settings in NSCLC along with ICIs have failed to show statistically significant clinical benefits (J Clin Invest.2022;132(l ):el48550). Specifically, the inconsistency of results on combined radio-immunotherapy in NSCLC could be due to the unclear biological mechanisms involved, and to a poor selection of patients that can benefit from this combination treatment It is therefore clinically relevant to investigate the underlying cellular mechanisms, such as mechanistic regulation of the glycolytic pathway and its downstream effectors, for example glycolytic activity and its relation to DNA repair.

[0031] One plausible connection between glucose metabolic dysregulation in cancer and resistance to cancer therapy is DNA damage response (DDR). DDR deficiency affects the immune response in cancers, as effective immune responses depend on the DNA rearrangement and repair processes required for antigen receptor generation in B / T cells.

[0032] Consequently, the efficacy of combination therapies (for example, RT+ICI therapy) in combination with other therapeutic modalities requires further investigation. For example, the optimal combination of radiation therapy and immunomodulation varies between patients. This discrepancy must be addressed in relation to the activity of glycolysis, since patient selection and identification of a group of patients suitable for a specific type of treatment are crucial to improve treatment efficacy and to avoid unnecessary toxicity and expenses.

[0033] In light of the above, present inventors present a unique approach to enhance the effect of combination therapies comprising immunotherapy (for example, RT+ICI therapy), by combining them with glycolytic inhibition, specifically HK1 and / or HKlb inhibition, since increased glycolytic activity causing poor response and / or resistance to therapy (for example to radio- or immune-therapies) is related to both HK1 and HKlb isoforms.

[0034] HKlb isoform is known to be a critical enzyme in the glucose metabolism of NSCLC cells. HKlb was characterized as the soluble form for HK1, lacking the mitochondrial binding domain (N- terminal). It is predominantly expressed in NSCLC cells (A549 WT) and NSCLC tumors, and it distinguishes NSCLC cells from the surrounding normal tissue, indicating a higher dependency on glycolysis and resistance to cell death in these tumor cells. It was shown that higher expression of HKlb is associated with poor NSCLC clinical outcomes. HKlb deletion inhibits glycolysis, proliferation andtumorigenesis in vivo, whereas higher expression of HKlb promotes DNA repair and might confer resistance against DNA damaging agents (Yasemin Yozgat, Emre Karakoc, Ozgur Sahin, Seyma Cimen, Wael M Rabeh, Mehmet Serif Aydin, Adil Mardinoglu, Ihsan Gursel, et al. Hexokinase lb is a novel target for Non-small-cell lung cancerbio Rxiv, 2022.06. 27.497447).

[0035] It was observed as a result of the studies performed in the scope of the invention, that the combination of glycolytic modulation with immune checkpoint inhibitors and at least one further co-therapeutic agent enhances the efficacy of a combination treatment, for example IM+RT treatment. More specifically, use of metformin to inhibit glycolysis via its effects on HK1 and / or HKlb enhances the efficacy of a combination therapy, especially ICI+RT treatment, in NSCLC patients having high-glycolytic activity and / or glucose uptake in their respective tumor tissues. Without being bound by a theory, metformin is thought to weaken the tumoral cell’s ability to repair DNA damage caused by radiotherapy. With the present invention, combination therapy comprising immunotherapy, for example an immune checkpoint inhibitor, is tested for the first time in combination with glycolytic inhibition in NSCLC. It is also a novel approach in the relevant technical field that glycolysis inhibition is achieved through HK-lb inhibition in pre-clinical trials.

[0036] Therefore in an aspect of the present invention, a combination therapy comprising at least one immune checkpoint inhibitor, at least one further co-therapeutic agent, and optionally metformin, for use in treatment of a subject diagnosed with cancer is provided, wherein

[0037] - the subject is diagnosed with a glycolytic activity of a tumor tissue, which is equal to or lower compared to a control sample, as measured based on a level of HK1 and / or HKlb isoform (s) in the tumor tissue, whereby the combination therapy does not comprise administration of metformin, or

[0038] - the subject is diagnosed with a glycolytic activity of a tumor tissue, which is higher compared to a control sample, as measured based on a level of HK1 and / or HKlb isoform (s) in the tumor tissue, whereby the combination therapy comprises administration of metformin.

