Combination therapy using tumor treatment fields (TTField)

Abstract

A method for reducing the survival of cancer cells in a subject is provided by applying an alternating current electric field to the cancer cells and delivering at least one of an E2F inhibitor and a CDK4 / 6 inhibitor to the cancer cells. In some examples, the alternating current electric field is applied to the cancer cells at a frequency between 80 and 300 kHz. In some examples, at least a portion of the applying step is performed simultaneously with at least a portion of the delivering step. In some embodiments, the alternating current electric field is applied to the cancer cells for at least 72 hours.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 993,603, filed on March 23, 2020, which is hereby incorporated by reference in its entirety.

[0002] All references cited herein, for example, but not limited to, patents and patent applications, are hereby incorporated by reference in their entirety.

Background Art

[0003] Lung cancer is the second most common cancer and a major cause of cancer - related deaths in the United States. Non - small cell lung cancer (NSCLC) is the most common type, accounting for approximately 80% of new cases. There are numerous treatment options for lung cancer, such as surgical resection, chemotherapy, radiation therapy, and immunotherapy. The 5 - year survival rates for patients with stage I and II NSCLC are approximately 50% and 30%, respectively. However, despite the available treatment options, the 5 - year survival rates for patients with later stages IIIA, IIIB, and IV are 14%, 5%, and 1%, respectively, highlighting the need for new treatment modalities that can be used alone or in combination with conventional therapies to increase survival rates.

[0004] In addition, many cancers, including lung cancer, are associated with deficiencies in genes that function in the DNA repair machinery. These genes are members of various gene regulatory pathways related to cancer development (e.g., the BRCA pathway, the Fanconi anemia (FA) / BRCA pathway).

[0005] TTField is a non-invasive physical modality of cancer treatment approved in combination with temozolomide for recurrent and newly diagnosed glioblastoma multiforme (GBM), and in combination with platinum-based chemotherapy for unresectable locally advanced or metastatic malignant pleural mesothelioma (MPM). Clinical trials are underway for other cancers, including lung cancer, pancreatic cancer, and ovarian cancer. [Overview of the project] [Means for solving the problem]

[0006] This disclosure provides enhancements and augmentations to existing treatment mechanisms utilizing TTField, for example, through combination treatments using TTField with an E2F inhibitor (e.g., HLM006474) and / or a CDK4 / 6 inhibitor (e.g., abemaciclib). Embodiments of this disclosure target specific aspects of the CDK-RB-E2F signaling pathway. Embodiments of this disclosure selectively downmodulate and upmodulate aspects of the CDK-RB-E2F signaling pathway. Embodiments described herein combine TTField with agents that target cancer by reducing DNA repair capacity via multiple pathways that rely on specific DNA repair pathways (e.g., homologous recombination, non-homologous end joining, mismatch repair, replication fork maintenance, and chromosome maintenance). E2F is a universal transcription factor involved in cell cycle regulation, DNA repair, and chromosome maintenance routines in all cell types. By combining TTField with, for example, an E2F inhibitor, embodiments described herein target a wide range of cancers and cell types.

[0007] Embodiments described herein provide a method for reducing the survival of cancer cells in a subject, comprising the steps of delivering at least one of an E2F inhibitor and a CDK4 / 6 inhibitor to cancer cells, and applying an alternating electric field to cancer cells at a frequency between 80 and 300 kHz.

[0008] Embodiments described herein provide a method for killing cancer cells in a target, comprising the steps of delivering at least one of an E2F inhibitor and a CDK4 / 6 inhibitor to the cancer cells, and applying an alternating electric field to the cancer cells at a frequency between 80 and 300 kHz. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows the conserved domains present in the E2F protein. [Figure 2] This figure shows an exemplary signaling pathway of the proteome after exposure to TTField. [Figure 3A] This figure shows an exemplary change in E2F target expression levels after exposure to TTField. [Figure 3B] This is a continuation of Figure 3A, and shows an exemplary change in the expression level of the E2F target after exposure to TTField. [Figure 3C] This is a continuation of Figure 3B, and shows an exemplary change in the expression level of the E2F target after exposure to TTField. [Figure 3D] This is a continuation of Figure 3C, and shows an exemplary change in E2F target expression levels after exposure to TTField. [Figure 4] This figure shows exemplary gene signature markers for E2F-RB dysfunction. [Figure 5] This figure shows an exemplary diagram illustrating the effects of combining TTField with an E2F inhibitor and a CDK4 / 6 inhibitor, according to the embodiments described herein. [Figure 6A] This figure shows the results of exemplary clonal survival assays in four cell lines using the indicated combinations of TTField, an E2F inhibitor, and a CDK4 / 6 inhibitor, according to the embodiments described herein. [Figure 6B]This is a continuation of Figure 6A, and shows the results of exemplary clonal survival assays in four cell lines using the indicated combinations of TTField, an E2F inhibitor, and a CDK4 / 6 inhibitor, according to the embodiments described herein. [Figure 7A] Figures 6A-6B show combined index values ​​from exemplary clonality assays according to some embodiments of the present disclosure. [Figure 7B] This is a continuation of Figure 7A, and shows combined index values ​​from the exemplary clonality assays in Figures 6A-6B, according to some embodiments of the present disclosure. [Figure 8A] This figure shows the time-dependent mRNA and protein expression levels in the Fanconi anemia / BRCA pathway gene in the indicated cell lines. [Figure 8B] This is a continuation of Figure 8A, and shows the time-dependent mRNA and protein expression levels in the indicated cell lines for the Fanconi anemia / BRCA pathway gene. [Figure 8C] This is a continuation of Figure 8B, and shows the time-dependent mRNA and protein expression levels in the indicated cell lines for the Fanconi anemia / BRCA pathway gene. [Figure 8D] This is a continuation of Figure 8C, and shows the time-dependent mRNA and protein expression levels in the indicated cell lines for the Fanconi anemia / BRCA pathway gene. [Figure 8E] This is a continuation of Figure 8D, and shows the time-dependent mRNA and protein expression levels in the indicated cell lines for the Fanconi anemia / BRCA pathway gene. [Figure 8F] This is a continuation of Figure 8E, and shows the time-dependent mRNA and protein expression levels in the indicated cell lines for the Fanconi anemia / BRCA pathway gene. [Figure 9A] This figure shows the transcriptional activity and gene expression of FA pathway genes, cell cycle genes, and DNA replication genes after exposure of cancer cells to TTField. [Figure 9B] Continuation of FIG. 9A, showing the transcriptional activity and gene expression of FA pathway genes, cell cycle genes, and DNA replication genes after exposure of cancer cells to TTField. [Figure 10] Exemplary combination index values obtained from a clonogenic assay for various drug combinations shown at 24, 48, and 72 hours after exposure of H1299 cells to TTField and to TTField containing an E2F inhibitor and a CDK inhibitor, according to aspects described herein. **DETAILED DESCRIPTION**

