Combination therapy using tumor treatment fields (TTField)

Abstract

This invention provides a method for reducing the survival of cancer cells in a target organism. [Solution] A method is provided comprising the steps of applying an alternating current electric field to 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 part of the application step is performed simultaneously with at least part of the delivery 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 leading 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. Among the numerous treatment options for lung cancer are, for example, surgical resection, chemotherapy, radiotherapy, 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]

[0005] TTField has been shown in combination with temozolomide to treat relapsed and newly diagnosed cases. Unresectable due to glioblastoma multiforme (GBM) and in combination with platinum-based chemotherapy. It is approved for locally advanced or metastatic malignant pleural mesothelioma (MPM), but It is a non-invasive physical modality of cancer treatment, including lung cancer, pancreatic cancer, and ovarian cancer. Clinical trials for this cancer are underway. [Overview of the project] [Means for solving the problem]

[0006] This disclosure, for example, describes how TTField can be used with an E2F inhibitor (e.g., HLM006474). combinations used with and / or CDK4 / 6 inhibitors (e.g., abemaciclib) This treatment provides enhancement and improvement to existing treatment mechanisms utilizing TTField. Embodiments of this disclosure target specific aspects of the CDK-RB-E2F signaling pathway. Embodiments of the present disclosure selectively modify the CDK-RB-E2F signaling pathway. Adjust downwards and upwards. The embodiments described herein specify the TTField to DNA repair pathways (for example, homologous recombination, non-homologous end joining, mismatch repair, and multiple end joining) Reduced DNA repair capacity through multiple pathways (reliant on fork maintenance and chromosome maintenance) It is used in combination with cancer-targeting drugs. E2F is present in the pericellular space of all cell types. It is a universal transcription factor involved in phase regulation, DNA repair, and chromosome maintenance routines. By combining TField with, for example, an E2F inhibitor, as described herein, The methods used target a wide range of cancers and cell types.

[0007] The embodiments described herein are methods for reducing the survival of cancer cells in a subject, The procedure involves delivering at least one of the following to cancer cells: an E2F inhibitor and a CDK4 / 6 inhibitor. Step, and step, which applies an alternating electric field to cancer cells at a frequency between 80 and 300 kHz. We provide a method using the app.

[0008] The embodiments described herein are methods for killing cancer cells in a subject, E2 Steps to deliver at least one of the F inhibitors and CDK4 / 6 inhibitors to cancer cells The steps include applying an alternating electric field to cancer cells at a frequency between 80 and 300 kHz. This provides a method. [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]A diagram showing an exemplary graphical representation of the effect of combining TTFields with an E2F inhibitor and a CDK4 / 6 inhibitor, according to the aspects described herein. [Figure 6A] A diagram showing the results of an exemplary clonogenic survival assay in four cell lines with the indicated combinations of TTFields, an E2F inhibitor, and a CDK4 / 6 inhibitor, according to the aspects described herein. [Figure 6B] A continuation of FIG. 6A, showing the results of an exemplary clonogenic survival assay in four cell lines with the indicated combinations of TTFields, an E2F inhibitor, and a CDK4 / 6 inhibitor, according to the aspects described herein. [Figure 7A] A diagram showing combination index values from the exemplary clonogenic assays of FIGS. 6A - 6B, according to some embodiments of the present disclosure. [Figure 7B] A continuation of FIG. 7A, showing combination index values from the exemplary clonogenic assays of FIGS. 6A - 6B, according to some embodiments of the present disclosure. [Figure 8A] A diagram showing the time - dependent mRNA expression levels and protein expression levels in the indicated cell lines for genes in the Fanconi anemia / BRCA pathway. [Figure 8B] A continuation of FIG. 8A, showing the time - dependent mRNA expression levels and protein expression levels in the indicated cell lines for genes in the Fanconi anemia / BRCA pathway. [Figure 8C] A continuation of FIG. 8B, showing the time - dependent mRNA expression levels and protein expression levels in the indicated cell lines for genes in the Fanconi anemia / BRCA pathway. [Figure 8D] A continuation of FIG. 8C, showing the time - dependent mRNA expression levels and protein expression levels in the indicated cell lines for genes in the Fanconi anemia / BRCA pathway. [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] This is a continuation of Figure 9A, and 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 10] This figure shows exemplary combination index values ​​obtained from clonality assays for H1299 cells, for TTField and various drug combinations, including E2F inhibitors and CDK inhibitors, 24, 48, and 72 hours after exposure to TTField. [Modes for carrying out the invention]

[0010] This disclosure describes therapeutic agents that target pathways related to the growth and survival ability of cancer cells. Regarding the application of TTField in combination with [the other method].

[0011] Embodiments of this disclosure relate to E2, which is known to play an active role in the replication of cancer cells. To dysregulate members of the F family, apply TTField, and cancer cells. The target is the delivery of therapeutic agents to cells. For example, disclosed combination therapy using TTField The method involves combining TTField with an E2F inhibitor and / or a CDK4 / 6 inhibitor. Adjust E2F1 and E2F2 of the CDK-RB-E2F axis downwards. In addition, several In this embodiment, the application of combination therapy involves TTField as an E2F inhibitor and / or Combined with a CDK4 / 6 inhibitor, it upregulates E2F6 on the CDK-RB-E2F axis. .

[0012] TTField dysregulates the E2F family of transcription factors, and RB-E To make the 2F-CDK4 / 6 axis susceptible to drugs targeting it, thus, TTField TTField alone, or in combination with DNA repair, replication stress, and RB-E2F-CD Substantially increases the tumor-killing effect of drugs that target other pathways regulated by the K4 / 6 axis. To make someone do it.

[0013] TTField is a doctor specializing in recurrent and newly diagnosed glioblastoma multiforme (GBM). It is used for pleural mesothelioma. TTField disrupts mitosis and replication stress. By adding and downregulating DNA repair and cell cycle checkpoint genes, It is used to induce cell death.

