Statin, alone or in association at least another anticancer agent, for use in the treatment of tumors characterized by aberrant expression of HERG1

Abstract

The present invention describes a cholesterol synthesis inhibitor alone or in association with at least another anticancer agent for use in the treatment of cancers characterized by aberrant expression of hERG1. Further the present invention describes also a kit for a simultaneous, separate or sequential use in the treatment of cancers characterized by aberrant expression of hERG1, said kit comprising a cholesterol lowering agent and at least an anticancer agent, preferably a cholesterol synthesis inhibitor, an anti-hERG1 / β1 integrin complex bispecific antibody and / or chemotherapy agent.

Description

[0001]TITLE: STATIN, ALONE OR IN ASSOCIATION AT LEAST ANOTHER ANTICANCER AGENT, FOR USE IN THE TREATMENT OF TUMORS CHARACTERIZED BY ABERRANT EXPRESSION OF HERG1 FIELD OF THE INVENTION The present invention refers to the field of treatments of tumors, in particular those tumors characterized by an aberrant expression of hERG1. STATE OF THE ART Many cellular functions are regulated by a complex array of local signals often triggered by the formation of macromolecular complexes on the plasma membrane which can recruit either cytoskeletal elements or signaling molecules. LRs are highly dynamic molecular assemblages within membranes, enriched in cholesterol, sphingolipids, gangliosides (peculiar is the presence of GM1) and proteins, such as caveolin-1 and flotillin-1. LRs are characterized by high lateral fluidity and a constant flux of assembly / disassembly, which make them pivotal membrane hubs to modulate signaling pathways implicated in different biological processes such as apoptosis, proliferation and autophagy. In cancer, LR-mediated signaling has been implicated in all the phases of tumor progression, from primary carcinogenesis to the late metastatic steps. LRs represent pivotal molecular devices in cancer (Lavie et al., 2000; Mollinedo et al., 2010; Gajate and Mollinedo et al., 2014) as well as an innovative target for anticancer drug development. LRs also contribute to integrin-mediated signaling, as activated integrins are specifically translocated into these microdomains, where they can specifically interact with signaling molecules to regulate the integrin bi-directional signaling. This role of integrin localization in LRs has a clear relevance in the cancer context, directing e.g. the localization and functioning of the small GTPase Rac1, one of the main drivers of tumor cell migration. Among integrins interactors within macromolecular plasma membrane complexes, ion channels are increasingly recognized. Different ion channels are involved but evidence is especially wide for the K+channel encoded by the human ether-à-go- go-related gene 1 (hERG1). hERG1 is often aberrantly expressed in cancer cells, in particular in aggressive carcinomas, where it regulates different aspect of cancer cell behavior that depend on cell adhesion to the Extra Cellular Matrix (ECM). This occurs through the formation of a hERG1 / ^1 integrin macromolecular complex, which has been observed only in cancer and not in normal cells. Overall, hERG1 and consequently hERG1 / β1 integrin complex represent cancer biomarkers in different tumours such as Neuroblastoma, Endometrial Cancer, Colorectal Cancer, Glioblastoma Multiforme, Pancreatic Ductal Adenocarcinoma, Breast Cancer, Clear Cell Renal Carcinoma, Esophageal Squamous Cell Carcinoma, Gastrointestinal Cancer, Neuroendocrine Ileum and Pancreas Cancer, Lymphoma, Melanoma and non-melanoma skin cancers and Leukemia [Expert Opin Ther Targets.2024, 1-13; Biomed Res Int.2015, 2015, 896432; Cancer Res. 1998, 58, 815–822; Br. J. Cancer 2000, 83, 1722–1729; J. Biol. Chem.2003, 278, 2947–2955; Cancer Res.2004, 64, 606–6; Br. J. Cancer 2005, 93, 781–792; Cancer Biol.2008, 7, 45–50; Br. J. Cancer 2015, 112, 1076–1087; Cancer Cell Int.2018, 18, 93; Eur. J. Surg. Oncol.2020, 46, 209–215; J. Surg. Oncol.2008, 97, 57–62; J. Physiol. 2023, 601.9, 1597–1610; Int J Mol Sci. 2022, 23(18),10623; Picchi M. Biotechnology Bachelor Thesis: Immunohistochemical evaluation of hERG1 and hERG1 / β1 Integrin complex expression in a case series of MALT lymphomas: clinical correlations and response to clarithromycin therapy”. Academic year 2020 / 2021. University of Florence; Melanoma Res.2013, 23, 185–190; Blood.2007, 110(4), 1238–1250]. WO2019015936A1 describes an anti-hERG1 Ab and anti-hERG1 / β1 integrin complex bispecific Ab, said anti-hERG1 Ab comprising a Heavy chain Variable (VH) domain having SEQ ID NO:1, and a Light chain Variable (VL) domain having SEQ ID NO:2, preferably said Ab being an anti-hERG1-scFv having SEQ ID NO:3; said anti-hERG1 / β1 integrin complex bispecific Ab comprising a Heavy chain Variable (VH) domain of anti-hERG1 Ab with SEQ ID No: 1; and the Light chain Variable (VL) domain of anti-hERG1 Ab with SEQ ID No: 2; and the Heavy chain Variable (VH) domain and a Light chain Variable (VL) domain of an anti-βΐ integrin Ab having VH and VL with respectively SEQ ID N: 4 and 5 of TS2 / 16 or SEQ ID N: 6 and 7 of BV7, preferably said anti-hERG1 / β1 integrin complex bispecific Ab is a scDb (scDb- hERG1-β1) having SEQ ID No:8.Said anti-hERG1 Ab being selective in binding and an anti-hERG1 / β1 integrin complex bispecific antibody (scDb-hERG1-β1) effective as anticancer agent for the treatment of cancers characterized by aberrant expression of hERG1. Lottini T, et al. (Cancers (Basel).2023, 15(7):2013) tested the effect of Gemcitabine combined with scDb-hERG1-β1 for PDAC treatment. Aim of the present invention is to provide a novel and improved therapeutic strategy for the treatment of cancers characterized by aberrant expression of hERG1 and consequently hERG1 / β1 integrin complex. SUMMARY OF THE INVENTION It was surprisingly found that inhibition of cholesterol synthesis by a statin