[0010] The present disclosure relates to the application of TTField in combination with therapeutic agents to target pathways related to the growth and viability of cancer cells.

[0011] Embodiments of the present disclosure are directed to the application of TTField and the delivery of therapeutic agents to cancer cells to dysregulate members of the E2F family, which are known to play an active role in cancer cell replication. For example, the disclosed combination therapies using TTField combine TTField with an E2F inhibitor and / or a CDK4 / 6 inhibitor to downregulate E2F1 and E2F2 of the CDK-RB-E2F axis. Additionally, in some embodiments, the application of the combination therapy combines TTField with an E2F inhibitor and / or a CDK4 / 6 inhibitor to upregulate E2F6 of the CDK-RB-E2F axis.

[0012] TTField dysregulates the E2F family of transcription factors and renders tumor cells sensitive to agents that target the RB-E2F-CDK4 / 6 axis, thus substantially increasing the antitumor effect of TTField alone or in combination with agents that target other pathways regulated by DNA repair, replication stress, and the RB-E2F-CDK4 / 6 axis.

[0013] TTField is used for relapsed and newly diagnosed glioblastoma multiforme (GBM), as well as pleural mesothelioma. TTField is used to induce cell death by disrupting mitosis, adding replication stress, and downregulating DNA repair and cell cycle checkpoint genes.

[0014] TTField is used for the treatment of solid, treatment-resistant primary and recurrent tumors. TTField electrodes are non-invasive and deliver a low-intensity (e.g., 1–3 V / cm) medium-frequency (e.g., 100–300 kHz) alternating electric field across the entire tumor bed. TTField creates a heterogeneous intracellular environment that induces dielectrophoretic migration of polar molecules towards areas of higher electric field intensity, efficiently preventing polymerization and other important biochemical functions. In this way, TTField preferentially targets cancer cells by utilizing cell proliferation, efficiently leaving non-dividing normal cells. In addition, TTField does not stimulate nerves and muscles due to its high frequency, and does not generate high levels of heat due to its low intensity.

[0015] TTField induces conditional vulnerability to ionizing radiation (IR) in non-small cell lung cancer cells (NSCL cell lines). TTField induces a conditional susceptibility state that results in enhanced sensitivity to ionizing radiation, supporting its use as a combination modality therapy with radiation, PARP inhibitors, other DNA damaging agents, E2F inhibitors, and CDK4 / 6 inhibitors.

[0016] TTField reduces cell proliferation and induces incomplete apoptosis in dividing cancer cells across various human and rodent tumor cell lines. Prevention of proper spindle apparatus formation and activation of spindle checkpoints have been proposed as mechanisms by which TTField kills dividing cells. Specifically, exposure to TTField causes microtubule depolymerization and septin mislocalization. This can lead to cell membrane instability and bleb formation that disrupts cytokinesis, resulting in abnormal chromosome segregation, abnormal mitotic termination, and the generation of confused cells that subsequently undergo apoptosis.

[0017] Embodiments described herein provide a method for reducing the survival of cancer cells in a subject, comprising the steps of delivering at least one of an E2F inhibitor and a CDK4 / 6 inhibitor to cancer cells, and applying an alternating electric field to cancer cells at a frequency between 80 and 300 kHz.

[0018] In some examples, at least part of the application step is performed simultaneously with at least part of the delivery step. In some examples, the application step has a period of at least 72 hours. In other examples, the application step has a period of at least 24 hours or 48 hours.

[0019] In some examples, the frequency of the alternating electric field is between 100 and 200 kHz. In another example, the alternating electric field has an electric field strength of at least 1 v / cm in at least some of the cancer cells.

[0020] The concentration of E2F inhibitors in cancer cells may be approximately 10 μM to 50 μM, or approximately 20 μM to 40 μM. The concentration of CDK4 / 6 inhibitors in cancer cells may be approximately 0.1 μM to 5 μM, or approximately 0.5 μM to 2 μM.

[0021] In some cases, the IC25 values ​​for CDK inhibitors (e.g., HLM006474) may be approximately 0.5 μM, 1 μM, and 1.5 μM. In another embodiment, concentrations of approximately 0.5 to 1.5 μM of CDK inhibitors may be used in cancer cells.