[0014] TTField is used for the treatment of solid, treatment-resistant primary and recurrent tumors. The TTField electrode is non-invasive and provides low-intensity (e.g.,) treatment across the entire tumor bed. For example, delivering an AC electric field of medium frequency (e.g., 100-300 kHz) with a voltage of 1-3 V / cm. TTField induces the dielectrophoretic migration of polar molecules towards regions of higher electric field strength. It creates a heterogeneous intracellular environment that efficiently prevents polymerization and other important biochemical functions. In this way, TTField preferentially targets cancer cells by utilizing cell proliferation. This efficiently preserves non-dividing normal cells. In addition, TTField uses those high frequencies Therefore, they do not stimulate nerves and muscles, and their low intensity does not generate high levels of heat. do not have.

[0015] TTField uses ionizing radiation (I) in non-small cell lung cancer cells (NSCL cell lines). It induces conditional vulnerability to R). TTField is resistant to ionizing radiation. It induces a conditional sensitivity state that leads to increased sensitivity to radiation, PARP Combination modalities with inhibitors, other DNA damage agents, E2F inhibitors, and CDK4 / 6 inhibitors. Support the use of TTField as a therapeutic agent.

[0016] TTField reduces cell proliferation and is effective in various human and rodent tumor cell lines. It induces incomplete apoptosis in dividing cancer cells. Spindle apparatus The proper formation of the spindle checkpoint is prevented, and the TTField splits. It has been proposed as a mechanism to kill cells that are exposed to TTField. Specifically, exposure to TTField This leads to microtubule depolymerization and septin mislocalization. This causes instability in the cell membrane. This can lead to qualitative and bleb formation that disrupts cytokinesis, resulting in abnormal chromosome segregation. This leads to the generation of disordered cells that undergo abnormal termination of mitosis and subsequent apoptosis. Wake up.

[0017] The embodiments described herein are methods for reducing the survival of cancer cells in a subject, The procedure involves delivering at least one of the following to cancer cells: an E2F inhibitor and a CDK4 / 6 inhibitor. Step, and step, which applies an alternating electric field to cancer cells at a frequency between 80 and 300 kHz. We provide a method using the app.

[0018] In some examples, at least part of the application step is a fraction of the delivery step. At the very least, it is performed simultaneously with some of the other steps. In some examples, the application step is at least It also has a period of 72 hours. In another example, the application step is at least 24 hours Or it has a period of 48 hours.

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

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

[0021] In some cases, ICs for CDK inhibitors (e.g., HLM006474) The 25 values ​​may be approximately 0.5 μM, approximately 1 μM, and approximately 1.5 μM. In another embodiment Regarding the concentration of CDK inhibitor in cancer cells, approximately 0.5 to 1.5 μM may be used.

[0022] In some cases, E2F inhibitors (e.g., LY2835219, also known as Abemacic) The IC25 value for the rib can be approximately 20 μM, 25 μM, or 40 μM. In another embodiment, the concentration of the E2F inhibitor in cancer cells is approximately 20 μM to approximately 40 μM. It is possible.

[0023] In yet another example, E2F inhibitors are HLM006474, MRT0003365 9. One or more of YKL-5-124-TFA and YKL-5-124 Selected from the following group.

[0024] In further examples, CDK4 / 6 inhibitors include abemaciclib, ribociclib, and trilla. Ciclib, Ibrance, Lerocyclib, Arbocilib, Roniclib, Ribiclib, Mi Lucicrib, RGB-286638, NSN3106729, PHA-793887, R 547, Indirubin, NU6102, Bohemin, CDK9-IN-7, CGP6047 4. Pulvaranol A, PF-06873600, Nimboride, FN-1501, AG- 024322, ON123300, G1T28, G1T38, AMG925, SHR-6 390, BPI-1178, BPI-16350, FCN437, birocilib, BEB T-209, Ty-302, TQB-3616, HS-10342, PF-068428 74, CS-2002, MM-D37K, CDK4 / 6-IN-2, SU9516, oyo Selected from a group consisting of one or more of the following:

[0025] In some cases, the delivery step involves E2F inhibitors and CDK4 / 6 inhibitors. The step includes administering or delivering to cancer cells. In some examples, the delivery The step involves administering or delivering E2F inhibitors and CDK4 / 6 inhibitors. Includes TEP. In one embodiment, the E2F inhibitor is HLM006474. Further In this embodiment, the CDK4 / 6 inhibitor is abemaciclib. In yet another example, The E2F inhibitor is HLM006474, and the CDK4 / 6 inhibitor is abemaciclib. be.

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

[0027] E2F transcription factors are active in all cell types, for example, in cell cycle regulation, D Related to NA repair and chromosome maintenance routines. HLM00647 is an E2F inhibitor. 4 inhibits DNA binding for all E2F complexes and is used in breast cancer and melanoma models. It is used in [location]. CDK4 / 6 inhibitors are used to treat glioblastoma and metastatic breast cancer. It is used for that purpose.

[0028] In some cases, cancer cell survival was observed when the cells were not exposed to an alternating electric field, and E Compared to cancer cells that did not receive 2F inhibitors and CDK4 / 6 inhibitors, 20- It is reduced to 1 / 100th.

[0029] In some cases, cancer cell survival was observed when the cells were not exposed to an alternating electric field, and E Compared to cancer cells that have not received delivery of 2F inhibitors and CDK4 / 6 inhibitors, (i) (ii) Exposure of cancer cells to an alternating electric field for 72 hours, (ii) E2F inhibition at concentrations of 10 μM to 50 μM. (iii) Delivery of harmful agents, and delivery of CDK4 / 6 inhibitors at concentrations of 0.1 μM to 2 μM. After that, it is reduced to about 1 / 100th. In some cases, E2F inhibitors are reduced by about 20 CDK4 / 6 inhibitors are delivered to cancer cells at a concentration of μM, and at a concentration of approximately 5 μM, they reach cancer cells. It will be delivered.

