Mitotic cell phase enrichment and ionizing radiation

Abstract

The invention relates to a combination therapy comprising administering a mitotic-phase (M-phase) cell cycle arresting compound and a modulator of an interphase cell cycle checkpoint to a subject and delivering ionizing radiation to the same subject for the treatment of a tumor. Provided is for (M-phase) cell cycle arresting compounds, modulators of an interphase cell cycle checkpoint and ionizing radiation for use in such treatments, as well as methods of the treatment involving the use of (M-phase) cell cycle arresting compounds and modulators of an interphase cell cycle checkpoint and ionizing radiation.

Description

[0001] Title: Mitotic cell phase enrichment and ionizing radiation

[0002] FIELD OF THE INVENTION

[0003]

[0001] This invention pertains in general to the field of the treatment of tumors, and more in particular to a combination therapy comprising at least administering mitotic- phase cell arresting compounds and delivering ionizing radiation for the treatment of several kinds of tumors, and with special focus on brain tumors.

[0004] BACKGROUND OF THE INVENTION

[0005]

[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006]

[0003] Cancer is one of the major concerns faced by Health Authorities. Cancer therapies are diverse and are evolving fast over the last three decades, which has improved overall survival. However, within the heterogenous group of cancer there are several malignancies that are still far from good prognosis and survival. Some of these malignancies display for one or more reasons reluctancy or insensitivity to ionizing radiation therapy.

[0007]

[0004] As a typical example reference can be made to brain malignancies, including primary brain tumors or secondary brain tumors. Despite that over the past decades there have been major improvements in understanding the biology of brain tumors, the prognosis and survival of subjects with brain tumors remains poor and has not improved substantially. Thus, there is a great and unmet need for better therapies.

[0008]

[0005] The international patent application with publication number WO 2021 / 150109 (Stichting het Nederlands Kanker I nstitituut-Antoni van Leeuwenhoek Ziekenhuis) proposes a new and versatile approach for treatment of tumors that are in the brain (primary or secondary tumors) based on the administration of the compound ABT-751 prior to exposure to ionizing radiation. This achieves effective radiosensitization and is, moreover, compatible with hyperfractionated radiotherapy and chemoradiotherapy.

[0009]

[0006] There is a continuous need for further or more efficient treatments of tumors, as well as alternative treatments. At the same time, the reduction of side-effects associated with treatments, including treatments that comprise the use of ionizing radiation is still a need in the field.

[0010]

[0007] Moreover, in the particular cases of tumors that are managed with the administration of ionizing radiation, optionally in combination with chemotherapeutic compounds, there is an additional limitation inherent to the treatment, namely the maximal dose of radiation a subject can receive during life. Thus, it is of paramount importance to assure each cycle of ionizing radiation is able to provide the highest efficiency possible.

[0011]

[0008] In light of the limitations of present treatment options, novel products, compositions, methods and uses to treat a tumor, such as a brain tumor, in a subject are highly desirable yet are not readily available. In particular there is a clear need for reliable, efficient, and reproducible products, compositions, methods and uses that allow to effectively treat tumors, such as brain tumors, while aiming to keep sideeffects at a minimum. Accordingly, the technical problem underlying the present invention can been seen in the provision of such products, compositions, methods and uses for complying with any of the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and is provided herein below.

[0012] SUMMARY OF THE INVENTION

[0013]

[0009] As embodied and broadly described herein, the present invention is directed to the surprising finding that when a mitotic-phase (M-phase) cell cycle arresting compound, such as the microtubule inhibitor ABT-751 , is combined with a modulator, preferably an inhibitor, of an interphase cell cycle checkpoint, such as with one or more inhibitors of interphase cell cycle checkpoints (e.g., inhibitors of ATR, CHK1 or Wee1), an efficient and surprising accumulation of cancer cells in the mitotic phase can be induced, and wherein the compounds may act in a synergistic mode. It was also found that as a consequence of the treatment, the treated cancer cells are very vulnerable to ionizing radiation. This therapeutic approach thus provides for the radiosensitization of tumors to radiotherapy, improving thereby the efficiency of the treatment.