dissociates the hERG1 / β1 integrin complex in LRs in PDAC cells. It was also surprisingly found that a statin in combination with an anti-hERG1 / β1 integrin complex bispecific antibody and / or a chemotherapy agent (like Gemcitabine or Oxaliplatin) exert a synergic anticancer effect. Subject-matter of the present invention is therefore a cholesterol lowering agent alone or in association with at least another anticancer agent for use in the treatment of a cancer characterized by aberrant expression of hERG1 and hERG1 / β1 integrin complex. Further subject-matter of the present invention is a kit for a simultaneous, separate or sequential use in the treatment of cancers characterized by aberrant expression of hERG1, said kit comprising a cholesterol lowering agent and at least one anticancer agent, preferably comprising a cholesterol synthesis inhibitor and an anti- hERG1 / β1 integrin complex bispecific antibody and / or a chemotherapy agent, and instructions for use thereof. DETAILED DESCRIPTION OF THE INVENTION Types of cancer characterized by aberrant expression of hERG1 and consequently hERG1 / β1 integrin complex are Neuroblastoma, Endometrial Cancer, Colorectal Cancer, Glioblastoma Multiforme, Pancreatic Ductal Adenocarcinoma, Breast Cancer, Clear Cell Renal Carcinoma, Esophageal Squamous Cell Carcinoma, Gastrointestinal Cancer, Neuroendocrine Ileum and Pancreas Cancer, Lymphoma, Melanoma and non-melanoma skin cancers, Leukemia or any other cancer whose primary lesion or a body fluid of a patient thereof results positive to the biding with an anti-hERG1 antibody and / or anti-hERG1 / β1 integrin complex bispecific antibody, for example using the protocols for IHC described herein below and also in Duranti C, et al, Oncotarget.2018, 9(79):34972-34989 and Arcangeli A, et al. Expert Opin Ther Targets.2024, 1-13. According to the invention anti-hERG1 antibody and anti- hERG1 / β1 integrin complex bispecific antibody are those described in WO2019015936A1: said anti-hERG1 Ab is comprising a Heavy chain Variable (VH) domain having SEQ ID NO:1, and a Light chain Variable (VL) domain having SEQ ID NO:2, preferably said Ab is an anti-hERG1-scFv having SEQ ID NO:3; said anti- hERG1 / β1 integrin complex bispecific Ab is comprising a Heavy chain Variable (VH) domain of anti-hERG1 Ab with SEQ ID No: 1; and the Light chain Variable (VL) domain of anti-hERG1 Ab with SEQ ID No: 2; and the Heavy chain Variable (VH) domain and a Light chain Variable (VL) domain of an anti-βΐ integrin Ab having VH and VL with respectively SEQ ID N: 4 and 5 of TS2 / 16 or SEQ ID N: 6 and 7 of BV7, preferably said anti-hERG1 / β1 integrin complex bispecific Ab is a scDb (scDb- hERG1-β1) having SEQ ID No:8. According to the present invention said cholesterol lowering agent is an inhibitor of cholesterol synthesis thus preferably a HMG-CoA reductase inhibitor, more preferably the HMG-CoA reductase inhibitor is a statin. Preferably the statin is selected from the group consisting of Simvastatin, Fluvastatin, Lovastatin, Atorvastatin. According to the invention said cholesterol lowering agent can also be Inclisiran, Inclisiran is the first small interfering RNA molecule currently in clinical trials developed to help lower cholesterol by preventing production of PCSK9. According to the present invention said at least another anticancer agent is selected from the group consisting of chemotherapy agents, antibodies or any other known anticancer agent. According to the present invention the anticancer agent is preferably anti-hERG1 / β1 integrin complex bispecific antibody and is preferably as described in WO2019015936A1, more preferably the therein described scDb-hERG1-β1 having SEQ ID No:8. According to the present invention the anticancer agent is preferably a chemotherapy agent and preferably is selected from the group consisting of gemcitabine, oxaliplatin or another platinum derivates, 5-Fluorouracil or the antibiotic clarithromycin used as in Petroni et al., (Cell Death and Desease), 2020. According to the present invention the cholesterol synthesis inhibitor is preferably administered orally. Preferably the dosage of the cholesterol synthesis inhibitor is 20-450 mg / die when administered alone or when administered in association with the at least another anticancer agent. According to the present invention the anti-hERG1 / β1 integrin complex bispecific antibody is preferably administered by parenteral route or oral route, wherein the parenteral administration is preferably intravenous or subcutaneous and the oral administration is preferably by way of a gastroresistant pharmaceutical formulation. Preferably the dosage of the anti-hERG1 / β1 bispecific antibody is 25-300 mg (Reviews in Pharmacy Vol 11, Issue 8, Aug-Sept 2020 A Guide for Estimating the Maximum Safe Starting Dose and Conversion it between Animals and Humans). According to the present invention the chemotherapy agent is preferably administered according to its known administration route which can be intravenous or oral depending on the chemotherapy agent. So, for example Gemcitabine is administered by intravenous infusion route, and Oxaliplatin is administered by intravenous infusion route. Preferably the dosage of the chemotherapy agent in association with the cholesterol lowering agent, preferably a statin, is halved with respect to the dose normally administered as alone chemotherapy agent thus reducing its adverse effects. Therefore, the kit according to the present invention preferably comprises a pharmaceutical composition of a cholesterol synthesis inhibitor and a pharmaceutical composition of an anti-hERG1 / β1 bispecific antibody as described above and / or a pharmaceutical composition of a chemotherapy agent and instructions for use thereof. The kit according to the invention preferably comprises: a cholesterol lowering agent selected from the group consisting of Simvastatin, Fluvastatin, Lovastatin, Atorvastatin and