[0039] In a preferred embodiment, a combination therapy comprising at least one immune checkpoint inhibitor and at least one further co-therapeutic agent is provided, wherein

[0040] - the subject is diagnosed with a glycolytic activity of a tumor tissue, which is equal to or lower compared to a control sample, as measured based on a level of HK1 and / or HKlb isoform(s) in the tumor tissue.

[0041] In another preferred embodiment, a combination therapy comprising at least one immune checkpoint inhibitor, at least one further co-therapeutic agent and metformin is provided, wherein

[0042] - the subject is diagnosed with a glycolytic activity of a tumor tissue, which is higher compared to a control sample, as measured based on a level of HK1 and / or HKlb isoform(s) in the tumor tissue.

[0043] In more detail, patients with low- or normal- glycolytic activity in their respective tumors will receive at least one immune checkpoint inhibitor and at least one further co-therapeutic agent, while those with high-glycolytic activity in their respective tumors will be treated with a glycolysis inhibitor like metformin in addition to at least one immune checkpoint inhibitor and at least one further co-therapeutic agent, as determined by a level of HK1 and / or HKlb isoform(s). This way, by reducing the frequency of ineffective treatments and the severity of side effects, the healthcare system could save significant costs associated with hospital stays and second-line treatments.

[0044] Preferably, the level of HK1 and / or HKlb isoform(s) in the tumor tissue is obtained from the results of FDG-PET, which is routinely used in case of suspicion of cancer. Herein, the level of HK1 and / or HKlb isoform(s) is considered higher compared to a control sample when the level of HK1 and / or HKlb isoform(s) is at least 1.1-fold, for example 1.2-fold, 1.3-fold, 1.4 fold, 1.5 fold, 1.6- fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 4.0-fold or 5.0-fold as compared to a control sample. Immune checkpoint inhibitor according to the invention can be selected from a group comprising ipilimumab, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab or dostarlimab. Immune checkpoint inhibitor according to the invention is preferably pembrolizumab. Present inventors noticed that the combination therapy according o the present invention is most effective when used with pembrolizumab, especially in terms of increased response to treatment.

[0045] At least one further co-therapeutic agent according to the invention maybe selected from a group comprising radiotherapy, chemotherapy and targeted therapy.

[0046] Chemotherapic agent herein may be selected from alkylating agents, and can be for example altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide or trabectedin. Chemotherapic agent herein may further be selected from antimetabolites, and can be for example 5 -fluorouracil, 6- mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin or pralatrexate. Chemotherapic agent herein may further be selected from plant alkaloids, and can be for example vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan or topotecan. Chemotherapic agent herein may further be selected from antitumor antibiotics, and can be for example daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin or valrubicin. Specifically, combination therapy comprising at least one immune checkpoint inhibitor and cisplatin was proved to be effective in treating the disease and alleviating the symptoms. targeted therapy herein may be selected from parp inhibitors, for example olaparib, rucaparib or niraparib. targeted therapy herein may further be crizotinib, sotorasib, osimertinib or bevacizumab. targeted therapy herein may further be selected from monoclonal antibodies, and can be for example trastuzumab, pertuzumab, bevacizumab or rituximab, targeted therapy herein may further be selected from cancer growth blockers, and can be for example axitinib, dasatinib, erlotinib, imatinib, nilotinib, pazopanib, sunitinib, bortezomib, carfilzomib, ixazomib, temsirolimus, everolimus, vorinostat, romidepsin, vemurafenib, dabrafenib or encorafenib. targeted therapy herein may further be selected from anti angiogenics, and can be for example aflibercept, ramucirumab, sunitinib, sorafenib, axitinib, regorafenib or cabozantinib. most preferably, targeted therapy herein may be selected from a group comprising bevacizumab, ramucirumab, sotorasib, adagrasib, osimertinib, afatinib, erlotinib, dacomitinib, gefitinib, lorlatinib, alectinib, brigatinib, ceritinib, crizotinib, entrectinib, repotrectinib, selpercatinib, pralsetinib, cabozantinib, capmatinib, tepotinib and larotrectinib. In a preferred embodiment, at least one further co-therapeutic agent is radiotherapy.

[0047] Present inventors further found out that cancer types where metabolic reprogramming plays a critical role in poor response and / or resistance to treatment can be effectively treated by the combination therapy of the invention. Cancer according to the invention can be selected from a group comprising non-small-cell lung cancer, breast cancer, high grade sereous over carcinoma, colorectal cancer and glioblastoma. More preferably, the cancer is non-small-cell lung cancer.