[0030] The embodiments described herein are methods for killing cancer cells in a subject, E2 Steps to deliver at least one of the F inhibitors and CDK4 / 6 inhibitors to cancer cells The steps include applying an alternating electric field to cancer cells at a frequency between 80 and 300 kHz. This provides a method.

[0031] In some examples, at least part of the application step is a fraction of the delivery step. At the very least, it is performed simultaneously with some other steps. The application step has a period of at least 72 hours. Obtain. In another embodiment, the step of applying is performed for at least 24 hours or 48 hours. It may have a gap.

[0032] E2F inhibitors include HLM006474, MRT00033659, and YKL-5-124. - Selected from a group consisting of one or more of TFA and YKL-5-124. ru.

[0033] CDK4 / 6 inhibitors include abemaciclib, ribociclib, trilaciclib, and Ibrance. , rerocyclib, arbocidicib, roniciclib, ribiclib, miliclib, RGB- 286638, NSN3106729, PHA-793887, R547, Indirubin NU6102, Bohemin, CDK9-IN-7, CGP60474, Purvalanol A PF-06873600, Nimbolid, FN-1501, AG-024322, ON1 23300, G1T28, G1T38, AMG925, SHR-6390, BPI-11 78, BPI-16350, FCN437, bilocyclib, BEBT-209, Ty-3 02, TQB-3616, HS-10342, PF-06842874, CS-2002 Among MM-D37K, CDK4 / 6-IN-2, SU9516, and AT7519 One or more of these can be selected from the group.

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

[0035] The experimental data presented below shows that TTField and combination therapy can check the cell cycle. This presents points and DNA repair, as well as therapeutic challenges to conventional treatment systems. To identify mechanisms that can downregulate important genes related to other survival pathways. Therefore, we will discuss the use of proteomics analysis.

[0036] As discussed herein, quantitative proteomics experiments have shown that the transcription activator E We identified a reduction in the expression of 2F1 and E2F2. In addition, quantitative proteomics experiments showed that We identified an increase in the expression of the transcription repressor E2F6. Therefore, as described herein This combination therapy affects the CDK-RB-E2F axis, and E2F4 and E2F6 signals. DNA repair genes (e.g., RAD51, BRCA1, and BRCA2) are transmitted through DNA transfer. ) causes dysregulation.

[0037] Figure 1 shows the conserved domains present in the E2F protein (i.e., conserved and designated domains). This provides an illustrative outline of the domains (which have the function of) the E2F protein has eight functions. It consists of mille members (E2F1~8), and based on their functions, transcription activators ( They are divided into E2F1~E2F3a) and transcription repressors (E2F3b~E2F8). The 2F protein is involved in cell cycle progression, DNA replication, DNA damage checkpoints, and It regulates thousands of genes crucial for DNA repair and plays a central role in cell proliferation. The levels of activated proteins (E2F1, E2F2, and E2F3A) are in the G1-S phase. The transition peaks, and atypical inhibitor (E2F7 and E2F8) levels are in the late S phase. It peaks in the latter half, but classical inhibitors (E2F3B, E2F4, E2F5 and E2 F6) 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 are linked to DNA. To combine, members of the transcription factor dimerization partner (TFDP) family (TFD This requires dimerization with P1 or TFDP2. This bond is a leucine zipper (L The dimerized domain consists of a Z) domain and a marked box (MB) domain. Therefore, it is promoted. E2F1, E2F2, E2F3, E2F4 and E2F5, pocket The transactivating proteins (RB, p107, and p130) bind via their transactivation domains. .

[0039] The minimum site required for interaction with pocket proteins is shown in (RB). RB is coupled to all of 2F1, E2F2, E2F3, E2F4 and E2F5, but p1 07 and p130 are coupled only to E2F4 and E2F5. E2F6 is pocket tongue It does not bind to proteins, but instead is regulated by Polycomb proteins. The E2F protein also has a nuclear localization sequence (NLS), a nuclear export sequence (NES), and or it has a cyclin A (CCNA) regulatory domain. E2F7 and E2F8 are dimers. Lacking the embodying domain and transactivation domain, it is a TFDP or pocket protein. They do not bind to DNA. Instead, they have two tandem DNA-binding domains.

[0040] Exemplary upstream analysis of differentially expressed proteins upon cell exposure to TTField This is because activators such as E2F1 and E2F2 are inhibited, and inhibitory factors such as E2F6 are activated. It demonstrated that sexualization occurs.

[0041] Figure 2 provides an exemplary upstream analysis of the proteome after cell exposure to the TTField. The proteomics results showed that, as a result of TTField exposure, the transcription activator (E2 This suggests that F1 and E2F2 are inhibited, and the inhibitory factor (E2F6) is activated. .

[0042] The E2F-RB axis is involved in mitosis, DNA damage and repair, DNA replication, and chromatin remodeling. The ring, as well as the central dynamo that controls many pathways in cancer cells, including apoptosis. It functions as a junction. Gene expression studies and direct function studies are conducted using E2F-R. We have shown that pathway B targets several members of the FA (Fanconi anemia) pathway. Furthermore, TTField acts as an upstream regulator of the FA pathway and the cell cycle. Dysregulation of E2F-RB causes downregulation of members of the FA pathway.

[0043] Figures 3A-3D show the results of exposure to TTField based on different proteomic analyses. Compared to cells without exposure, E2F target expression in lung cancer cells after exposure to TTField was increased. This provides exemplary results for Issey.

[0044] The E2F targets examined in Figures 3A-3D are members of the FA pathway (e.g., BRC) A1, FANCD2, RAD51), members regarding duplicate forks (e.g., MCM6) RFC3, RFC4), and proteins related to mitosis (e.g., BUB3, CC) This includes NE2 and EZH2. As shown in Figures 3A-3D, the expression levels are log As indicated by the current value, it changes upon exposure to TTField. Changes in expression levels. The transformation was also classified as being inhibited, affected, or activated.