[0022] In some cases, the IC25 value for an E2F inhibitor (e.g., LY2835219, also known as abemaciclib) may be approximately 20 μM, 25 μM, or 40 μM. In another embodiment, the concentration of the E2F inhibitor in cancer cells may be approximately 20 μM to 40 μM.

[0023] In yet another example, the E2F inhibitor is selected from a group consisting of one or more of HLM006474, MRT00033659, YKL-5-124-TFA, and YKL-5-124.

[0024] In further examples, CDK4 / 6 inhibitors include abemaciclib, ribociclib, trilaciclib, Ibrance, rerocyclib, arbocidibu, roniciclib, ribiciclib, miliclib, RGB-286638, NSN3106729, PHA-793887, R547, indirubin, NU6102, bohemin, CDK9-IN-7, CGP60474, purvalanol A, PF-06873600, nimboride, and FN-1501. Selected from one or more of the following: AG-024322, ON123300, G1T28, G1T38, AMG925, SHR-6390, BPI-1178, BPI-16350, FCN437, bilocyclib, BEBT-209, Ty-302, TQB-3616, HS-10342, PF-06842874, CS-2002, MM-D37K, CDK4 / 6-IN-2, SU9516, and AT7519.

[0025] In some examples, the delivery step includes administering or delivering an E2F inhibitor and a CDK4 / 6 inhibitor to cancer cells. In some examples, the delivery step includes administering or delivering an E2F inhibitor and a CDK4 / 6 inhibitor to a target. In one embodiment, the E2F inhibitor is HLM006474. In a further embodiment, the CDK4 / 6 inhibitor is abemaciclib. In yet another embodiment, the E2F inhibitor is HLM006474 and the CDK4 / 6 inhibitor is abemaciclib.

[0026] In some cases, cancer cells are selected from a group consisting of lung cancer cells, breast cancer cells, pancreatic cancer cells, glioblastoma cells, prostate cancer cells, liver cancer cells, fallopian tube cancer cells, peritoneal cancer cells, skin cancer cells, and ovarian cancer cells.

[0027] E2F transcription factors are active in all cell types and are involved in, for example, cell cycle regulation, DNA repair, and chromosome maintenance routines. The E2F inhibitor HLM006474 inhibits DNA binding for all E2F complexes and has been used in breast cancer and melanoma models. CDK4 / 6 inhibitors have been used to treat glioblastoma and metastatic breast cancer.

[0028] In some cases, cancer cell survival is reduced to 1 / 20 to 1 / 100 compared to cancer cells that have not been exposed to an alternating electric field and have not received delivery of E2F inhibitors and CDK4 / 6 inhibitors.

[0029] In some cases, cancer cell survival is reduced to approximately 1 / 100th compared to cancer cells that have not been exposed to an alternating electric field and have not received delivery of E2F inhibitors and CDK4 / 6 inhibitors, after (i) 72 hours of exposure of cancer cells to an alternating electric field, (ii) delivery of E2F inhibitors at concentrations of 10 μM to 50 μM, and (iii) delivery of CDK4 / 6 inhibitors at concentrations of 0.1 μM to 2 μM. In some cases, E2F inhibitors are delivered to cancer cells at a concentration of approximately 20 μM, and CDK4 / 6 inhibitors are delivered to cancer cells at a concentration of approximately 5 μM.

[0030] Embodiments described herein provide a method for killing cancer cells in a target, comprising the steps of delivering at least one of an E2F inhibitor and a CDK4 / 6 inhibitor to the cancer cells, and applying an alternating electric field to the cancer cells at a frequency between 80 and 300 kHz.

[0031] In some examples, at least part of the application step is performed simultaneously with at least part of the delivery step. The application step may have a period of at least 72 hours. In another embodiment, the application step may have a period of at least 24 hours or 48 hours.

[0032] The E2F inhibitor may be selected from one or more of the following: HLM006474, MRT00033659, YKL-5-124-TFA, and YKL-5-124.

[0033] CDK4 / 6 inhibitors include abemaciclib, ribociclib, trilaciclib, Ibrance, rerocyclib, arbocidibu, roniciclib, ribiciclib, milcilib, RGB-286638, NSN3106729, PHA-793887, R547, indirubin, NU6102, bohemin, CDK9-IN-7, CGP60474, purvalanol A, PF-06873600, nimboride, FN-1501, and AG-0. The group may be selected from one or more of the following: 24322, ON123300, G1T28, G1T38, AMG925, SHR-6390, BPI-1178, BPI-16350, FCN437, bilocyclib, BEBT-209, Ty-302, TQB-3616, HS-10342, PF-06842874, CS-2002, MM-D37K, CDK4 / 6-IN-2, SU9516, and AT7519.

[0034] In some cases, the E2F inhibitor is HLM006474. In some cases, the CDK4 / 6 inhibitor is abemaciclib. In yet another case, the E2F inhibitor is HLM006474 and the CDK4 / 6 inhibitor is abemaciclib.

[0035] The experimental data presented below discuss the use of proteomics analysis to identify the mechanisms by which TTField and combination therapies can downregulate key genes related to cell cycle checkpoints and DNA repair, as well as other survival pathways that present therapeutic challenges to conventional treatment systems.

[0036] As discussed herein, quantitative proteomics experiments identified reduced expression of the transcription activators E2F1 and E2F2. In addition, quantitative proteomics experiments identified increased expression of the transcription repressor E2F6. Therefore, the combination therapies described herein affect the CDK-RB-E2F axis and dysregulate DNA repair genes (e.g., RAD51, BRCA1, and BRCA2) through E2F4 and E2F6 signaling.