Inclisiran; and an anti-hERG1 / β1 integrin complex bispecific antibody as described above; and / or a chemotherapy agent selected from the group consisting of Gemcitabine or Oxaliplatin or platinum derivates or clarithromycin or 5-Fluorouracil. More preferably the kit of the invention comprises a statin oral pharmaceutical composition, a parenteral or gastroresistant pharmaceutical composition of anti- hERG1 / β1 bispecific antibody and / or a parenteral or oral pharmaceutical composition of a chemotherapy agent. According to a preferred embodiment of the kit of the invention the statin is selected from the group consisting of Simvastatin, Fluvastatin, Lovastatin, Atorvastatin, and the anti-hERG1 / β1 bispecific antibody is scDb-hERG1-β1 having SEQ ID No:8; the chemotherapy agent is Gemcitabine or Oxaliplatin. For an aspect the present invention relates to a method of treatment of a cancer characterized by aberrant expression of hERG1 said method comprising administering to a patient in need thereof a cholesterol lowering agent as above described. For an aspect the present invention relates to a method of treatment of a cancer characterized by aberrant expression of hERG1 said method comprising administering to a patient in need thereof a cholesterol lowering agent and at least one further anticancer agent as above described. The present invention can be better understood in light of the following experimental section. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Simvastatin reduced the expression of caveolin-1 and the formation of the hERG1 / β1 integrin complex. (A) IF performed on PANC-1 following 90 min adhesion onto FN with or without treatment with Simvastatin, scDb-hERG1-β1 and their combination. Quantitative analyses and MOC are reported in the graphs. a.u.= arbitrary units. At least a total of 20 cells per condition from three independent experiments (n=3) were analyzed. All data are presented as mean values ± s.e.m. (n=3). (B) IF on PANC-1 cells stained with anti-ARP2 / 3 antibody after treatment with scDb-hERG1-β1 (20ug / ml), Simvastatin (4.7μM) and their combination onto FN for 90 mins. At least a total of 20 cells per condition from three independent experiments (n=3) were analyzed. Quantification graphs of ARP2 / 3 fluorescent intensity, F-actin stress fibers length and cortical F-actin density were reported. *P < 0.05; **P < 0.01, and ***P < 0.001 (One-Way ANOVA). CTR= control; MOC= Mander’s Overlapping Coefficient. Figure 2: All the statins reduced cell vitality of PDAC cells and the effect was significantly potentiated by the combination with scDb-hERG1-β1. (A) Curves of percentage live cells, PANC-1 (top panel), MiaPaCa2 (middle panel) and HEK293 (bottom panel), treated for 24h with Simvastatin, Fluvastatin, Lovastatin and Atorvastatin are reported, cells. Data are presented as mean values ± s.e.m. (n=3). (B) Graph of percentage live cells, PANC-1 (left panel), MiaPaCa2 (middle panel) and HEK293 (right panel) cells, treated for 24h with IC50 of Simvastatin, Fluvastatin, Lovastatin, Atorvastatin, scDb-hERG1-β1 and combination of each statins with scDb-hERG1-β1 are reported. Data are presented as mean values ± s.e.m. (n=3). (C) Curves of percentage live cells, hERG1 silenced (pink) and lipofectamine (blue) PANC-1 (left panel) and MiaPaCa2 (right panel) cells, treated for 24h with Simvastatin or Atorvastatin are reported, Data are presented as mean values ± s.e.m. (n=3). *P < 0.05; **P < 0.01, and ***P < 0.001 (One-Way ANOVA). Figure 3: All the statins significantly reduced FN-induced motility in PDAC cells and the effect was significantly potentiated by the combination with scDb-hERG1-β1(A) Lateral motility experiments onto FN were performed on PANC-1, MiaPaCa2 and HEK-293 cells treated with Simvastatin, Fluvastatin, Lovastatin, Atorvastatin, scDb-hERG1-β1 (at IC50 values for 90 min) and combinations of each statin and scDb-hERG1-β1 (at IC50 values for 90 min). The motility is reported as graph of percentage of cell motility. (n=3). (B) Lateral motility experiments onto FN performed on hERG1 silenced PANC-1 and MiaPaCa2 cells treated with Simvastatin, Fluvastatin, Lovastatin, Atorvastatin (at IC50 values for 90 min). The motility is reported as graph of percentage of cell motility. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 4. The effect of the combined treatment of the three compounds is significantly higher compared to effect of the single treatment and to any of the combination of only two compounds. (A) LDH assay of Simvastatin, Oxaliplatin and scDb-hERG1-β1 treatments in PANC-13D culture cells (spheroids). (B) LDH assay of Simvastatin, Oxaliplatin and scDb-hERG1-β1 treatments in hERG1 silenced (siRNA hERG1) PANC-13D culture cells (spheroids). *P < 0.05, **P < 0.01, ***P < 0.001. Figure 5: in vivo effects of simvastatin and scDb-hERG1-β1 on PDAC tumor masses showing that treatment of the combination of simvastatin and scDb- hERG1-β1 is able to significantly reduce tumor masses better than simvastatin alone (A) Representative US images of tumor masses from PANC-1 cells at day 51. (B) Time course of the volume of orthotopic masses of PANC-1 cells, treated with simvastatin and simvastatin plus scDb-hERG1-β1 and schedule of treatment. hERG1 / β1 integrin complex and its impact on survival in PDAC patients. (C) EXPERIMENTAL SECTION MATERIALS AND METHODS Antibodies and reagents. The following primary antibodies were used: scDb-hERG1 / β1 having SEQ ID No:8 (Single chain diabody, MCK Therapeutics Srl, Pistoia, Italy), 20 μg / ml for cell. scDb- hERG1 / β1-alexa-48820 μg / ml for IF; anti-caveolin-1 pAb (Abcam, Cambridge, UK) at 1:1000 for WB. m-mAb. Anti-caveolin-1 (610406, BD Biosciences, Franklin Lakes, NJ) at 1:50 for IF; Anti-ARP2 / 3 complex (BS-12524R, Thermo Fisher Scientific, Waltham, MA) at 1:100 for IF. CellMaskTM Deep Red Actin staining reagent was (A57245, Invitrogen, Waltham, MA, USA) used according to manufacturer’s