[0045] As shown in Figures 3A-3D, E2F1 and E2F2 are activators, and E2F4 E2F6 is an inhibitor, and upon exposure to TTField, inhibition and Activated. Consequently, the expression of their targets decreased (e.g., BRCA1, MC). (M6, CCNE2, EZH2)

[0046] Figure 4 provides exemplary gene signature markers for E2F-RB dysfunction. Genetic signature analysis identifies genes that are deregulated by E2F-RB dysregulation / loss. The functional groups are shown. Figure 4 shows DNA replication, DNA damage and repair, apoptosis, and mitosis. Identify exemplary gene targets associated with fissures and chromatin regions.

[0047] Figure 5 shows the TTField, E2F inhibitors, and CDK4 / 6 inhibitors described herein. An illustrative diagram of the described combination therapy is provided. As illustrated, TTField and The inventors observed the following mechanisms of action of TTField: cell cycle, DNA damage, and The combination with inhibitors of upstream regulators of replication stress (E2F and CDK4 / 6) is This would be very beneficial. The adjustment of E2F and CDK4 / 6 as disclosed herein Factors include E2F inhibitors (e.g., HLM006474) and CDK4 / 6 inhibitors (e.g., It can be targeted by abemaciclib. Although not bound by theory, as illustrated in Figure 5. Thus, exposure of cancer cells to TTField and E2F inhibitors affects the E2F gene target. While CDK4 / 6 inhibitors target dysregulation, CDK4 / 6 inhibitors target cell cycle progression. This pair The mismatch leads to abnormal cell division, DNA damage, replication stress, and ultimately cell death. It can be caused.

[0048] As shown in Figure 5, TTField causes E2F to become dysregulated, thus affecting cancer cells. It can be applied to cells. Additional E2F inhibitors (e.g., HLM006474) suppress It modulates E2F targets, causing increased expression of inhibitors, decreased expression of activators, or inhibition of activators. It can be applied to cancer cells to cause insufficiency. CDK4 / 6 inhibitors (e.g., A Bemaciclib also inhibits CDK4 / 6 activity and cell cycle expression, leading to abnormal cell division and D Cell cycle, FA, DNA damage, and replication that cause NA damage and increased replication stress. These can be applied to target genes involved in mitosis. This can lead to an increase in abnormal cells, resulting in increased fission / catastrophic cancer cell death.

[0049] Figures 6A-6B show TTField and E2F signaling at 24, 48, and 72 hours. Alternatively, various combinations with drugs that dysregulate CDK4 / 6 signaling are being tested. Four lung cancer cell lines, H1299, A549, H157, and H400, under various conditions. This provides the results of an exemplary clonal survival assay in 6 cells. A549 is a non-small cell lung cancer cell that is resistant to conventional treatment using TTField. It is a strain. In contrast, H157 and H4006 are more responsive to TTField. It is a known cell line. TTField alone, or TTField and E2F Inhibitors, CDK4 / 6 inhibitors, or a combination of E2F inhibitors and CDK4 / 6 inhibitors This shows cell survival after increased exposure in combination with the wasotherapy.

[0050] As shown in Figures 6A-6B, lower doses of E2F inhibitors and CDK4 / 6 inhibitors When the agent is used in combination with TTField, 72 hours after TTField exposure, the fine Cell survival is reduced by 1 / 20 to 1 / 100. In one case, 72 hours of TTField exposure... Russia, 20 μM of the E2F inhibitor HLM006474, and 0.5 μM of abemaciclib This combination resulted in a reduction of approximately 1 / 100th in cell survival. However, this is not bound by theory. The unexpected reduction in cell survival is thought to have resulted from the transient targeting of several pathways. It can be done.

[0051] Figures 7A–7B show the results from the exemplary clonal survival assay described in Figures 6A–6B. Provides a combined metric index (CI) value. In some examples, a CI greater than 1.0 is provided. The I value indicates a synergistic effect or a surprisingly favorable effect on antitumor activity. Figure 7 As shown in A-7B, the H1299 and A549 cell lines are TTField C When administered in combination with a DK+E2F inhibitor, it shows a significant synergistic effect after 72 hours (for example) For example, the H1299 index value after 72 hours was 11.65, and the A549 index value after 72 hours was 11.65. (The CI index value is 13.91).

[0052] In some aspects, the application of combination therapy is the application of TTField, TTField Along with or immediately after the application of d, the application of one or more therapeutic agents to cancer cells or Combine it with delivery.

[0053] In some aspects, the application of combination therapy is the application of TTField, TTField After a specified period following the application of d, it is combined with the application of one or more therapeutic agents to cancer cells. The predetermined period is determined based on the observed vulnerability of cancer cells after TTField is applied. It can be determined. For example, in some embodiments, cancer cells are treated with TTField. Approximately 24, 48, or 72 hours later, the patient may be treated with or exposed to concomitant therapy.

[0054] Figures 8A-8F show the results after 24, 48, and 72 hours of exposure to TTField with H400. In cells 6, H157, A549, H1299, and H1650, FA-BRAC Route A (BRCA1, FANCE, FANCC, FANCB, FANCA, and RFC) 3) Exemplary examples of mRNA and protein-level expression of genes The data is provided. As shown in Figures 8A-8F, TTField is these genes It reduced mRNA and protein expression, showing the greatest decrease in expression after 72 hours.

[0055] Figures 9A-9B show the FA routes (BRCA1, FANCD2, MLH1, RBL1, RFC 3, RFC4), mitosis / cell cycle (CCNE2, DUSP1, EZH2, MAD2L 1) and DNA replication (CDC45, DHFR, MCM6, POLA2, RRM2) Regarding the indicated target genes, transcription activators (E2F1, E2F2) and transcription Exemplary activities of the inhibitors (E2F4, E2F6) are illustrated. In summary, see Figures 9A-9B. The data in TTField shows that for these three classes of genes, transcription activity It inhibits sexing factors (E2F1, E2F2) and activates transcriptional repressors (E2F4, E2F6). This indicates transformation.