[0048] Also in the scope of the present invention, a subject’s response to cancer treatment can be anticipated by a method according to the invention. Therefore in another aspect, present invention provides a method for determining the treatment response of a subject diagnosed with cancer to a cancer treatment Herein, a change in the level of HK1 and / or HKlb isoform(s) of a tumor tissue compared to a control sample is a predictive of the subject's response to treatment. Level of HK1 and / or HKlb isoform(s) being equal to or lower compared to a control sample indicates a potential for a sufficient response to therapy, wherein level of HK1 and / or HKlb isoform (s) being higher compared to a control sample indicates a potential for a poor response and / or resistance to therapy. In the latter case, metformin may be added to the combination therapy in order to overcome the poor response and / or resistance to therapy.

[0049] Said cancer treatment may be combination therapy selected from a group comprising immunotherapy + chemotherapy combination therapy, immunotherapy + targeted therapy combination therapy and immunotherapy + radiotherapy combination therapy. Said cancer treatment is preferably immunotherapy + radiotherapy combination therapy. Preferably, said immunotherapy comprises at least one immune checkpoint inhibitor, which may be selected as pembrolizumab.

[0050] In another aspect, present invention provides an inhibitor of HK-lb for use as an inhibitor of glycolysis. Inhibitor herein could be any molecule or sequence that inhibits the function and / or expression of HK-lb isoform. Present inventors observed that inhibition of HK-lb provides for an efficient inhibition of glycolysis.

[0051] In another aspect, a method of screening comprising [18F]FDG-PET is provided. Herein, the method identifies the existence and / or level of HKlb isoform. As a novel approach, present invention makes use of [18F]FDG-PET for the detection of HKlb isoform. HKlb isoform is preferably detected in a NSCLC-PDO.

[0052] In a further aspect, a method for immune cell distribution profiling in NSCLC patient-derived organoids (PDOs) is provided. Herein the method comprises the identification at least one marker selected from a group comprising CD4+, CD8+, immune checkpoint receptor PD-1 and immune checkpoint receptor PD-L1.

[0053] With the present invention, it is further aimed to generate pre-clinical data on the combination of ICI+RT with HK1 and / or HKlb inhibition to avoid poor response and / or resistance to treatment and improve efficacy. This data will be translationally compared with retrospective clinical FDG- PET data, to extract predictive biomarkers. Hence, new biomarkers / therapeutic targets that interlink glycolysis with resistance to cancer therapies, especially to ICI and RT, are aimed to be disclosed.

[0054] With the help of the present invention, clinicians will be able to use FDG-PET scans, which are already routinely used in case of suspicion of cancer, to stratify patients and personalize their treatment to avoid poor response and / or resistance to therapy. Personalization entails the use of metformin, to enhance treatment efficacy and to reverse the resistance induced by high glycolysis. This will result in a dramatic reduction in poor response and / or resistance to treatment for NSCLC without increasing treatment complexity or costs.

[0055] Present invention makes use of NSCLC co-culture PDOs to test the treatments. These PDOs mimic the tumor environment and replicate the variability of individual patient tumors, providing a more accurate prediction of results during clinical trials and reducing the risk of translational failure.

[0056] Furthermore, immune cell distribution profiling in N CLC PDOs is performed. This technique allows for a detailed mapping of immune cell localization and observation of cell-cell interactions within the tumor microenvironment, enabling the identification of molecular mechanisms that complement / go beyond those identified through multi-omics.

[0057] The experiments conducted in scope of the invention are explained in detail below for the better understanding of the invention.

[0058] EXAMPLES

[0059] Collection and storage of NSCLC tumor, normal tissue, and blood samples

[0060] 80 cases of NSCLC will be recruited across 3 centres: 28 from Istanbul Medipol University Hospital (Medipol); 27 from Dokuz Eyliil University Hospital (IBG); 25 from Riga East University Hospital (UL, after obtaining patient informed consent Primary inclusion criteria were: diagnosis of IA- IIIA operable NSCLC, absence of neo-adjuvant treatment, being >18 years old and available pre- operative FDG-PET scan. All studies were conducted in accordance with the Declaration of Helsinki and guidelines on Good Clinical Practice.