instructions. Alexa Fluor 546 goat anti-mouse, and CY2 goat anti-rabbit antibodies (Thermo Fisher Scientific, Waltham, MA) were used 1:500. Hoechst was used for staining nuclei in IF experiments (1:1000 in PBS, 45 minutes; Merck Sigma, Burlington, MA). Geneticin (0.8 mg / ml final concentration; G418, Thermo Fisher Scientific, Waltham, MA). Fibronectin (FN) was purchased from Sigma-Aldrich, Darmstadt, Germany, human plasma. Rhodamine-conjugated phalloidin was purchased from Invitrogen (Waltham, MA, USA) and used according to manufacturer’s instructions. Simvastatin, Atorvastatin, Lovastatine and Fluvastatin were purchased from Merck Millipore. Statins were dissolved in DMSO at 12 mM concentration (Simvastatin), 250 mM concentration (Fluvastatin), 2,75 M concentration (Atorvastatin) and 12 mM concentration (Lovastatin). Dulbecco's Modified Eagle's Medium High Glucose w / o Sodium Pyruvate w / o L-Glutamine (DMEM, Euroclone, Milan, Italy). L-glutamine (Euroclone, Milan, Italy). Fetal bovine serum (FBS, Fetal Bovine Serum EU Approved, Euroclone, Pero, Italy). Dulbecco's Phosphate Buffer Saline w / o Ca w / o Mg (PBS, Euroclone, Milan, Italy). Trypsin- EDTA 1X in PBS w / o Ca w / o Mg w / o Ph Red (Euroclone, Milan, Italy). Trypan blue (Sigma, Darmstad, Germany). Bovine Serum Albumin (BSA, Sigma-Aldrich, Darmstadt, Germany). Prolong Diamond antifade mountant (Invitrogen, Waltham, Massachusetts, USA). Pierce 16% Formaldehyde Solution (w / v) Methanol-Free (PFA, 4% final concentration, Thermo scientific, Rockford, IL, U.S.A.). Cells and culture. The human PANC-1, MiaPaCa2, HEK293 cell line were obtained from the American Type Culture Collection (ATCC). HEK293 cells expressing the hERG1 construct (HEK293-hERG1) were prepared as previously described in (Becchetti, A. et al. The conformational state of hERG1 channels determines integrin associa-tion, downstream signaling, and cancer progression. Sci Signal. 10(473):eaaf3236 (2017). and maintained in complete culture medium supplemented with 0.8 mg / ml of Geneticin. Cells were routinely cultured at 37°C with 5% CO2 in a humified atmosphere. PANC-1, MiaPaCa2 and HEK293 were cultured in DMEM supplemented with 4 mM of L-glutamine and 10% FBS. We certify that all the cell lines used in the present study were routinely screened for Mycoplasma contamination, and only Mycoplasma negative cells were used. Silencing of hERG1 in PANC-1 and MiaPaCa2 cells, was carried out with siRNAs as previously described in (Crociani, O. et al. hERG1 Channels Modulate Integrin Signaling To Trigger Angiogenesis And Tumor Progression In Colorectal Cancer. Sci Rep. 3:3308 (2013). Cell treatments. All the experiments were performed on cells seeded on Fibronectin (FN). The coating with FN was performed following the standard protocol provided with the product. FN was diluted in sterile PBS at 5 µg / cm2 concentration. The culture surface was coated with a minimal volume. The dishes were left air-drying for 1 h at room temperature before introducing cells and medium. Cells were starved overnight prior to seeding onto fibronectin coatings and the kept in serum-free medium for the entire duration of the experiments. After treatment, cells were collected and prepared for experimental procedures. Treatment with scDb-hERG1- β1 antibody: scDb-hERG1-β1 was added in the serum-free-BSA medium at the final concentration of 20 μg / ml for 90 minutes for IF and motility experiments. For viability experiments was add at IC50: HEK293: 200µM; MiaPaCa2: 20.40µM; PANC-1: 18.13µM for 24 h. Treatment with statins: Following the procedure in (Chun, Y.S. et al. Cholesterol modulates ion channels via down-regulation of phosphatidylinositol 4,5-bisphosphate. J Neurochem. 112(5):1286-94 (2010), cells were pretreated overnight with statins (20 μM) the day before the experiments. The treatment with Simvastatin was performed at IC50 concentration for the different cell lines for viability experiments for 24 h. The treatment with Simvastatin was performed at IC50 concentration for the different cell lines for IF and motility experiments for 90 min in the serum-free-BSA medium. HEK293: 7.57µM; MiaPaCa2: 4.16µM; PANC-1: 4.47µM. The treatment with Simvastatin was performed at 20µM for cholesterol levels for 90 min in the serum-free-BSA medium. The treatment with Fluvastatin was performed at IC50 concentration for the different cell lines for viability experiments for 24 h. The treatment with Fluvastatin was performed at IC50 concentration for motility experiments for 90 min in the serum-free-BSA medium HEK293: 3.45µM; MiaPaCa2: 3.45µM; PANC-1: 2.47µM. The treatment with Lovastatin was performed at IC50 concentration for the different cell lines for viability experiments for 24 h. The treatment with Lovastatin was performed at IC50 concentration for motility experiments for 90 min in the serum-free-BSA medium HEK293: 3.26µM; MiaPaCa2: 2.07µM; PANC-1: 2.78µM. The treatment with Atorvastatin was performed at IC50 concentration for the different cell lines for viability experiments for 24 h. The treatment with Atorvastatin was performed at IC50 concentration for motility experiments for 90 min in the serum-free-BSA medium HEK293: 2.09µM; MiaPaCa2: 3.13µM; PANC-1: 1.80µM. Different dose of Gemcitabine and Oxaliplatin were used as indicated in Table 2 and Figure 4. Cell viability assay. Cell viability was measured by the trypan blue exclusion test as in Iorio J, Antonuzzo L, et al. Prognostic role of hERG1 Potassium Channels in Neuroendocrine Tumours of the Ileum and Pancreas. Int J Mol Sci. 2022 Sep 13;23(18):10623. doi: 10.3390 / ijms231810623. After incubation with the drug and the scDb-hERG1-β1 antibody, the trypan blue dye was added to the cells and live cells were counted using LUNA-II™ Automated Cell Counter (Logos Biosystems, Villeneuve d’Ascq, France). The 50% inhibitory concentration (IC50) was calculated using the equation Y=Min+Max−min1−(XIC50) Hill coefficient, as in71. Combination