[0014]

[0010] The cell cycle consists of a series of growth- and development phases comprising the life cycle of a cell. The phases of a cell cycle are in brief: the G1-phase or first gap phase, the S-phase, wherein the cell synthesizes a copy of its DNA, the G2-phase or second gap phase, and the M-phase or mitosis. During mitosis cells undergo a duplication. The DNA comprised in the mother cells is replicated (S phase) and cells are duplicated and divided into two daughter cells. Generally, one mother cell divides in two, preferably identical, daughter cells. In general, the cell cycle of one cell is not synchronized with the cell cycle of another cell. In other words, in for example a specific tissue the cells present are undergoing various phases of the cell cycle at a given moment. M-phase is the most sensitive cell cycle phase for ionizing radiation, as little to no DNA repair can occur during this phase. However, cells typically spend very little time in M-phase, leading to only a small population of cells being in this most vulnerable phase at any given time.

[0015]

[0011] Therefore, the invention provides, a mitotic-phase (M-phase) cell cycle arresting compound for use in the treatment of a tumor in a subject, wherein the treatment comprises administering the M-phase cell cycle arresting compound to the subject, administering a modulator of an interphase cell cycle checkpoint to the subject, , and delivering ionizing radiation to the subject; and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

[0016]

[0012] Also provided by the invention is a modulator of an interphase cell cycle checkpoint for use in the treatment of a tumor in a subject, wherein the treatment comprises administering a M-phase cell cycle arresting compound to the subject, administering the modulator of an interphase cell cycle checkpoint to the subject, and delivering ionizing radiation to the subject; and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

[0017]

[0013] Both, mitotic-phase (M-phase) cell cycle arresting compound for use in the treatment of a tumor in a subject and the modulator of an interphase cell cycle checkpoint for use in the treatment of a tumor in a subject, can also be (re)formulated as the use of a mitotic-phase (M-phase) cell cycle arresting compound, in combination with the use of a modulator of an interphase cell cycle checkpoint, for the preparation of a medicament for the treatment of a tumor in a subject, wherein the treatment comprises administering the M-phase cell cycle arresting compound to the subject, and administering the modulator of an interphase cell cycle checkpoint to the subject, and delivering ionizing radiation to the subject; and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

[0018]

[0014] The invention also refers to a method for the treatment of a tumor in a subject, wherein the treatment comprises administering a therapeutically effective amount of a mitotic-phase (M-phase) cell cycle arresting compound to a subject in need thereof (i.e., suffering from a tumor), and wherein the treatment further comprises administering a therapeutically effective amount of a modulator of an interphase cell cycle checkpoint, and delivering ionizing radiation to the subject; and wherein the M- phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject. / nip

[0019]

[0015] The invention also provides for a combination, namely for a drug combination, comprising:

[0020] (a) a M-phase cell arresting compound, preferably a pharmaceutically acceptable M-phase cell arresting compound, preferably the compound ABT- 751 ; and (b) a modulator of an interphase cell cycle checkpoint, preferably a pharmaceutical acceptable modulator of an interphase cell cycle checkpoint, preferably selected from one or more of adavosertib, zedoresertib, berzosertib, rabusertib, prexasertib, tuvusertib and lunresertib; wherein (a) and (b) are comprised in a single pharmaceutical composition or, alternatively, (a) and (b) are comprised in separate pharmaceutical compositions.

[0021] The compounds in (a) and (b) are provided, preferably, in separate pharmaceutical compositions when the mode and route of administration differs, or when due to the particular schedule and timing of administration of each of the compounds it is not possible to include them in a single pharmaceutical composition.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