[0061] The sample size was calculated based on the power analysis using GPower software, with the following input parameters: 0.80 statistical power; 2-sided alpha (significance) level of 0.05. To take into account the potential dropouts in PDOs generation (~70% success rate), the adjusted sample was determined as 80.

[0062] Clinically and immunohistologically verified fresh NSCLC cells available from surgical resection of the primary lesions and normal lung tissue were collected from the patients. Immunostaining was performed on paraffin-embedded archival tissue at pathology department of each hospital.

[0063] From the same population of patients, peripheral blood were also obtained and processed into peripheral blood mononuclear cells (PBMCs) and plasma. PBMCs were obtained by Ficoll® density gradient centrifugation, and were analyzed using Flow cytometry (Fluorescence- Activated Cell Sorting, FACS). Excess PBMCs were frozen in cryovials ata concentration of 3 x 106cells / mL in liquid nitrogen.

[0064] Generation and characterization of NSCLC PDO co-culture models

[0065] Medipol, IBG and UL undertook on-site generation of PDOs. PDOs were integrated with Tumor- Infiltrating Lymphocytes (TILs) and with Cancer Associated Fibroblasts (CAFs) in order to obtain co-cultures representing the Tumor Microenvironment.

[0066] For organoid cultures, tumor tissues were processed into 4-mm-diameter pieces and washed with ice-cold PBS. Tumor pieces were dissociated into single cells using Tumor Dissociation Kit (Miltenyi Biotec) from primary cells obtained from NSCLC patient tumor and normal lung tissues. Hematopoietic cells were first separated from general tumor cells using the CD45 cell marker, and then the CD45-cells were isolated. Cells were counted and resuspended in 100% growth factor- reduced Matrigel (VWR), plated in 24-tissue culture plates as Matrigel domes and maintained in 37°C 5% CO2 with media overlaying the Matrigel dome. The media was prepared as described by Shi et. al. (Ruoshi Shi, Nikolina Radulovich, Christine Ng, Ni Liu, et al. Organoid Cultures as Preclinical Models ofNon-Small Cell Lung Cancer. Clin Cancer Res., 20201;26(5):1162-1174).

[0067] Establishment of co-culture tumor microenvironment (TME) model

[0068] Tumor-infiltrating lymphocytes (TILs) are most representative of the in vivo tumor situation due to their origin from the Tumor Microenvironment Therefore, establishment of optimal culture medium is needed in which immune cells are not harmed and organoids are still able to proliferate.

[0069] Isolation of CD45+ TILs and cancer associated-fibroblasts (CAFs) were performed using proper antibodies and magnet bead separation.

[0070] Pre- and post-enrichment by flow cytometry gated on CD3 positivity. CD4+ and CD8+ populations were measured by flow cytometry. T-cells were seeded into 24-well plates at a concentration of 1 x 106cells per well in RPMI in a humidified CO2 incubator at 37 °C. For lymphocyte co-culture, 500,000 CD3+ T-lymphocytes per well were suspended in 500 pL RPMI and added to organoids in Matrigel domes or empty Matrigel domes. For fibroblast co-culture, 5 x 105patient matched CAFs per well were suspended in Matrigel and plated with organoids.

[0071] Characterization of NSCLC organoid cultures

[0072] The generated co-cultures of PDOs were subsequently characterized, with a three-fold purpose: i) to assess the concordance with the original tumor. H&E staining and immunohistochemistry (including TTF-1, CK7, P40, Napsin-A) were first performed to compare the PDOs (i.e LCOs, Lung Cancer Organoids) morphologically / histologically with parental NSCLC tissue. PBMCs were subjected to FACS for quantitative analysis of markers at single-cell level, including measurements. Next Generation Sequence (NGS) was performed to verily the genetic identity between PDOs and the original samples, and to ensure the analytical validity to detect clonally dominant alterations. ii) to profile immune cell distribution in PDOs. Advanced tissue clearing and fluorescent microscopy were performed, allowing for detailed mapping of immune cell localization and interactions within the tumor microenvironment Key immune cell markers such as CD4+, CD8+, and the immune checkpoint receptors PD-1 and PD-L1 were investigated by scanning individual PDOs with confocal and light sheet microscopy. Al-based quantification was used to analyze the distribution, cell-cell interactions, and activation states. iii) to assess glucose metabolism in PDOs. Seahorse Fuel-Flex test was performed, using Seahorse XF Mito Fuel Flex Test Kit, after generation of PDOs co-culture, assessing in vitro rate of glucose uptake in each PDO. The dependency, capacity, and flexibility of cells to oxidize three critical mitochondrial fuels, i.e. glucose, glutamine and long chain fatty acids were measured. This test served as an indirect measure of HKl / HKlb activity. In addition, the uptake of [18F]FDG into PDOs was assessed using gamma-counting. Metabolic phenotype was analyzed for each PDO in 96- well plate formats. Changes in glycolysis and respiration were measured using the Seahorse metabolic analyzer. Based on the results and concordance between the two measurements, PDOs were categorized into high-glycolytic group (n~ 15), low-glycolytic group (n~ 15), and non-conclusive group (n~ 20).