Index (CI) calculation were performed as previously described in71. Lateral motility assay. Lateral motility was determined using 35 mm dishes and drawing 15 horizontal lines and 3 perpendicular lines on the dish bottom to generate a grid system. Plates were coated with FN and 5x105 cells were seeded and allowed to attach for 90 min. Three wounds were drawn following the 3 horizontal lines. Then, the distances between cells were measured at each mark point (where the 3 horizontal lines crossed the 15 vertical lines) using a light microscope. The widths measured at time 0 correspond to the W0 parameter. These different 45 points were measured again after 90′. Motility Index (MI) was assessed using the following formula: MI = 1 – Wt / W0, where Wt is the width of the wounds after 90 min. Immunofluorescence (IF). Staining for hERG1 / β1 integrin complex on cells was performed following the protocol previously described in (Duranti, C., et al. Harnessing the hERG1 / β1 Integrin Complex via a Novel Bispecific Single-chain Antibody: An Effective Strategy against Solid Cancers. Mol Cancer Ther.20(8):1338-1349 (2021).72. Duranti, C., et al. Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging. Oncotarget. 9(79):34972-34989 (2018). After 2 hours of blocking in PBS with 10% BSA, slides were incubated for further 2 hours with scDb-hERG1 / β1-alexa 488 (20 µg / ml final concentrations). Nuclei were stained with Hoechst and slides were mounted using Prolong Diamond antifade mountant (see “Reagents and antibodies” section). Staining with anti-caveolin monoclonal antibody (1:50) was followed by secondary Alexa-546-anti-mouse antibody (1:500) (see “Reagents and antibodies” section). Staining with anti-Arp2 / 3 complex antibody. Cells were fixed using 4% PFA, followed by permeabilization with 0.1 % Triton-X (see “Reagents and antibodies” section) and blocking with 10% bovine serum albumin (BSA) (see “Reagents and antibodies” section) in PBS. Staining with anti-Arp2 / 3 complex (1:100 in 5% BSA) (see “Reagents and antibodies” section) was performed O / N at 4°C and followed by incubation with secondary goat anti-rabbit CY2 antibody (1:500 in PBS) for 60 minutes in the dark at room temperature. CellMaskTM Deep Red Actin (1:1) staining was then performed for 15 minutes in the dark at room temperature. For all IF experiments, nuclei were stained with Hoechst (1:1000 in PBS, 5 minutes) and slides were mounted using Prolong Diamond antifade mountant. All images were captured using confocal microscope, Nikon Eclipse TE2000-U (Nikon, Tokyo, Japan) and analyzed using Image J software. Cortical actin density was quantified using the dedicated PlasMACC Fiji Plugin (Kurps, J., et al. Quantitative image analysis tool to study the plasma membrane localization of proteins and cortical actin in neuroendocrine cells. J Neurosci Methods.236, 1-10 (2014). while actin stress fibers length was quantified following the method described in (Manoli, S., et al. The Activity of hERG1 Potassium Channel Modulates F-Actin Or-ganization During Cell Migration of Pancreatic Ductal Adenocarcinoma Cells. Cancers (Ba-sel).11-135 (2019). A total of 20 cells per condition from three independent experiments were analyzed for IF experiments. 3D cultures PANC-1 cells were cultured in an up-to-date 3D conformation, which is that of a spheroid. The latter are usually non-scaffold-based cultures formed by rounded cellular aggregates. Cells were routinely cultured in a humidified incubator at 37 °C with 5% CO2using DMEM Dulbecco’s Modified Eagle Medium (DMEM) (Fisher Scientific Italia, Rodano (MI), Italy), High Glucose (Euroclone S.p.A, Pero (MI), Italy) supplemented with 4 mM L-Gln (Glutamine) and 10% FBS (Fetal Bovine Serum) (Euroclone S.p.A, Pero (MI), Italy). For 3D spheroid cultures, cells were detached with trypsin and counted using trypan blue. Trypan blue dye exclusion test was used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue. The 103cells were seeded on an agarose base layer (1.5 g / l) in a 96-well plate and grown for 96 h in a humidified incubator at 37 °C and 5% CO2. LDH Assay Samples of the culture medium were collected at the desired experimental time points by removing 2–5μl of medium and diluting into 48–95μl LDH Storage Buffer (Promega). Optional: If a Maximum LDH Release Control is required, it’s possible to add 2μl of 10% Triton X-100 (per 100μl original volume) to the vehicle-only wells, mix and incubate for at least 10–15 minutes before sample removal. After collecting and diluting all samples 50μl of diluted sample were transferred into a 96-well opaque-walled, non-transparent assay plate (with clear or opaque bottom). 50μl of LDH Detection Reagent (Promega) were added to each well. Plates were then incubated for 60 minutes at room temperature. Luminescence was recorded after 30– 60 minutes after adding LDH Detection Reagent. (Experimental LDH Release−Medium Background) Cytotoxicity (%) = 100 ^^ (Maximum LDH Release Control−Medium Background) In vivo experiments. Panc-1 cells were resuspended in PBS and injected subcutaneously (1X106cells / injection) into both lateral flanks of Athymic Nude-Foxn1nu / nu(Envigo, Indianapolis, ID, USA). Once tumors reached an average size of mm3, mice were randomly assigned to four groups of treatments and administered by oral gavage with Simvastatin (80 mg / kg daily), intravenously (iv) with scDb (16 mg / kg daily), combination of Simvastatin and scDb, or vehicle for 51 days. Simvastatin was dissolved in 0.5 % methyl cellulose (PRODOTTI GIANNI Srl) freshly prepared every week. Mice belonging to the vehicle group were treated with methyl cellulose by gavage daily. Mice were euthanized after 3 weeks of treatment, and the tumors were harvested for immunohistochemical analysis. Ultrasound Imaging. After anesthesia with isoflurane (induction dosage of 4% and a maintenance dose of 2%), mice were placed in a prone position on a heated pad at 37°C, and body