[0016] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0017] Figure 1 . Schematic overview of two Mitotic Enrichment strategies, one in which an M-phase cell arresting compound is administered in combination with ionizing radiation (herewith also termed first-generation Mitotic Enrichment strategy, and widely disclosed in the international patent application with publication number WO 2021 / 150109); and one strategy in which an M-phase cell arresting compound in combination with a modulator of an interphase cell cycle checkpoint are administered, in turn, in combination with ionizing radiations (herewith also termed Next-generation Mitotic Enrichment strategy). (A) First-generation Mitotic Enrichment strategy. Cells progress sequentially trough different phases of the cell cycle: G1 , S and G2 phase, which together make up interphase, and M phase or mitosis. In G1 phase cells prepare for DNA replication, which occurs subsequently in S phase. In G2 phase cells prepare for actual division, which occurs in mitosis. Each phase is equipped with its own checkpoints, or stop signs, which allow temporary shutdown of the cell cycle when critical events such as damage occur. Cells are much more sensitive to ionizing radiation during M phase compared to interphase. Unfortunately, cells spend but a fraction of their time in mitosis. Mitotic Enrichment is a radiosensitization strategy that exploits the intrinsic vulnerability of mitotic cells to ionizing radiation. It aims to trap cells during mitosis for a prolonged period of time prior to delivering radiotherapy, allowing cells to progress naturally through interphase and accumulate in mitosis, thus enriching a tumor for the sensitive mitotic population. Irradiation of a mitotically enriched tumor is shown to lead to increased tumor cell death compared to when irradiating a non-enriched tumor. First-generation Mitotic Enrichment, which has now successfully completed pre-clinical development for glioblastoma, utilized the tubulin polymerization inhibitor ABT-751 to induce a mitotic arrest and trap cells during mitosis. (B) Next-generation Mitotic Enrichment combines M-phase cell cycle arresting compounds, such as the ABT-751 illustrated in the figure with inhibitors of the interphase cell cycle checkpoints, such as S phase or G2 phase checkpoints illustrated in the figure, to achieve superior mitotic enrichment and subsequent radiosensitization. While progressing through interphase, cancer cells are frequently halted by cell cycle checkpoints to repair damage, diminishing the amount of cells that can accumulate in mitosis in a given timeframe. Inhibition of these checkpoints was found to drive cells through interphase (arrow along S and G2 phases), resulting in superior mitotic enrichment by ABT-751 , as an example of M-phase cell arresting compound in this figure, and superior radiosensitization. Candidate drugs include ATR and ATM inhibitors to inhibit S phase checkpoints and Wee1 and CHK1 inhibitors to inhibit the G2 checkpoint. Inhibition of Wee1 would not only abrogate the G2 checkpoint but also further enforce mitotic arrest, as Wee1 also plays a role in driving mitotic exit.

[0024]

[0018] Figure 2. Next-generation Mitotic Enrichment using, for example, ABT-751 in combination with, for example, Wee1 inhibition (using adavosertib) achieves superior mitotic enrichment and radiosensitization. Flow cytometric cell cycle analysis using propidium iodide (PI; DNA staining) and phospho-histone H3 staining (pHH3; mitotic marker) demonstrates superior induction of mitotic enrichment by combining ABT-751 with a Wee1 inhibitor compared to ABT-751 alone in colorectal (HCT-116) and brain cancer cells (11251 , glioblastoma (serum-cultured); U3086MG, classical glioblastoma; VUmc-ATRT-01 , SHH subtype AT / RT; Vllmc-HGG-15, BRAF mutant pHGG). The Wee1 inhibitor adavosertib dose-dependently increases the percentage of mitotic cells (DNA 4N, pHH3+, four column in the sets showed for each dose in the bar graphics) in combination with ABT-751. G1 cells (DNA 2N, pHH3-, first column in the sets showed for each dose in the bar graphics) are already severely depleted by ABT-751 alone and S phase cells (DNA 2N-4N, pHH3-, second column in the sets showed for each dose in the bar graphics) stay unaffected. However, in line with its mode of action to inhibit the G2 checkpoint, addition of adavosertib to ABT-751 also reduces the amount of G2 cells (DNA 4N, pHH3-, third column in the sets showed for each dose in the bar graphics). These combined effects result in superior mitotic enrichment compared to using ABT-751 alone. ***p < 0.001 , ****p < 0.0001 (Extra sum-of-squares F test)

[0025]

[0019] Figure 3. Next-generation Mitotic Enrichment using, for example ABT-751 , in combination with ATR inhibition, for example by berzosertib, achieves superior mitotic enrichment. Flow cytometric cell cycle analysis using propidium iodide (PI; DNA staining) and phospho-histone H3 staining (pHH3; mitotic marker) demonstrates superior induction of mitotic enrichment by combining ABT-751 with an ATR inhibitor compared to ABT-751 alone in colorectal (HCT-116) and brain cancer cells (11251 , glioblastoma (serum-cultured). The ATR inhibitor berzosertib dose-dependently increases the percentage of mitotic cells (DNA 4N, pHH3+; four column in the sets showed for each dose in the bar graphics) in combination with ABT-751 . G1 cells (DNA 2N, pHH3-, first column in the sets showed for each dose in the bar graphics) are already severely depleted by ABT-751 alone and are unaffected by berzosertib. However, in line with its mode of action to inhibit the intra-S and S-G2 checkpoints, addition of berzosertib to ABT-751 also reduces the combined amount of S phase cells (DNA 2N-4N, pHH3-, second column in the sets showed for each dose in the bar graphics) and G2 cells (DNA 4N, pHH3-, third column in the sets showed for each dose in the bar graphics). These combined effects result in superior mitotic enrichment compared to using ABT-751 alone.