[0056] Figure 10 shows the effects of exposure to TTField on H1299 cells at levels 24, 48, and 72. Exemplary combinations of TTField and various drug combinations after several hours. We provide CI (Compound Index) data. Using combination index studies, we analyze drug combinations. Determine the additive or synergistic effects of biological effects. For example, Chou et al., Drug c ombination studies and their synergy qua ntification using the Chou-Talalay method d. Cancer Res, January 15, 2010; Vol. 70 (No. 2): pp. 440-446. Please be illuminated. In one embodiment, 72 hours of TTField exposure, 20 μM of E2F inhibitor The combination of HLM006474 and 0.5 μM abemaciclib yielded 8.72 Unexpectedly and surprisingly, it yielded a high combination index. [Examples]

[0057] Materials and methods for the experiment cell culture Human NSCLC cell lines (H157, H4006, A549, and H1299) are used in A I bought it from the American Tissue Culture Collection. All of these cell lines were tested using 10% (v / v) fetal bovine serum (Atlanta Biol). (Psychogicals, Flowery Branch, Ga., USA) and penicillin / Streptoavidin (final concentration 50 μg / ml; Sigma-Aldrich, St.L) All cells were grown in RPMI medium supplemented with ouis, Mo., U29SA. The plants were grown at 37°C in a humidified incubator with a constant supply of 5% CO2.

[0058] Tumor Therapy Electric Field inovitro system (NovoCure Ltd, Haifa, Israel) Using high dielectric constant ceramics (magnesium niobate lead-titanium (lead Printed vertically on the outer wall of a petri dish made of titanite (PMN-PT) A TTField was generated using two pairs of electrodes. Transducer array This was connected to a sinusoidal waveform generator that generates a low-intensity electric field at a desired frequency in the culture medium. TTF The direction of the ield is switched 90° every second, and in this way the large axis of the cell division direction is The entire area was covered. The temperature was maintained at 19°C and generated by the inovtro system. By placing the plate in a refrigerated incubator that has had the heat removed, the plate temperature The temperature was maintained at 37°C. The temperature was controlled by two thermistors (Omega) attached to the ceramic wall. Measured by ga Engineering, Stamford, Conn., USA I did that. I put all the cell suspensions into an inovitro dish (NovoCure Ltd). The plants were grown on coverslips within the ) and treated with TTField for the time shown in the figure.

[0059] Cell growth assay Human NSCLC (H157, H4006, A549, and H1299) cell lines, 2 For 4, 48, and 72 hours, treat with different frequencies of the indicated TT field to promote cell growth. For each sample, three times, the Beckman Coulter counter (Beckman Coulter Inc. (Indianapolis, hid., USA) It installed. GraphPad Prism V.6 (GraphPad Software) Using re Inc., La Jolla, Calif, USA, count at each point in time Using the average cell number and a given TTField frequency, a graph of the growth curve is drawn. there was.

[0060] Cell cycle analysis Cells were collected at specific times and treatments, and stored in 75% ice-cold ethanol at -20°C for 2 minutes. The cells were fixed for 4 hours. The fixed cells were washed with PBS, 500 μl of PI staining solution, and then... This includes 1 mg / ml of RNAse A (Sigma-Aldrich) and 0.05% of t Riton X-100 and 30 μg / ml PI (Sigma-Aldrich) The cells were incubated in PBS at 37°C for 30 minutes. The cell cycle distribution was analyzed using FACS. Calibur System (BD Biosciences, San Jose, Cali The determination was made using f., USA). More than 10,000 cells were counted per sample. The result is FlowJo software v8.7.1 (Tree Star Inc, Ashley Star Inc.). The analysis was performed using (and, Oreg., USA).

[0061] RNA labeling and hybridization for gene expression analysis Illumina Whole Genome HumanWG6 v4 Expre ssion BeadChip (Illumina Inc, San Diego, Ca. (lif., USA) was used. Each RNA sample (0.5 μg) was treated with biotin UTP (En Zo Life Sciences, Inc., Farmingdale, NY, U The Illumina TotalPrep RNA amplification kit uses SA labeling. The data was amplified using T7 oligo(dT) primers to generate single-stranded cDNA. Next, double-stranded cDNA was generated by double-stranded synthesis, and then purified by column chromatography. T7 Using RNA polymerase, in vitro transcription is performed to obtain biotin-labeled cRNA. It was synthesized. Next, the cRNA was purified using a column filter, and then the Bio-Rad Experion system... (Bio-Rad Laboratories, Hercules, Calif., US) A) was used to check the size and yield. Then, cRNA (1.5 μg) was used. g) streptavidin-Cy3 (Amersham Bi) is used for detection. Standard Il (using osciences, Piscataway, NJ, USA) We used the Lumina protocol to hybridize each array. (Slide) At Illumina Beadstation (Illumina Inc.) I scanned it.

[0062] Data processing and significance analysis of differential gene expression Summary expression values ​​for each probe set are available in BeadStudio 3.1(I Created using Ilumina Inc. The data was processed using the MBCB algorithm. Then, background subtraction and displacement-displacement-normalized across the entire sample. A plot for comparison was created using normalized gene expression values. Genes differentially expressed in cell lines were analyzed using SAM. FDR<0 A value of 0.05 was considered statistically significant. Cluster analysis and heatmaps were performed using P. Artek Genomic Suite software (Partek Incorporated) Created using rated (St. Louis, Mo., USA). Gene ontology G and path analysis, IPA (QIAGEN, Redwood City, Calif This was done using (USA).