temperature, respiration rate, and ECG continuously were continuously monityored. Ultrasonic transmission gel was used on the mice skin. The VevoLAZR-X imaging platform (Fujifilm VisualSonics) was used for ultrasonic imaging on B-mode modality and axial 3D acquisition of the tumor masses were carried out with a 55-MHz transducer. The Vevo LAB software was then used to define the regions of interest (ROI) for each frame, allowing to rendering 3D the tumors and measuring the volumes. Overall survival (OS) and disease free survival (DFS) in pancreatic cancer patients. In silico analysis. Clinical and gene expression data of 184 pancreatic cancer patients were obtained from the TCGA database, and survival curves were drawn employing the of R (version 4.4.0) packages ‘survival’ according to gene expression of KCNH2, ITGB1 and co-expression of both genes. IHC analysis. hERG1 / β1 integrin complex expression has been evaluated in a case series of PDAC samples (46 samples obtained from Campus biomedico of Rome and 36 obtained from AOUC of Florence). Survival curves were drawn employing the of R (version 4.4.0) packages Statistical analysis. For western blot analysis the statistical procedures were performed by GraphPad Prism software Inc. (San Diego, CA, USA). D’Agostino-Pearson omnibus normality test was used to assess the normal distribution of the data. Normally distributed variables were summarized using the mean ± standard deviation (SD). Differences between numerical variables were tested using Paired t-test. *p≤0.05, **p≤0.001 ***p≤0.001. RESULTS The inhibition of cholesterol synthesis by statins dissociates the hERG1 / β1 integrin complex in LRs in PDAC cells. Statins acts on the rate-limiting step and serves as HMG-CoA reductase inhibitors, consequently leading to decreased cholesterol concentrations. Different Statins were tested on PDAC cells and their effects analyzed. Simvastatin reduced the expression of caveolin-1 (Fig.1A left graph) and the formation of the hERG1 / β1 integrin complex (Fig.1A middle graph), and the co-localization of the hERG1 / β1 integrin complex with caveolin-1 in PANC-1 cells (Fig.1A right graph). Furthermore, Simvastatin decreased Arp 2 / 3 FI, increased f-actin stress fiber length and decreased cortical f-actin density (Fig. 1B). These effects were similar to those obtained dissociating the hERG1 / β1 integrin complex through the treatment with scDb-hERG1-β1 (Fig. 1A an B, bars labeled as “scDb-hERG1-β1 Treatment”). Consistently, the combination of the two treatments (Simvastatin+scDb-hERG1-β1) decreased hERG1 / β1 integrin complex formation, caveolin-1 expression, co- localization with the hERG1 / β1 integrin complex, and modulated f actin organization at significantly higher extent compared to single treatments (Fig.1B related bar graphs). It can be concluded that Statins (Simvastatin) disassembles LRs in PDAC cells and dissociates the hERG1 / β1 integrin complex from LRs, hence decreasing the signaling pathway triggered by the complex. It was then tested the effects of different Statins (Simvastatin, Fluvastatin, Lovastatin and Atorvastatin) on cell vitality and migration of PDAC cells, alone and in combination with the scDb-hERG1-β1. The IC50 concentrations of the different Statins were first determined in two PDAC cell lines (PANC-1 and MiaPaCa2) and in normal HEK 293 cells as control (Fig.2A and Table 1A). All the statins reduced cell vitality of PDAC cells (both PANC-1 and MiaPaCa2). Their effect was significantly potentiated by the combination with scDb-hERG1-β1 in both PANC-1 and MiaPaCa2 cells (Fig. 2B and Table 1A). The effect of the combination of scDb-hERG1-β1 with either Simvastatin or Atorvastatin was synergic in both PANC-1 and MiaPaCa2 cells (Table 1B). All the statins also reduced cell viability in HEK 293 cells; but no further potentiation with scDb-hERG1-β1 was observed since these cells do not express the hERG1 channel (Fig.2B and Table 1A). Notably, the IC50 of both Simvastatin and Atorvastatin was significantly higher in PDAC cells (either PANC-1 and MiaPaCa2) where hERG1 was silenced (Fig.2C and Table 1C), indicating that their effect on cell vitality depended on the presence of the hERG1 / β1 integrin complex on the plasma membrane. Table 1. IC50values, effects on vitality reduction and combination index of Simvastatin, Fluvastatin, Lovastatin, Atorvastatin and scDb-hERG1-β1 in PANC-1, MiaPaCa2 and HEK-293 cells. (A) IC50values were determined after 24 h of treatment by the Trypan Blue exclusion test, using the Origin Software, in PANC- 1, MiaPaCa2 and HEK-293 cells. (B) Combination index in PANC-1 and MiaPaCa2 cells after different treatment combinations. All the drugs were used at drug concentrations indicated in the first column. Data are means±s.e.m. of three independent experiments, each carried out in triplicate. CI values were calculated using the Calcusyn software Version 2 (Biosoft). For statistical analysis, Student’s t- test was applied.(C) IC50 values were determined after 24 h of treatment by the Trypan Blue exclusion test, using the Origin Software, in PANC-1 and MiaPaCa2 hERG1 silenced cells. ns: no significant. A IC50 Statin IC50 Statin IC50 + P value (μM) % vitality scDb- Statin IC50% vitality reduction reduction hERG1-β1 Vs (24h) IC50Statin IC50+ scDb-hERG1-β1 IC50% vitality % vitality reduction reduction (24h) (24h) PANC-1 Simvastatin 4.47 ± 0.48 45.42 ± 1.56 68.87 ± 1.12 0.02 Fluvastatin 2.47 ± 1.06 36.64 ± 2.45 67.56 ± 1.12 0.01 Lovastatin 2.79 ± 0.84 44.82 ± 2.24 64.28 ± 2.54 0.02 Atorvastatin 1.81 ± 1.12 51.16 ± 1.97 72.98 ± 1.34 0.02 MiaPaCa2 Simvastatin 4.16 ± 1.02 49.00 ± 1.34 70.00 ± 2.45 0.02 Fluvastatin 3.45 ± 1.05 47.07 ± 2.45 64.88 ± 1.45 0.02 Lovastatin 2.07 ± 1.01 42.32 ± 4.56 61.28 ± 2.23 0.03 Atorvastatin 3.13 ± 1.13 53.88 ± 1.12 69.98 ± 1.76 0.04 HEK-293 Simvastatin 7.57 ± 1.22 43.89 ± 4.23 43.91 ± 5.3 ns Fluvastatin 3.45 ± 0.98 52.88 ± 1.23 52.97 ± 1.12 ns Lovastatin 3.26 ± 