[0026]

[0020] Figure 4. Next-generation Mitotic Enrichment using, for example ABT-751 , in combination with CHK1 inhibition, for example by rabusertib or prexasertib, achieves superior mitotic enrichment. Flow cytometric cell cycle analysis using propidium iodide (PI; DNA staining) and phospho-histone H3 staining (pHH3; mitotic marker) demonstrates superior induction of mitotic enrichment by combining ABT-751 with a CHK1 inhibitor compared to ABT-751 alone in brain cancer cells (11251 , glioblastoma (serum-cultured)). The CHK1 inhibitors rabusertib and prexasertib dose-dependently increase the percentage of mitotic cells (DNA 4N, pHH3+; four column in the sets showed for each dose in the bar graphics) in combination with ABT-751 . G1 cells (DNA 2N, pHH3-, first column in the sets showed for each dose in the bar graphics) are already severely depleted by ABT-751 alone and are unaffected by rabusertib or prexasertib. However, in line with its mode of action to inhibit the G2-M checkpoint, addition of rabusertib or prexasertib to ABT-751 also reduces the amount of G2 phase cells (DNA 4N, pHH3-, third column in the sets showed for each dose in the bar graphics). These combined effects result in superior mitotic enrichment compared to using ABT-751 alone.

[0027]

[0021] Figure 5. Superior in vitro radiosensitization by next-generation Mitotic Enrichment using ABT-751 in combination with the Wee1 inhibitor adavosertib compared to first-generation Mitotic Enrichment using ABT-751 alone. First-generation Mitotic Enrichment using ABT-751 alone radiosensitized 11251 glioblastoma cells (left panel; see also Figure 2 data with 11251 glioblastoma cells). However, next-generation Mitotic Enrichment using addition of 1 pM adavosertib resulted in superior radiosensitization of 11251 glioblastoma cells compared to ABT-751 alone, in line with the superior mitotic enrichment achieved by this combination (right panel; see also see also Figure 2 data with 11251 glioblastoma cells). Importantly, adavosertib did not radiosensitize 11251 cells by itself in a Mitotic Enrichment setup. ***p < 0.001 , ****p < 0.0001 (Extra sum-of-squares F test).

[0028]

[0022] Figure 6. Enforcing a G2 checkpoint arrest by CDK1 inhibition, for example by RO-3306 (CAS No. : 872573-93-8), prevents mitotic enrichment by ABT-751. Flow cytometric cell cycle analysis using propidium iodide (PI; DNA staining) and phosphohistone H3 staining (pHH3; mitotic marker) demonstrates loss of mitotic enrichment by combining ABT-751 with a CDK1 inhibitor compared to ABT-751 alone in brain cancer (U251 , glioblastoma (serum-cultured), melanoma (Mel57) and cervical cancer cells (HeLa). The CDK1 inhibitor RO-3306 dose-dependently decreases the percentage of mitotic cells (DNA 4N, pHH3+; lines with solid capsized triangle points) in combination with ABT-751. G1 cells (DNA 2N, pHH3-, blue lines with solid circle points) are depleted by ABT-751 alone but recover by combined inhibition of CDK1. The amount of S phase cells (DNA 2N-4N, pHH3-, lines with solid square points) and G2 phase cells (DNA 4N, pHH3-, lines with solid upside triangles) remained largely unaffected. These combined effects result in loss of mitotic enrichment compared to using ABT- 751 alone.