[0063] Immunoblot Laemmli sample buffer (4×; Bio-Rad Laboratories) 30 μg of each protein sample was added, and the mixture was boiled at 95°C for 10 minutes. The protein mixture was loaded onto a 10% SDS-PAGE gel, followed by 90V, 4 The mixture was transferred to a PVDF membrane at 1 hour at °C. The membrane was then heated using 5% nonfat milk in PBST at room temperature. Block for 1 hour, then 2% bovine serum albumin (Thermo Fisher S Contains Scientific Inc. (Bridgewater, NJ, USA) Anti(3-actin(1:5000; Cell Signaling, Dan) vers, Mass., USA), anti-BRCA1 (1:1000), anti-FANCD2 (1 :2000) and anti-FANCA (1:500; Novus Biologicals The membrane was examined overnight at 4°C using a method (LLC, Littleton, Colo, USA). Wash with phosphate-buffered saline containing 0.1% Tween-20 (TBST; each 3 x 10 minutes), followed by horseradish peroxidase (GE Healthcare, B Secondary antibody (1:5000) conjugated with (buckinghamshire, UK) The membrane was incubated at room temperature for 1 hour. ProteinSimple (San Jose, Calif, USA) is involved in chemistry. Luminescence detection kit (Thermo Scientific, Rockford, Ill.) Developed using (USA). Quantification was performed using ImageJ software (NIH, Bet). Performed using HESDA (Md., USA) and normalized using the corresponding actin density. did.

[0064] Immunofluorescence The cells were seeded onto glass coverslips, and after processing, the cells were washed and ice-cold methanol was used. The sample was fixed with phosphate. The sample was blocked for 1 hour with 10% normal goat serum, and phosphodiester was used. -Histone-γ-H2AX antibody (Ser139; Upstate Biotechnol) (ogy, Temecula, Calif, USA) and p53-binding protein 1 (53 The sample was incubated with BP1 antibody (Cell Signaling). Wash in PBS for 5 minutes, three times, then Alexa Fluor 488 conjugate Anti-rabbit antibody and Alexa Fluor 555 conjugate anti-mouse antibody (I nvitrogen (Carlsbad, Calif., USA) along with 1 hour ink The nucleus was embedded in Vecatshield (Vector Laboratories). DAPI contained in ries Inc., Burlingame, Calif, USA Counterstaining was performed using [a specific method]. Next, the stained cells were each placed in a 0.2 μM thick z-stack. Using five sections of the sample, a fluorescence microscope equipped with a 63x objective lens (oil immersion, numerical aperture 1.3) was used. Mirror (Axio Imager M2, Carl Zeiss, Thornwood, N. The analysis was performed under Y., USA. Quantitative image analysis of 40 nuclei from each experiment was performed using Imaris. Software version 8.0 (Bitplane, Concord, Mass., US This was performed using the cell module described in A).

[0065] Radiation exposure and clonal cell survival To study the effect of radiation sensitivity on NSCLC cells, exponentially growing cells The cells are irradiated using the Mark II Cs irradiator (JL Shepherd and Asso). Using ciates, the dose rate was 3.47 Gy / min, treated with IR, followed by 24. TTField was immediately applied for 48 and 72 hours. Then, the cells were placed in a 60 mm dish. The cells were reseeded in sorghum and incubated for up to two weeks. Colonies containing more than 50 cells were observed. — was considered to be alive. Data are expressed as the mean ± SEM of three independent experiments. The radiation sensitization effect of TTField is calculated as shown below, and the CI is calculated accordingly. Evaluated according to The Highest Single Agent approach did.

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

[0067] Combination effects are considered to be enhancing / synergistic when CI > 1, and additive when CI = 1. The statistical significance of the positive effect was determined given the combination (TTField and IR). A dual-field comparison of dose and time after IR to the single agent showing the highest cell death. The p-value was determined by the ANOVA multiple comparison statistical test. [Examples]

[0068] Proteometric analysis of lung cancer cells exposed to TTField In some aspects, proteomic analysis of lung cancer cells exposed to a tumor therapeutic electric field. Dysregulation of the E2F-Rb-CDK4 / 6 axis affects tumor cells, affecting CDK4 / 6 and / or Alternatively, it was identified that the cells become sensitive to novel combination therapies that target E2F. One mechanism described for the death-inducing TTField is through the disruption of mitosis. More recent studies suggest that TTField causes replication stress and DNA repair This suggests that it also downregulates genes related to cell cycle checkpoints. However, Therefore, the exact cause of the downregulation of DNA repair and cell cycle checkpoint genes is, There is no way to explain it. For that purpose, the technology disclosed is Tandem Mass Tag (TMT) Relative quantitative proteomics analysis was performed using [specific method / tool]. All samples were tested for trypsin elimination. They underwent chemical and labeling with different TMT reagents. Then they were mixed, and the mixture was formed. The data was processed using an Orbitrap Fusion mass spectrometer.

[0069] Peptide quantification was achieved by comparing the intensities of the TMT reporter ion. STRING DB analysis of differentially expressed proteins reveals cell cycle and DNA damage repair mechanisms. We revealed interaction networks including replication, transcription, and translation regulation. Upstream analysis of key genes related to cell cycle checkpoints and DNA repair is a transcriptional analysis. Reduced expression of the activators E2F1 and E2F2, and the transcription repressor E2F6 We identified increased expression and demonstrated that TTField affects the CDK-RB-E2F axis. This was instigated. For example, important DNA repair genes including RAD51, BRCA1, and BRCA2. The downregulation of this child is, for example, the transcription repressors E2F4 and E2F6 (known for BRCA1). This could be explained by the upregulation of the inhibitory factor.