1.21 45.32 ± 5.6 44.67 ± 4.3 ns Atorvastatin 2.09 ± 1.26 50.88 ± 1.21 50.77 ± 1.78 ns B Combination Index Effect PANC-1 Simvastatin (μM) scDb-hERG1-β1 (μg / ml) 2,35 (IC25) 10,4 (IC25) 0.29 ± 0.02 synergy 4,47 (IC50) 20,8 (IC50) 0.27 ± 0.01 synergy Atorvastatin (μM) scDb-hERG1-β1 (μg / ml) 0,9 (IC 25) 10,4 (IC25) 0.94± 0.03 synergy 1,81 (IC50) 20,8 (IC50) 0.61± 0.02 synergy C IC50 IC50 (μM) (μM) PANC-1 siRNA hERG1 MIaPaCa2 siRNA hERG1 Simvastatin 11.61 ± 1.12 Simvastatin 12.01 ± 2.14 Atorvastatin 12.89 ± 1.61 Atorvastatin 13.87 ± 1.02 PANC-1 lipofectamine MiaPaCa2 lipofectamine Simvastatin 4.31 ± 1.13 Simvastatin 5.76 ± 1.43 Atorvastatin 1.85 ± 2.12 Atorvastatin 3.77 ± 0.73 All the statins also significantly reduced FN-induced motility in both PANC-1 and MiaPaCa2 cells This effect was significantly potentiated by the combination with scDb-hERG1-β1 (Fig.3A), but was weaker when hERG1 was silenced (Fig.3B). Overall, the pharmacological inhibition of cholesterol synthesis by statins and the subsequent disassembly of LRs produces a significant inhibition of both cell viability and cell migration in PDAC cells which is potentiated by the treatment with the scDb- hERG1-β1 and depends on the presence of the hERG1 / β1 integrin complex on the plasma membrane. It was evaluated the combination index (CI) between Gemcitabine and statins, and Oxaliplatin and statins in PANC-1 and MiaPaCa2 cells and in hERG1 silenced (siRNA hERG1) PANC-1 and MiaPaCa2 cells. In table 2 is reported 25% inhibitory concentration (IC25) of drugs. We observed a synergic effect for all combinations in PANC-1 and MiaPaCa2 cells and the synergy has not been observed in hERG1 silenced (siRNA hERG1) PANC-1 and MiaPaCa2 cells. It was evaluated the citotoxicity of Simvastatin, Oxaliplatin and scDb-hERG1-β1 treatments in PANC-13D culture cells with LDH assay. In Fig.4A we observed that the effect of the combined treatment of the three compounds is statistically significant higher compared to effect of the single treatment and to any of the combination of only two compounds. This result is not shown in hERG1 silenced (siRNA hERG1) PANC-13D culture cells (Fig.4B). Table 2. Combination index (CI) between Gemcitabin and statins, and Oxaliplatin and statins in PANC-1 and MiaPaCa2 cells and in hERG1 silenced (siRNA hERG1) PANC-1 and MiaPaCa2 cells. 25% inhibitory concentration (IC25) of drugs are reported. Data are means±s.e.m. of three independent experiments, each carried out in triplicate. CI values were calculated using the Calcusyn software Version 2 (Biosoft). PANC-1 CI values Gemcitabine 20.33 µM Oxaliplatin 1.53 µM Atorvastatin 0.90 µM 0.49± 0.02 0.12± 0.02 Simvastatin 2.35 µM 0.370± 0.01 0.48± 0.01 Lovastatin 1.40 µM 0.531± 0.03 0.79± 0.02 Fluvastatin 1.75 µM 0.56± 0.01 0.32± 0.03 PANC-1 siRNA hERG1 CI values Gemcitabine 19.87 µM Oxaliplatin 1.53 µM Atorvastatin 6.44 µM 1.20± 0.01 1.09± 0.02 Simvastatin 5.8 µM 1.00± 0.02 4.55± 0.01 Lovastatin 5.23 µM 1.3± 0.02 1.84± 0.02 Fluvastatin 3.64 µM 1.27± 0.03 1.23± 0.02 MiaPaCa-2 CI values Gemcitabine 23.95 µM Oxaliplatin 6.76 µM Atorvastatin 1.56 µM 0.77± 0.01 0.98± 0.03 Simvastatin 2.08 µM 0.46± 0.04 0.95± 0.01 Lovastatin 1.03 µM 0.39± 0.01 0.51± 0.02 Fluvastatin 1.75 µM 0.52± 0.02 0.41± 0.01 MiaPaCa-2 siRNA hERG1 CI values Gemcitabine 22.9 µM Oxaliplatin 6.43 µM Atorvastatin 6.93 µM 1.10± 0.03 1.71± 0.02 Simvastatin 6.00 µM 1.17± 0.02 1.12± 0.02 Lovastatin 5.72 µM 1.10± 0.03 1.67± 0.03 Fluvastatin 3.05 µM 1.07± 0.01 1.5± 0.02 It was then evaluated whether the combined treatment with statins and the antibody targeting and inhibiting the hERG1 / ^1 integrin complex would have an enhanced therapeutic effect in vivo. To this purpose an orthotopic PDAC mouse model was generated by ultrasound (US)-guided injection of 1 x 106 PANC1 cells into the pancreas 22 days after cell implantation, mice were randomized into 5 groups. Simvastatin was administrated daily at the dose of 40 mg / kg and 80 mg / kg, either alone or in combination with scDb-hERG1-β116 mg / kg. The doses used for in vivo treatment are obtained converting human doses as indicated in Nair, A. B et al., J. Basic Clin. Pharm., 2016. Treatment with simvastatin alone decreased the tumor masses and if administrated together simvastatin and scDb-hERG1-β1 the reduction of volume of tumor masses is higher (FIG 5A and 5B). Protocol for IHC (ImmunoHistoChemistry) on paraffin embedded samples with scFv-hERG1 and scDb-hERG1 / β1 (MCK Therapeutics S.r.l) IHC was carried out on 7-μm sections on positively charged slides. After dewaxing and rehydrating the sections, endogenous peroxidases were blocked with a 1% H2O2 solution in PBS. Subsequently, antigen retrieval was performed by treatment with proteinase K (5 μg / ml) in PBS at 37°C for 5 minutes. Sections were incubated with an UltraVBlock (LabVision) solution for 5 minutes at room temperature. The solution consists of a mixture of ubiquitous proteins necessary to saturate the non- specific bonds. The primary antibody (scFv-hERG1 or scDb-hERG1 / β1) is added at 20 μg / ml, and incubated overnight at 4°C. Sections were incubated with anti-6xHis antibody (Abcam, Cambridge, UK) for 90 min at RT (1:250 final dilution). Then the immunostaining was performed with a commercially available kit (e. g. PicTure max kit; Invitrogen) according to the manufacturer's instructions and nuclei were controstained with Mayer’s hematoxylin. In table 3 hERG1 / β1 integrin complex expression in tumor primary samples is reported. The table is also reported in Arcangeli A, et al. Expert Opin Ther Targets. 2024. Cancer type Expression Technique Number of (primary samples) positive samples BREAST + IHC 145 / 181 COLORECTAL + IHC 153 / 186 PANCREAS + IHC 132 / 172 NEUROENDOCRINE ILEUM + IHC 10 / 10 AND PANCREAS KIDNEY + IHC 32 / 38 LYMPHOMA + IHC 11 / 20 This protocol can be used to identify cancers characterized by aberrant expression of hERG1 thus expressing the hERG1 / β1 integrin complex.