[0029]

[0023] Figure 7. Loss of in vitro radiosensitization by using ABT-751 in combination with the CDK1 inhibitor RO-3306 compared to first-generation Mitotic Enrichment using ABT-751 alone. First-generation Mitotic Enrichment using ABT-751 alone radiosensitized 11251 glioblastoma cells (line with solid square points vs. line with solid circle points or control). However, combining ABT-751 with RO-3306 abrogated radiosensitization of 11251 glioblastoma cells compared to ABT-751 alone (line with empty circle points), in line with the loss of mitotic enrichment achieved by this combination (see also Figure 6). Importantly, RO-3306 did not radiosensitize 11251 cells by itself in a Mitotic Enrichment setup (line with solid triangles). Together, these data underline the importance of inhibiting interphase checkpoints and not enforcing them to achieve radiosensitization through Mitotic Enrichment. ***p < 0.001 , ****p < 0.0001 (Extra sum-of-squares F test).

[0030]

[0024] Figure 8. ABT-751 , Vincristine, Compound-20, and SB-743921 are able to significantly increase the mitotic fraction of 11251 cells without inducing cytotoxicity. (A) Effect of ABT-751 (5 pM), Vincristine (4 nM), Compound-20 (10 pM) and SB- 743921 (1 nM) on cell survival after various exposure times. The dotted line indicates the treatment duration in (B). No cytotoxic effects were observed for any of these drugs until after 8 hours of exposure. (B) FACS cell cycle analysis using propidium iodide and phospho-histone H3 staining demonstrates effective induction of mitotic enrichment at non-cytotoxic conditions by all compounds tested.

[0031]

[0025] Figure 9. Mitotic Enrichment radiosensitizes 11251 cells to fractionated radiation. (A) Schematic depiction of the radiosensitization schedule used. (B) Compound-20 and ABT-751 considerably radiosensitized 11251 cells to the schedule depicted in (A).

[0032]

[0026] Figure 10. Irradiation prior to exposure to mitotic enrichment inducers prevents radiosensitization. (A) Schematic depiction of the radiosensitization schedule used. (B) U251 cells could not be radiosensitized by ABT-751 or Compound-20 when ionizing radiation was given prior to exposure to the compounds.

[0033]

[0027] Figure 11. SB-743921 brain penetration and plasma pharmacokinetics in WT and ABC transporter knockout mice. (A) Brain penetration and plasma concentrations of wildtype and transporter knockout mice at 8 hours after receiving i.p. SB-743921. SB-743921 reaches concentrations in the brain of WT mice up to 8 hours after i.p. administration that are sufficient to induce mitotic enrichment in vitro, although it appears to be a substrate for Abcbl as indicated by higher brain penetration in Abcb1a / b- / - mice that did not increase further in Abcg2; Abcbl a / b- / - mice. (B) Plasma pharmacokinetics curves of the mice from (A). These plasma concentration time curves are in line with those in patients receiving clinically acceptable dose levels (Holen KD et al. Cancer Chemother Pharmacol. 2011. 67(2):447-54. doi: 10.1007 / S00280-010- 1346-5).

[0034]

[0028] Figure 12. The Eg5 / KIF11 / KSP inhibitors filanesib and litronesib and the CENP- E inhibitors GSK923295 and PF-2771 are able to significantly increase the mitotic fraction of cancer cells without inducing cytotoxicity. (A) Effect of filanesib (10 nM), litronesib (10 nM), and GSK-923295 (100 nM) on 11251 cell survival after various exposure times. No cytotoxic effects were observed for any of these drug until after 8 hours of exposure. (B) FACS cell cycle analysis using propidium iodide and phosphohistone H3 staining demonstrates effective induction of mitotic enrichment of various cancer cells after 6 hours at non-cytotoxic conditions by all compounds tested.

[0035]