[0037] Figure 1 provides an illustrative overview of the conserved domains (i.e., domains that are conserved and have designated functions) present in E2F proteins. E2F proteins consist of eight family members (E2F1-8), which are divided into transcription activators (E2F1-E2F3a) and transcription repressors (E2F3b-E2F8) based on their function. E2F proteins play a central role in cell proliferation by regulating thousands of genes crucial for cell cycle progression, DNA replication, DNA damage checkpoints, and DNA repair. Activator protein (E2F1, E2F2, and E2F3A) levels peak during the G1-S phase transition, atypical repressor (E2F7 and E2F8) levels peak in the latter half of late S phase, while classical repressor (E2F3B, E2F4, E2F5, and E2F6) levels remain constitutively expressed throughout all stages of the cell cycle.

[0038] As illustrated, all E2Fs share a unique winged helical DNA-binding domain. E2F1, E2F2, E2F3, E2F4, E2F5, and E2F6 require dimerization with a member of the transcription factor dimerization partner (TFDP) family (TFDP1 or TFDP2) to bind to DNA. This binding is facilitated by a dimerization domain consisting of a leucine zipper (LZ) domain and a marked box (MB) domain. Pocket proteins (RB, p107, and p130) bind to E2F1, E2F2, E2F3, E2F4, and E2F5 via their transactivation domains.

[0039] The minimum site required for interaction with pocket proteins is indicated by (RB). RB binds to all E2F1, E2F2, E2F3, E2F4, and E2F5, while p107 and p130 bind only to E2F4 and E2F5. E2F6 does not bind to pocket proteins but is instead regulated by Polycomb proteins. Some E2F proteins also possess a nuclear localization sequence (NLS), a nuclear export sequence (NES), or a cyclin A (CCNA) regulatory domain. E2F7 and E2F8 lack dimerization and transactivation domains and do not bind to TFDP or pocket proteins. Instead, they possess two tandem DNA-binding domains.

[0040] Exemplary upstream analysis of differentially expressed proteins upon cell exposure to TTField showed that activators such as E2F1 and E2F2 were inhibited, while repressors such as E2F6 were activated.

[0041] Figure 2 provides an exemplary upstream analysis of the proteome after cell exposure to TTField. The proteomic results suggest that TTField exposure leads to inhibition of transcription activators (E2F1 and E2F2) and activation of a repressor (E2F6).

[0042] The E2F-RB axis functions as a central junction controlling numerous pathways in cancer cells, including mitosis, DNA damage and repair, DNA replication, chromatin remodeling, and apoptosis. Gene expression and direct function studies have shown that the E2F-RB pathway targets several members of the Fanconi anemia (FA) pathway. Furthermore, TTField downregulates members of the FA pathway through dysregulation of E2F-RB (which acts as an upstream regulator of the FA pathway and cell cycle).

[0043] Figures 3A–3D provide exemplary results of E2F target expression assays in lung cancer cells after exposure to TTField, compared to cells not exposed to TTField, based on different proteomic analyses.

[0044] The E2F targets examined in Figures 3A–3D include members of the FA pathway (e.g., BRCA1, FANCD2, RAD51), members related to replication forks (e.g., MCM6, RFC3, RFC4), and proteins related to mitosis (e.g., BUB3, CCNE2, EZH2). As illustrated in Figures 3A–3D, expression levels change upon exposure to TTField, as indicated by log expression values. Changes in expression levels were also classified as inhibited, affected, or activated.

[0045] As shown in Figures 3A-3D, E2F1 and E2F2 are activators, and E2F4 and E2F6 are inhibitors; they were inhibited and activated, respectively, upon exposure to TTField. Consequently, the expression of their targets decreased (e.g., BRCA1, MCM6, CCNE2, EZH2).

[0046] Figure 4 provides exemplary gene signature markers for E2F-RB dysfunction. Gene signature analysis revealed a set of gene functions deregulated by E2F-RB dysregulation / loss. Figure 4 identifies exemplary gene targets related to DNA replication, DNA damage and repair, apoptosis, mitosis, and chromatin regions.

[0047] Figure 5 provides a diagrammatic representation of exemplary combination therapies described herein with respect to TTField, an E2F inhibitor, and a CDK4 / 6 inhibitor. As illustrated, the combination of TTField with inhibitors of upstream regulators (E2F and CDK4 / 6) of the cell cycle, DNA damage, and replication stress, which the inventors observed as the mechanism of action of TTField, would be highly beneficial. As disclosed herein, the E2F and CDK4 / 6 regulators can be targeted by E2F inhibitors (e.g., HLM006474) and CDK4 / 6 inhibitors (e.g., abemaciclib). While not bound by theory, as illustrated in Figure 5, exposure of cancer cells to TTField and E2F inhibitors targets dysregulation of the E2F gene target, while CDK4 / 6 inhibitors target cell cycle progression. This combination can lead to abnormal cell division, DNA damage, replication stress, and ultimately cell death.

[0048] As illustrated in Figure 5, TTField can be applied to cancer cells to dysregulate E2F. Additional E2F inhibitors (e.g., HLM006474) can be applied to cancer cells to dysregulate E2F targets, causing increased expression of repressors, decreased or inhibited expression of activators. CDK4 / 6 inhibitors (e.g., abemaciclib) can also be applied to target genes involved in the cell cycle, FA, DNA damage, and replication, inhibiting CDK4 / 6 activity and cell cycle expression, leading to abnormal cell division, increased DNA damage, and increased replication stress. These effects can lead to an increase in abnormal cells, resulting in increased mitotic / catastrophic cancer cell death.