Claims

CLAIMS 1. A cholesterol lowering agent for use in the treatment of a cancer characterized by aberrant expression of hERG1.

2. The cholesterol lowering agent for use according to claim 1 wherein the cancer characterized by aberrant expression of hERG1 is selected in the group consisting of Neuroblastoma, Endometrial Cancer, Colorectal Cancer, Glioblastoma Multiforme, Pancreatic Ductal Adenocarcinoma, Breast Cancer, Clear Cell Renal Carcinoma, Esophageal Squamous Cell Carcinoma, Gastrointestinal Cancer, Neuroendocrine Ileum and Pancreas Cancer, Lymphoma, Melanoma and non-melanoma skin cancers, Leukemia.

3. The cholesterol lowering agent for use according to claim 1 or 2 wherein said cholesterol lowering agent is an inhibitor of cholesterol synthesis or a siRNA effective in lowering cholesterol levels.

4. The cholesterol lowering agent for use according to claim 3 wherein said inhibitor of cholesterol synthesis is a statin.

5. The cholesterol lowering agent for use according to claim 4 wherein said statin is selected from the group consisting of Simvastatin, Fluvastatin, Lovastatin, Atorvastatin.

6. An association of a cholesterol lowering agent with at least one further anticancer agent for use in the treatment of a cancer characterized by aberrant expression of hERG1 according to any one of claims 1-5.

7. The association for use according to claim 6 wherein said anticancer agent is an anti-hERG1 / β1 integrin complex bispecific antibody and / or a chemotherapy agent.

8. The association for use according to claim 7 wherein said chemotherapy agent is selected from the group consisting of gemcitabine, oxaliplatin or another platinum derivates, clarithromycin and 5-Fluorouracil.

9. The association according to any one of claims 6-8 comprising a cholesterol lowering agent selected from the group consisting of Simvastatin, Fluvastatin, Lovastatin, Atorvastatin and Inclisiran; and an anti-hERG1 / β1 integrin complex bispecific antibody having SEQ ID No:8; and / ora chemotherapy agent selected from the group consisting of gemcitabine and oxaliplatin.

10. A kit for a simultaneous, separate or sequential use in the treatment of cancers characterized by aberrant expression of hERG1, said kit comprising a cholesterol lowering agent, at least one further anticancer agent and instructions for use thereof.

11. The kit according to claim 10 wherein the cholesterol lowering agent is a cholesterol synthesis inhibitor and the at least one further anticancer agent is an anti-hERG1 / β1 integrin complex bispecific antibody and / or a chemotherapy agent.

12. The kit according to claim 10 or 11 comprising: a cholesterol lowering agent selected from the group consisting of Simvastatin, Fluvastatin, Lovastatin, Atorvastatin and Inclisiran; and an anti-hERG1 / β1 integrin complex bispecific antibody having SEQ ID No:8; and / or a chemotherapy agent selected from the group consisting of gemcitabine and oxaliplatin.

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