[0070] These proteins are involved in homologous recombination repair and nucleotide excision repair, but multiple It is also involved in the maintenance of the fork, the collapse of the replicated fork, and the overall replica stress, and these The latter can cause cell death. Therefore, in one example, TTField is C With or without the DK4 / 6 inhibitor abemaciclib, the E2F inhibitor HLM00647 It was combined with 4. TTField combined with any of the inhibitors is TTField Compared to each individual, it synergistically enhanced cell death, but the combination of the three determines the synergy. Clonogenicity assay and subsequent Highest Single Agent As measured by the approach, it is highly lethal (>90% within 72 hours). It was found that, in one case, this result suggests that TTFi is beneficial for cancer treatment. Targets that could lead to the development of novel drugs that can be used in combination with ELD The CDK-RB-E2F axis was identified. [Examples]

[0071] Further genomic and proteomics support for combination therapies including TTField te To capture the whole picture of biological processes in a non-targeting and unbiased manner, and a series of non-minor details The overall gene expression changes in alveolar lung cancer (NSCLC) cells upon TTField exposure. To understand this, the transcriptomics approach has been used by previous research groups. Ingenuity Pathway Analysis of TTField-responsive genes The results of sis(IPA) indicate that the changes occur in the cell cycle and mitotic regulatory pathways. This suggests that, while consistent with previous studies, TTField exposure significantly downmodulated it. The BRCA1 DNA damage response pathway (P<0.05) was also revealed. More precisely, what causes the downregulation of genes involved in DNA repair and cell cycle checkpoints? It was hard to pinpoint what was going on.

[0072] Therefore, how do changes in expression at the genomic level affect the function of biological activity? To determine whether the changes in protein expression, which are the key players, are translated, the proteo To investigate the TTField that induces changes in the thyroid level, we conducted experiments including proteomics analysis. An experiment was conducted using tandem mass tagging (TMT) for relative quantitative proteomics analysis. Using this method, the proteome of control and TTField treatment conditions in H1299 cells This was quantified. After TTField exposure, a total of 1 was observed at 24 hours and 48 hours, respectively. Differentially expressed proteins of 06 and 541 were present. Quality string database analysis is used for cell cycle, DNA damage repair and replication, and mitochondrial analysis. Elucidating Dorian dysfunction and the interaction network including transcription and translation regulation. The expression of members of the FA pathway, cell cycle, and DNA damage and replication pathways. The same pattern of change is seen in Figures 8A-8F, where proteins from proteomics This was observed at the crystalline level and at the mRNA level from transcriptomics analysis.

[0073] Figures 8A-8F show transient mRNA expression levels from transcriptomics data. Changes in protein expression levels from proteomics data, as well as downward regulation. Western blots of some of the FA pathway members identified as having been The graph illustrates the verification of changes in protein levels, showing that F was observed at both the gene and protein levels. This suggests similar expression patterns for members of pathway A.

[0074] In addition, these regulatory nodes exert maximum control over dysregulated pathways. However, it exerts minimal control over undisturbed paths, so several downstream paths, for example, For example, it acts as a regulatory node for the expression of the cell cycle, DNA damage repair, and replication. Experiments were conducted to identify important upstream regulatory network mechanisms. Differentially expressed Analytical analysis of upstream regulatory factors of TTField, which induces proteins, reveals upstream transcriptional regulatory factors. Nodal factors, for example, E2F1 and E, which are transcription activators and transcription repressors, respectively. We identified a decrease in 2F2 expression and an increase in E2F6 expression, as illustrated in Figures 9A-9B. Thus, TTField affects the CDK-Rb-E2F axis, and E2F4 and proteins involved in E2F6 signaling and replication forks (MCM6, RFC) 3. RFC4), proteins involved in mitosis (BUB3, CCNE2, EZH2) It regulates important DNA repair proteins, including RAD51, BRCA1, and BRCA2. This suggested that these proteins cause failure. These proteins are involved in the cell cycle, homologous recombination repair, and nucleation. Otid removal and repair, replication fork maintenance, replication fork collapse, and overall replication stress To be involved.

[0075] More specifically, Figures 9A-9B show upstream regulatory factor analyses from different proteomic analyses. The quantitative changes in E2F target expression are illustrated. TTField is a transcription factor E2F family Lee inhibits transcription activators (E2F1-3) and increases transcription repressors (E2F4-8). To add, Fanconi anemia (BRCA1, FANCD2, MLH1, RBL1) RFC3, RFC4) and DNA replication pathway proteins (CDC45, DHFR, MC) There is a decrease in the expression of M6, POLA2, and RRM2, and transcription to specific targets. The complexity of these activators / inhibitors is to be communicated to the inventors, etc., regarding the mitotic / cell cycle pathway. Different effects exist on proteins (CCNE2, DUSP1, EZH2, MAD2L1). It was present. Different pathway proteins, and the direction of transcription factors, and the development of the final target protein. The table provides the ratio (Log2) of the change in protein expression for the current state. Upstream regulatory factor analysis. This is a transcription style stored in the Ingenuity(registered trademark) Knowledge Base. Based on prior knowledge of the expected effects between nodal factors and their target genes, users We examined the number of known targets for each transcription regulator present in the dataset and the direction of their changes. (That is, the expression of the TTField sample compared to the control) will also be compared.

[0076] A comparative analysis of changes in transcriptome and proteome levels was performed, which indicates that The CDK-Rb-E2F axis is involved in the cell cycle, DNA damage repair, and replication stress pathways. Acting as an upstream regulatory node for the effects observed with TTField exposure This was shown. The CDK-Rb-E2F axis is something that could lead to the development of new drugs, and in fact, Currently, it is a major drug target in cancer treatment, and by doing so, this axis can be targeted. This allows for targeting DNA repair, cell cycle checkpoints, or proliferation and survival pathways. This includes becoming an integrated therapy that will enhance conventional radiotherapy and chemotherapy, including TTF This will likely change how ield is used in the future.

[0077] Figure 10 shows pairs of TTField with different drugs and ionizing radiation (IR). This provides a comparison of compatibility index values. In one example, the results show CDK inhibitors and E2F inhibitors. Both harmful agents are used in combination with TTField to block the CDK-E2F-RB axis. This suggests that it is highly effective compared to individual drugs or other drugs. The table provides P-values ​​for each pairing.