[0049] Figures 6A–6B provide the results of exemplary clonal survival assays in four lung cancer cell lines, H1299, A549, H157, and H4006 cells, under various conditions testing combinations of TTField with agents that dysregulate E2F signaling or CDK4 / 6 signaling at 24, 48, and 72 hours. H1299 and A549 are non-small cell lung cancer cell lines that are resistant to conventional treatments using TTField. In contrast, H157 and H4006 are cell lines known to be more responsive to TTField. Cell survival after increased exposure is shown for TTField alone or in combination with TTField and E2F inhibitors, CDK4 / 6 inhibitors, or combinations of E2F inhibitors and CDK4 / 6 inhibitors.

[0050] As shown in Figures 6A–6B, when lower doses of E2F inhibitors and CDK4 / 6 inhibitors are used in combination with TTField, there is a 20–100-fold reduction in cell viability 72 hours after TTField exposure. In one example, a combination of 72 hours of TTField exposure, 20 μM of the E2F inhibitor HLM006474, and 0.5 μM of abemaciclib resulted in approximately a 1 / 100-fold reduction in cell viability. While not bound by theory, this unexpected reduction in cell viability is thought to have resulted from the transient targeting of several pathways.

[0051] Figures 7A–7B provide combined index (CI) values ​​from exemplary clonal survival assays described in Figures 6A–6B. In some examples, CI values ​​greater than 1.0 indicate a synergistic effect or a surprisingly favorable effect on antitumor activity. As illustrated in Figures 7A–7B, the H1299 and A549 cell lines show a significant synergistic effect 72 hours after TTField was applied in combination with a CDK+E2F inhibitor (e.g., a CI of 11.65 for H1299 and a CI of 13.91 for A549 at 72 hours).

[0052] In some embodiments, the application of combination therapy involves combining the application of TTField with the application or delivery of one or more therapeutic agents to cancer cells, either together with or immediately after the application of TTField.

[0053] In some embodiments, the application of combination therapy involves combining the application of TTField with the application of one or more therapeutic agents to cancer cells after a predetermined period following the application of TTField. The predetermined period may be determined based on the observed vulnerability of the cancer cells after the application of TTField. For example, in some embodiments, cancer cells may be treated with or exposed to the combination therapy approximately 24, 48, or 72 hours after the application of TTField.

[0054] Figures 8A–8F provide exemplary data on mRNA and protein expression levels for genes in the FA-BRACA pathway (BRCA1, FANCE, FANCC, FANCB, FANCA, and RFC3) in H4006, H157, A549, H1299, and H1650 cells after 24, 48, and 72 hours of exposure to TTField. As shown in Figures 8A–8F, TTField reduced mRNA and protein expression of these genes, with the greatest decrease in expression observed after 72 hours.

[0055] Figures 9A-9B illustrate the exemplary activity of transcription activators (E2F1, E2F2) and transcription repressors (E2F4, E2F6) for the indicated target genes in the FA pathway (BRCA1, FANCD2, MLH1, RBL1, RFC3, RFC4), mitosis / cell cycle (CCNE2, DUSP1, EZH2, MAD2L1), and DNA replication (CDC45, DHFR, MCM6, POLA2, RRM2). Taken together, the data in Figures 9A-9B show that TTField inhibits transcription activators (E2F1, E2F2) and activates transcription repressors (E2F4, E2F6) for these three classes of genes.

[0056] Figure 10 provides exemplary combination index (CI) data for various combinations of TTField and drugs in H1299 cells at 24, 48, and 72 hours after exposure to TTField. Combination index studies are used to determine the additive or synergistic effects of the biological effects of drug combinations. See, for example, Chou et al., Drug combination studies and their synergy quantification using the Chou-Talalay method, Cancer Res, January 15, 2010; Vol. 70 (No. 2): pp. 440-446. In one embodiment, the combination of 72 hours of TTField exposure, 20 μM of the E2F inhibitor HLM006474, and 0.5 μM of abemaciclib resulted in an unexpectedly high combination index of 8.72. [Examples]

[0057] Materials and methods for the experiment cell culture Human NSCLC cell lines (H157, H4006, A549, and H1299) were purchased from the American Tissue Culture Collection. All of these cell lines were grown in RPMI medium supplemented with 10% (v / v) fetal bovine serum (Atlanta Biologicals, Flowery Branch, Ga., USA) and penicillin / streptavidin (final concentration 50 μg / ml; Sigma-Aldrich, St. Louis, Mo., U29SA). All cells were grown at 37°C in a humidified incubator with a constant supply of 5% CO2.

[0058] Tumor Therapy Electric Field An inovitro system (NovoCure Ltd, Haifa, Israel) was used to generate a TTField using two pairs of electrodes printed perpendicularly on the outer wall of a petri dish made of high dielectric constant ceramics (magnesium niobate lead titanate (PMN-PT)). The transducer array was connected to a sinusoidal generator that produced a low-intensity electric field at a desired frequency in the culture medium. The direction of the TTField was switched 90° every second, thus covering most of the directional axes of cell division. The plate temperature was maintained at 37°C by placing the plate in a refrigerated incubator where the temperature was maintained at 19°C and the heat generated by the inovitro system was dissipated. The temperature was measured by two thermistors (Omega Engineering, Stamford, Conn., USA) mounted on the ceramic wall. All cell suspensions were grown on coverslips in inovitro dishes (NovoCure Ltd) and treated with the TTField for the times shown in the figure.