[0073] In short, fully characterised NSCLC PDO co-cultures, showing morphologic, histologic, immunologic and genetic concordance with parent tissue were generated and classified based on glycolytic activity.

[0074] Treatment applications and anti-tumoral effect assessment

[0075] Organoids were dissociated into single cells, counted, and plated in Matrigel-coated 96 well plates (3,000 cells per well) in triplicate for 24 hours prior to drug treatment. The NSCLC PDOs from high-glycolytic and low-glycolytic group (n ~30) were tested for below treatment options. Radiotherapy consisted of X-ray irradiation (only doses <2Gy were applied). The focus size of 5.5 mm was used with an YXLON reference irradiator. Dose rate was measured in a cross calibrated ionization chamber. Immunotherapy comprised ICI pembrolizumab and administered at a concentration of 100 nM during a 72h incubation. Metformin (Metf) was administered at concentrations varying from 0.375 mM to 10 mM. Dose-response curves were built to determine IC50 for the pharmacologic treatments and the linear-quadratic model for radiotherapy.

[0076] After establishing the dosage, the following interventions are administered on PDOs (n= 30): i) Triple combination treatment (RT+ICI+Metf), ii) Double combination treatment (RT+ICI), iii) No treatment (control).

[0077] The following measurements for anti-tumoral effect were compared across the three conditions: i) Analysis of cell death mechanism, using Annexin V / Propidium Iodide staining assessed by FACS ii) Proliferation analysis, using KI-67 staining and subsequent immunohistochemistry

[0078] In addition, xCELLigence analysis can be performed.

[0079] As a general approach, two-way ANOVA statistics was carriedoutto test for the effect of treatment (double vs. triple vs. control) and group (low vs. high-glycolytic). ROC curves were built to determine the predictive power and best cut-offs for biomarkers from FDG-PET and multi-omics. Significantly higher cell death and lower proliferation indexes in the group treated with triple combination therapy compared to double combination therapy group, in PDOs of the high- glycolytic group, but not in the low-glycolytic group, were observed.

[0080] Response analysis and biomarker extraction

[0081] Treated PDOs underwent a comprehensive post-treatment exploration to determine the downstream molecular / cellular effects of the interventions, and to correlate them with the successful / unsuccessful anti-tumoral response. The following techniques were applied on selected molecular substrates from PDO co-cultures (respectively, isolated proteins, cell lysate, RNA, tissue sections): i) Proteomics: Identification and quantification of the proteins were performed by Liquid Cromatography (LC) and Mass Spectrometry (MS). Samples were acquired in data- independent acquisition (DIA) mode on the TimsTOF Ultra (Bruker), coupled to a nanoElute 2 system for the LC-MS / MS analysis. Protein digestion and peptide cleaning were executed on the robot Agilent AssayMAP Bravo. The acquired DIA data was analyzed with Spectronaut. ii) Metabolomics: Prepared samples were processed by the nanoElute 2 LC-MS system and analysed through Spectronaut for peak detection, alignment, and quantification. Principal Component Analysis (PCA) was employed to visualize group separations and pinpoint metabolites contributing to these differences. iii) Transcriptomics: RNA-seq and GeoMx analysis were performed, to quantify and profile spatially the transcriptome changes induced by treatment, a) RNA-seq: After library preparation, sequencing was performed using Illumina NovaSeq 6000. DESeq260 was used to compute differential gene expression between groups; b) GeoMx analysis: RNA probes were used for hybridization, and subsequent precise localization and quantification of RNA transcripts. UV light was used to release the barcoded oligonucleotides and then the same was quantified using NGS.