[0078] The systems and methods described herein apply after primary therapy has been administered to TTFie ld can be used in combination with drugs. TTField can be used in combination with drugs on the RB-E2F axis. By combining these, integrative therapy can be used with radiation, chemotherapy, or DNA repair and cell cycle checking. Enhance downstream therapies such as other therapies that target cytoplasm or proliferation and survival pathways. obtain.

[0079] The systems and methods described herein are for treating glioblastoma. Mesothelioma, pancreatic cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, skin cancer, peritoneal cancer It can be used for treatment and / or mitigation of such conditions. The system and The method involves improving the conventional system of applying TTField through combination therapy. It may be used for this purpose.

[0080] Although the present invention has been disclosed with reference to certain embodiments, many embodiments beyond those described are available. Modifications, alterations, and changes to the number are within the scope of the invention as defined in the appended claims. And it is possible without departing from the scope. Therefore, the present invention is possible without departing from the described embodiment Not limited to the form, but including the wording of the claims listed below and their equivalents. It is intended to have the full range defined by [the specified method / function].

Claims

1. A method for reducing the survival of cancer cells in a target, comprising an E2F inhibitor and CDK4 / The step of delivering at least one of the six inhibitors to cancer cells; and 80-300 A method comprising the step of applying an alternating electric field to cancer cells at a frequency between kHz.

2. At least part of the application step is performed simultaneously with at least part of the delivery step. The method according to claim 1.

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

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

5. An alternating electric field provides an electric field strength of at least 1 v / cm in at least some cancer cells. The method according to claim 1, having a degree.

6. The claim 1 states that the concentration of the E2F inhibitor in cancer cells is approximately 10 μM to approximately 50 μM. The method.

7. Claim 1, wherein the concentration of the E2F inhibitor in cancer cells is approximately 20 μM to approximately 40 μM. The method.

8. Claim 1, wherein the concentration of the CDK4 / 6 inhibitor in cancer cells is approximately 0.1 μM to approximately 5 μM. Methods used.

9. Claim 1, wherein the concentration of the CDK4 / 6 inhibitor in cancer cells is approximately 0.5 μM to approximately 2 μM. Methods used.

10. E2F inhibitors include HLM006474, MRT00033659, and YKL-5-124. - Selected from the group consisting of one or more of TFA and YKL-5-124. The method according to claim 1.

11. CDK4 / 6 inhibitors include abemaciclib, ribociclib, trilaciclib, and Ibrance. , lerociclib, arbociciclib, roniciclib, ribiciclib, miliciclib, RGB- 286638, NSN3106729, PHA-793887, R547, Indirubin NU6102, Bohemin, CDK9-IN-7, CGP60474, Pulvalanol A PF-06873600, Nimbolid, FN-1501, AG-024322, ON1 23300, G1T28, G1T38, AMG925, SHR-6390, BPI-11 78. BPI-16350, FCN437, birocilib, BEBT-209, Ty-3 02, TQB-3616, HS-10342, PF-06842874, CS-2002 Among MM-D37K, CDK4 / 6-IN-2, SU9516, and AT7519 The method according to claim 1, selected from one or more of the group.

12. The delivery step is the step administered to target E2F inhibitors and CDK4 / 6 inhibitors. The method according to claim 1, comprising p.

13. The method according to claim 12, wherein the E2F inhibitor is HLM006474.

14. The method according to claim 12, wherein the CDK4 / 6 inhibitor is abemaciclib.

15. The E2F inhibitor is HLM006474, and the CDK4 / 6 inhibitor is abemaciclib The method according to claim 12.

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

17. Cancer cell survival is not affected by exposure to alternating electric fields, and is not affected by E2F inhibitors and CDK Compared to cancer cells that did not receive 4 / 6 inhibitor delivery, the number was reduced to 1 / 20 to 1 / 100. The method according to claim 1.

18. A method for killing cancer cells in a target, comprising an E2F inhibitor and a CDK4 / 6 inhibitor. The step of delivering at least one of the harmful agents to cancer cells; and 80-300 kH A method comprising the step of applying an alternating electric field to cancer cells at a frequency between z.

19. At least part of the application step is performed simultaneously with at least part of the delivery step. The method according to claim 18.

20. The method according to claim 18, wherein the step of applying the solution has a period of at least 72 hours.

21. E2F inhibitors include HLM006474, MRT00033659, and YKL-5-124. - Selected from the group consisting of one or more of TFA and YKL-5-124. The method according to claim 18.

22. CDK4 / 6 inhibitors include abemaciclib, ribociclib, trilaciclib, and Ibrance. , lerociclib, arbociciclib, roniciclib, ribiciclib, miliciclib, RGB- 286638, NSN3106729, PHA-793887, R547, Indirubin NU6102, Bohemin, CDK9-IN-7, CGP60474, Pulvalanol A PF-06873600, Nimbolid, FN-1501, AG-024322, ON1 23300, G1T28, G1T38, AMG925, SHR-6390, BPI-11 78. BPI-16350, FCN437, birocilib, BEBT-209, Ty-3 02, TQB-3616, HS-10342, PF-06842874, CS-2002 Among MM-D37K, CDK4 / 6-IN-2, SU9516, and AT7519 The method according to claim 18, selected from one or more of the group.

23. The method according to claim 21, wherein the E2F inhibitor is HLM006474.

24. The method according to claim 22, wherein the CDK4 / 6 inhibitor is abemaciclib.

25. The E2F inhibitor is HLM006474, and the CDK4 / 6 inhibitor is abemaciclib The method according to claim 18.

26. The method according to claim 18, wherein the cancer cells include a defect in the BRCA pathway.

27. Cancer cells include lung cancer cells, breast cancer cells, pancreatic cancer cells, glioblastoma cells, and prostate cancer cells. Cells, liver cancer cells, fallopian tube cancer cells, peritoneal cancer cells, skin cancer cells, and ovarian cancer cells The method according to claim 18, selected from the group.

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