[0059] Cell growth assay Human NSCLC (H157, H4006, A549, and H1299) cell lines were treated for 24, 48, and 72 hours at different TTField frequencies as indicated, and cell growth was counted three times for each sample using a Beckman Coulter counter (Beckman Coulter Inc, Indianapolis, hid., USA). Growth curves were graphed using GraphPad Prism V.6 (GraphPad Software Inc, La Jolla, Calif, USA) with the average cell count at each time point and the given TTField frequency.

[0060] Cell cycle analysis Cells were collected at specific times and treatments and fixed in 75% ice-cold ethanol at -20°C for 24 hours. The fixed cells were washed with PBS and incubated for 30 minutes at 37°C in PBS containing 500 μl of PI staining solution, i.e., 1 mg / ml RNAse A (Sigma-Aldrich), 0.05% triton X-100, and 30 μg / ml PI (Sigma-Aldrich). Cell cycle distribution was determined using the FACSCalibur system (BD Biosciences, San Jose, Calif., USA). Over 10,000 cells were counted per sample, and the results were analyzed using FlowJo software v8.7.1 (Tree Star Inc., Ashland, Oreg., USA).

[0061] RNA labeling and hybridization for gene expression analysis The Illumina Whole Genome HumanWG6 v4 Expression BeadChip (Illumina Inc., San Diego, Calif., USA) was used. Each RNA sample (0.5 μg) was amplified using the Illumina TotalPrep RNA amplification kit with biotin UTP labeling (Enzo Life Sciences, Inc., Farmingdale, NY, USA). Single-stranded cDNA was generated using T7 oligo(dT) primers, followed by double-stranded synthesis to generate double-stranded cDNA, which was then purified by column. Biotin-labeled cRNA was synthesized by in vitro transcription using T7 RNA polymerase. The cRNA was then purified by column and checked for size and yield using the Bio-Rad Experion system (Bio-Rad Laboratories, Hercules, Calif., USA). Next, cRNA (1.5 μg) was hybridized to each array using the standard Illumina protocol with streptavidin-Cy3 (Amersham Biosciences, Piscataway, NJ, USA) used for detection. The slides were scanned using an Illumina Beadstation (Illumina Inc).

[0062] Data processing and significance analysis of differential gene expression Summary expression values ​​for each probe set were prepared using BeadStudio 3.1 (Illumina Inc). Data were background subtracted and displacement-displacement-normalized across the entire sample using the MBCB algorithm. Plots for comparison were created using the normalized gene expression values. Differential gene expression analysis in the treated cell lines was performed using SAM. An FDR < 0.05 was considered statistically significant. Cluster analysis and heatmaps were created using Partek Genomic Suite software (Partek Incorporated, St. Louis, Mo., USA). Gene ontology and pathway analysis were performed using IPA (QIAGEN, Redwood City, Calif., USA).

[0063] Immunoblot Laemmli sample buffer (4×; Bio-Rad Laboratories) was added to 30 μg of each protein sample, and the mixture was boiled at 95°C for 10 minutes. The protein mixture was then loaded onto a 10% SDS-PAGE gel and subsequently transferred to a PVDF membrane at 90 V, 4°C for 1 hour. The membranes were blocked at room temperature for 1 hour with 5% nonfat milk in PBST and examined overnight at 4°C with anti-(3-actin (1:5000; Cell Signaling, Danvers, Mass., USA), anti-BRCA1 (1:1000), anti-FANCD2 (1:2000), and anti-FANCA (1:500; Novus Biologicals LLC, Littleton, Colo., USA)) in PBST containing 2% bovine serum albumin (Thermo Fisher Scientific Inc, Bridgewater, NJ, USA). The membranes were washed with phosphate-buffered saline containing 0.1% Tween-20 (TBST; 3 × 10 mins each) and subsequently incubated with secondary antibodies (1:5000) conjugated with horseradish peroxidase (GE Healthcare, Buckinghamshire, UK) at room temperature for 1 hour. The membranes were then subjected to FluorChem M system (ProteinSimple, San The samples were developed using a chemiluminescence detection kit (Thermo Scientific, Rockford, Ill., USA) at Jose, Calif, USA. Quantification was performed using ImageJ software (NIH, Bethesda, Md., USA) and normalized using the corresponding actin density.

[0064] Immunofluorescence Cells were seeded on glass coverslips, processed, washed, and fixed with ice-cold methanol. Samples were blocked for 1 hour with 10% normal goat serum and incubated with phosphohistone-γ-H2AX antibody (Ser139; Upstate Biotechnology, Temecula, Calif, USA) and p53-binding protein 1 (53BP1) antibody (Cell Signaling). Samples were washed three times in PBS for 5 minutes each and then incubated for 1 hour with Alexa Fluor 488 conjugate anti-rabbit antibody and Alexa Fluor 555 conjugate anti-mouse antibody (Invitrogen, Carlsbad, Calif, USA). Nuclei were counterstained with DAPI contained in Vecatshield mounting medium (Vector Laboratories Inc, Burlingame, Calif, USA). Next, the stained cells were analyzed using five z-stack sections, each 0.2 μM thick, under a fluorescence microscope (Axio Imager M2, Carl Zeiss, Thornwood, NY, USA) equipped with a 63x objective lens (oil immersion, numerical aperture 1.3). Quantitative image analysis of 40 nuclei from each experiment was performed using the cell module in Imaris software version 8.0 (Bitplane, Concord, Mass., USA).