[0082] In addition to above, enzymatic assays (reduction of NADP+ followed at 340 nm) were performed on treated PDOs to verify function of HKlb. Immune profiling was executed to assess posttreatment immunologic changes in the PDOs.

[0083] Pre-operative clinical FDG-PET imaging, performed as standard-of-care before surgery, was assessed and measurements of tumoral glycolytic activity was derived for each patient (Standardized Uptake Values, SUV). SUV was correlated with the proliferation and cell death data obtained from related PDOs.

[0084] In summary, effects of the above treatments were compared in terms of cell death, proliferation and modulation of downstream pathways, by means of immune profiling, enzymatic and omics- wide analyses. Pre-operative clinical FDG-PET, performed as standard-of-care, collected from matched patients, was correlated with outcomes of treatment in the different groups.

[0085] As a result of multi-omics, significant changes differentiating PDOs responding well to triple combination therapy from those that did not respond were detected.

[0086] A suppression of HKl / HKlb function in PDOs responding well to triple combination therapy compared to those that did not respond was detected.

[0087] Regarding clinical FDG-PET comparison, high SUV was calculated in pre-operative FDG-PET of those patients for which matched PDOs showed high-glycolytic activity and whom successfully responded to triple combination therapy. This showed the potential predictive value of clinical FDG-PET in stratifying patients for additional treatment with glycolysis inhibitor.

[0088] With the present invention, enhanced efficacy of pembrolizumab and RT through glycolytic inhibition (metformin) is demonstrated. Furthermore, identification of glycolytic activity measured by FDG-PET as a predictive biomarker for treatment response in NSCLC is disclosed for the first time in scope of the present invention.

[0089] In conclusion, present invention develops and presents a unique strategy to overcome drug resistance for ICI+RT, thereby significantly improving patient outcomes and treatment response of NSCLC.

Claims

CLAIMS1. A combination therapy comprising at least one immune checkpoint inhibitor, at least one further co-therapeutic agent, and optionally metformin, for use in treatment of a subject diagnosed with cancer, wherein the subject is diagnosed with a glycolytic activity of a tumor tissue, which is equal to or lower compared to a control sample, as measured based on a level of HK1 and / or HKlb isoform(s) in the tumor tissue, whereby the combination therapy does not comprise administration of metformin, or the subject is diagnosed with a glycolytic activity of a tumor tissue, which is higher compared to a control sample, as measured based on a level of HK1 and / or HKlb isoform (s) in the tumor tissue, whereby the combination therapy comprises administration of metformin.

2. The combination therapy for use according to claim 1, wherein at least one immune checkpoint inhibitor is pembrolizumab.

3. The combination therapy for use according to claim 1, wherein at least one further co- therapeutic agent is radiotherapy, chemotherapy or targeted therapy.

4. The combination therapy for use according to claim 1, wherein the cancer is selected from a group comprising non-small-cell lung cancer, breast cancer, high grade sereous over carcinoma, colorectal cancer and glioblastoma.

5. The combination therapy for use according to claim 4, wherein the cancer is non-small-cell lung cancer.

6. The combination therapy for use according to claim 1, wherein the level of HK1 and / or HKlb isoform(s) in the tumor tissue is determined by FDG-PET.

7. A method for determining the treatment response of a subject diagnosed with cancer to a cancer treatment, wherein a change in the level of HK1 and / or HKlb isoform(s) of a tumor tissue compared to a control sample is a predictive of the subject's response to treatment.

8. The method according to claim 7, wherein the cancer treatment is a combination therapy comprising immunotherapy and at least one further co-therapeutic agent9. The method according to claim 8, wherein the at least one further co-therapeutic agent is radiotherapy, chemotherapy or targeted therapy.

10. The method according to claim 8, wherein the immunotherapy comprises at least one immune checkpoint inhibitor.

11. The method according to claim 10, wherein at least one immune checkpoint inhibitor is pembrolizumab.

12. An inhibitor of HKlb for use as an inhibitor of glycolysis.

13. A method of screening comprising [18F]FDG-PET, wherein the method identifies the existence and / or level of HKlb.

14. A method for immune cell distribution profiling in NSCLC patient-derived organoids, wherein the method comprises the identification at least one marker selected from a group comprising CD4+, CD8+, PD-1 and PD-L1.