[0065] Radiation exposure and clonal cell survival To study the effect of radiosensitization on NSCLC cells, exponentially growing cells were treated with IR at a dose rate of 3.47 Gy / min using a Mark II Cs irradiator (JL Shepherd and Associates), followed by immediate application of TTField for 24, 48, and 72 hours. The cells were then reseeded in 60 mm dishes and incubated for up to two weeks. Colonies containing more than 50 cells were considered viable. Data are expressed as the mean ± SEM of three independent experiments. The radiosensitizing effect of TTField was evaluated according to the Highest Single Agent approach by calculating CI as shown below.

[0066] CI=(SF IR ×SF TTField ) / SCIENCE FICTION IR + TTField (In the formula, SF = survival rate)

[0067] Combination effects were considered enhancing / synergistic if CI > 1, and additive if CI = 1. Statistical significance for positive effects was determined by the p-value of a two-way ANOVA multiple comparison test comparing the combination (TTField and IR) with a single agent showing the highest cell death rate for a given dose and time after IR. [Examples]

[0068] Proteometric analysis of lung cancer cells exposed to TTField In several embodiments, proteomics analysis of lung cancer cells exposed to tumor therapeutic electric fields identified dysregulation of the E2F-Rb-CDK4 / 6 axis as making tumor cells sensitive to novel combination therapies described that target CDK4 / 6 and / or E2F. One mechanism described for TTField inducing cell death is by disruption of mitosis, but more recent studies suggest that TTField induces replication stress and downregulates DNA repair and cell cycle checkpoint genes. However, the exact cause of the downregulation of DNA repair and cell cycle checkpoint genes remains elusive. For such purposes, the disclosed technique employed relative quantitative proteomics analysis using tandem mass tagging (TMT). All samples underwent trypsin digestion and labeling with different TMT reagents. They were then mixed, and the mixtures were processed in an Orbitrap Fusion mass spectrometer.

[0069] Peptide quantification was achieved by comparing the intensities of TMT reporter ions. STRING DB analysis of differentially expressed proteins revealed interaction networks including cell cycle, DNA damage repair and replication, and transcriptional and translational regulation. Upstream analysis of key genes associated with cell cycle checkpoints and DNA repair identified reduced expression of the transcription activators E2F1 and E2F2, and increased expression of the transcription repressor E2F6, suggesting that TTField influences the CDK-RB-E2F axis. For example, the downregulation of key DNA repair genes, including RAD51, BRCA1, and BRCA2, could be explained, for instance, by the upregulation of the transcription repressors E2F4 and E2F6 (known repressors of BRCA1).

[0070] These proteins are involved in homologous recombination repair and nucleotide excision repair, but are also involved in replication fork maintenance, replication fork collapse, and overall replication stress, the latter of which can lead to cell death. Therefore, in one example, TTField was combined with the E2F inhibitor HLM006474, with or without the CDK4 / 6 inhibitor abemaciclib. TTField combined with any of the inhibitors synergistically enhanced cell death compared to TTField alone, but the three combinations were found to be highly lethal (>90% by 72 hours), as measured by clonality assays and subsequent Highest Single Agent approaches to determine synergy. Taken together, in one example, these results identified the CDK-RB-E2F axis as a target that could lead to the development of novel drugs that can be used in combination with TTField for cancer treatment. [Examples]

[0071] Further genomic and proteomics support for combination therapies, including TTfields. Transcriptomics approaches have been used by previous research groups to capture the overall picture of biological processes in an untargeted and unbiased manner and to understand the overall changes in gene expression during TTField exposure of a series of non-small cell lung cancer (NSCLC) cells. Ingenuity Pathway Analysis (IPA) of TTField-responsive genes suggested that the changes occur in the cell cycle and mitotic regulatory pathways, which is consistent with previous studies, but also revealed a significantly downregulated BRCA1 DNA damage response pathway (P<0.05) upon TTField exposure. However, exactly what causes the downregulation of DNA repair and cell cycle checkpoint genes remained elusive.

[0072] Therefore, to determine how changes in genomic expression levels translate to changes in protein expression, which are functional players in biological activity, we conducted experiments including proteomic analysis to investigate TTField inducing changes at the proteome level. Relative quantitative proteomics analysis using tandem mass tagging (TMT) was used to quantify the proteome in H1299 cells under control and TTField-treated conditions. After TTField exposure, a total of 106 and 541 differentially expressed proteins were present at 24 and 48 hours, respectively. STRING database analysis of differentially expressed proteins revealed interaction networks including cell cycle, DNA damage repair and replication, mitochondrial dysfunction, and transcriptional and translational regulation. The same patterns of expression changes for members of the FA pathway, cell cycle, and DNA damage and replication pathways were observed at the protein level from proteomics and at the mRNA level from transcriptomics analysis, as illustrated in Figures 8A–8F.

[0073] Figures 8A–8F illustrate transient mRNA-level expression changes from transcriptomics data, protein-level expression changes from proteomics data, and verification of protein-level changes by Western blotting for some of the FA pathway members identified as downregulated, suggesting similar expression patterns for FA pathway members at both the gene and protein levels.

[0074] In addition, since these regulatory nodes exert maximum control over dysregulated pathways but minimal control over undisturbed pathways, we conducted experiments to identify key upstream regulatory network mechanisms that act as regulatory nodes for the expression of several downstream pathways, such as the cell cycle, DNA damage repair, and replication. Analytical analysis of TTField upstream regulators that induce differentially expressed proteins identified a decrease in the expression of upstream transcriptional regulators, such as the transcriptional activator and repressor E2F1 and E2F2, and an increase in the expression of E2F6. As illustrated in Figures 9A-9B, this suggests that TTField affects the CDK-Rb-E2F axis and dysregulates key DNA repair proteins, including RAD51, BRCA1, and BRCA2, through E2F4 and E2F6 signaling and replication fork-related proteins (MCM6, RFC3, RFC4) and mitotic proteins (BUB3, CCNE2, EZH2). These proteins are involved in the cell cycle, homologous recombination repair, nucleotide excision repair, replication fork maintenance, replication fork collapse, and overall replication stress.

[0075] More specifically, Figures 9A-9B illustrate upstream regulatory factor analysis and quantitative changes in E2F target expression from different proteomic analyses. TTField inhibits transcriptional activators (E2F1-3) and increases transcriptional repressors (E2F4-8) of the E2F family of transcription factors. Therefore, there was a decrease in the expression of Fanconi anemia (BRCA1, FANCD2, MLH1, RBL1, RFC3, RFC4) and DNA replication pathway proteins (CDC45, DHFR, MCM6, POLA2, RRM2), as well as different effects on mitotic / cell cycle pathway proteins (CCNE2, DUSP1, EZH2, MAD2L1), informing the inventors of the complexity of transcription by these activators / inhibitors towards specific targets. A table provides the ratio (Log2) of changes in protein expression for different pathway proteins, transcription factor directions, and the expression of the final target protein. Upstream regulatory factor analysis examines the number of known targets for each transcription factor present in the user's dataset, based on prior knowledge of expected effects between transcription factors and their target genes stored in the Ingenuity® Knowledge Base, and also compares the direction of their changes (i.e., expression in TTField samples compared to controls).

[0076] A comparative analysis of changes in transcriptome and proteome levels showed that the CDK-Rb-E2F axis acts as an upstream regulatory node for the effects observed with TTField exposure on cell cycle, DNA damage repair, and replication stress pathways. The CDK-Rb-E2F axis is potentially leading to the development of new drugs and is indeed a major drug target in cancer treatment today. In doing so, targeting this axis will change how TTField will eventually be used, including becoming an integrated therapy that would enhance conventional radiotherapy and chemotherapy targeting DNA repair, cell cycle checkpoints, or proliferation and survival pathways.

[0077] Figure 10 provides a comparison of combination index values ​​for different drugs and ionizing radiation (IR) in combination with TTField. In one example, the results suggest that blocking the CDK-E2F-RB axis using both CDK inhibitors and E2F inhibitors in combination with TTField is highly effective compared to using individual drugs or other drugs. The table provides the P-values ​​for each combination.

[0078] The systems and methods described herein may be used in combination with drugs after the application of first-line therapy. By combining TTField with drugs on the RB-E2F axis, the integrated therapy may enhance downstream therapies such as radiation, chemotherapy, or other therapies targeting DNA repair, cell cycle checkpoints, or proliferation and survival pathways.

[0079] The systems and methods described herein can be used for the treatment and / or relief of glioblastoma, mesothelioma, pancreatic cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, skin cancer, peritoneal cancer, and others. The systems and methods described herein may also be used to improve upon conventional systems that apply TTField in combination therapy.

[0080] While the present invention has been disclosed with reference to certain embodiments, numerous modifications, changes, and variations of the embodiments described are possible without departing from the scope and realm of the invention, as defined in the appended claims. Therefore, the present invention is not limited to the embodiments described and is intended to have a full scope as defined by the claims language and their equivalents listed below.

Claims

1. A composition comprising an E2F inhibitor and a CDK4 / 6 inhibitor for use in a method for reducing the survival of cancer cells, The method comprises the step of delivering the composition to cancer cells, wherein the E2F inhibitor comprises HLM006474 and the CDK4 / 6 inhibitor comprises abemaciclib; and the composition comprising the step of applying an alternating electric field to cancer cells at a frequency between 80 and 300 kHz for a period of time sufficient to dysregulate the E2F family of transcription factors and make the cancer cells sensitive to drugs targeting the CDK-RB-E2F axis.

2. The composition according to claim 1, wherein at least a portion of the application step is performed simultaneously with at least a portion of the delivery step.

3. The composition according to claim 1, wherein the step of applying the solution has a period of at least 72 hours.

4. The composition according to claim 1, wherein the frequency of the alternating electric field is between 100 and 200 kHz.

5. The composition according to claim 1, wherein the alternating electric field has an electric field strength of at least 1 V / cm in at least some of the cancer cells.

6. The composition according to claim 1, wherein the concentration of the E2F inhibitor in cancer cells is 10 μM to 50 μM.

7. The composition according to claim 1, wherein the concentration of the E2F inhibitor in cancer cells is 20 μM to 40 μM.

8. The composition according to claim 1, wherein the concentration of the CDK4 / 6 inhibitor in cancer cells is 0.1 μM to 5 μM.

9. The composition according to claim 1, wherein the concentration of the CDK4 / 6 inhibitor in cancer cells is 0.5 μM to 2 μM.

10. The composition according to claim 1, wherein the cancer cells are selected from the group consisting of lung cancer cells, breast cancer cells, pancreatic cancer cells, glioblastoma cells, prostate cancer cells, liver cancer cells, fallopian tube cancer cells, peritoneal cancer cells, skin cancer cells, liver cancer cells, and ovarian cancer cells.

11. The composition according to claim 1, wherein the survival of cancer cells is reduced to 1 / 20 to 1 / 100 compared to cancer cells that have not been exposed to an alternating electric field and have not been delivered with an E2F inhibitor and a CDK4 / 6 inhibitor.

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