[0029] Figure 13. Next-generation Mitotic Enrichment using ABT-751 in combination with Wee1 / Myt1 inhibitors achieves superior Mitotic Enrichment and radiosensitization. Flow cytometric cell cycle analysis using propidium iodide (PI; DNA staining) and phospho-histone H3 staining (pHH3; mitotic marker) demonstrates superior induction of mitotic enrichment by combining ABT-751 with a Wee1 inhibitor or Myt1 inhibitor compared to ABT-751 alone in (A) 11251 and (B) LN-751 brain cancer cells.; *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 (Two-way ANOVA with Dunnett’s multiple comparisons post-hoc test compared to ABT-751 alone). (C) Superior in vitro radiosensitization by next-generation Mitotic Enrichment using ABT- 751 in combination with the Wee1 inhibitor Debio123 (i.e. , zedoresertib) compared to first-generation Mitotic Enrichment using ABT-751 alone. First-generation Mitotic Enrichment using ABT-751 alone radiosensitized 11251 glioblastoma cells However, next-generation Mitotic Enrichment using addition of 1 pM Debio123 resulted in superior radiosensitization of 11251 glioblastoma cells compared to ABT-751 alone, in line with the superior mitotic enrichment achieved by this combination (see (A-B)). Importantly, Debio123 did not radiosensitize 11251 cells by itself in a Mitotic Enrichment setup. **p < 0.01 , ****p < 0.0001 (Extra sum-of-squares F test)

[0036] DESCRIPTION

[0037] Definitions

[0038]

[0030] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0039]

[0031] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0032] For purposes of the present invention, the following terms are defined below.

[0040]

[0033] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, a method for administrating a pharmaceutical agent includes the administrating of a plurality of molecules (e.g., 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).

[0041]

[0034] As used herein, the term “mitotic phase (M-phase) cell cycle arresting compound” is a compound of any nature with the capability to arrest in mitosis those cells of a pool or tissue that initiated or were in any of the phases of said mitosis, in such a way that the cells cannot exit the mitotic phase (i.e. , cannot prosecute / proceed to the G1-phase of the cell cycle). Particularly preferred M-phase cell cycle arresting compounds are enumerated and listed all along this description. The effect of a compound that arrests the M-phase is the retention or halt of the cell in the mitotic phase, because the mitosis / meiosis cannot be accomplished. After a prolonged period of mitotic arrest, the cells ultimately undergo apoptosis. The mechanism underlying the arrest of the cells in the mitotic phase can be any that ultimately does not allow the cells to go on beyond the mitosis and enter to G1 phase. Therefore, it includes those compounds that functionally do not allow the mitosis to progress. There are included compounds that ultimately cause the impairment or non-functioning of the microtubules (e.g., microtubule destabilizing agents or microtubule stabilizing agents). There may also be compounds that impair the microtubules to move closer together or further apart, both events ruled by kinesins and required for proper spindle functioning (e.g., kinesin inhibitors). There can also be included compounds that directly or indirectly modulate the checkpoint of the mitosis (i.e., spindle assembly checkpoint (SAC) in metaphase), in such a way that this is ultimately enhanced or activated to retain the cells in mitosis and not to continue through G1 phase. The later can also be referred to as a “enhancer or activator of M-phase cell cycle checkpoint” or as a “enhancer or activator of the metaphase checkpoint”. Using conventional of cellular and molecular biology techniques, a compound can be tested to determine if it is an M-phase cell cycle arresting compound. Some of these techniques are detailed in the examples of this description (e.g. flow cytometry analysis). The techniques primarily assess the presence of cells in the M phase. Example of these techniques are Flow Cytometry, which is a primary method used to determine cell cycle distribution; Microscopy (Immunofluorescence), where treated cells can be visualized under a microscope after staining specific cellular components (e.g., Stains for DNA to see condensed chromosomes that haven't separated) or directly show morphological changes indicative of mitotic arrest); Western Blotting used to measure the levels of specific proteins that regulate the cell cycle, such as Cyclin B or phosphorylated histone H3 (pH3, a specific marker for mitosis); and Mitotic Index Calculation, in which the percentage of cells that exhibit mitotic morphology (condensed chromosomes visible under a microscope) are counted in a treated sample compared to an untreated control.

[0042]

[0035] As used herein the term “ABT-751” refers to the chemical agent N-[2-(4- hydroxyanilino)-3-pyridinyl]-4-methoxybenzenesulfonamide, also referred to as N-(2- ((4-hydroxyphenyl)amino)pyrid-3-yl)-4-methoxybenzenesulfonamide and / or N-(2-((4- hydroxy-phenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide and any pharmaceutically acceptable salts, esters or ethers thereof. ABT-751 is described for the first time in US5250549, EP0472053 and equivalents. ABT-751 is a pharmaceutical agent. ABT-751 is classified as a tubulin inhibitor and / or an anti-mitotic agent, and it is responsible of the arrest of cells at the mitotic phase. ABT-751 is also known under the synonyms E7010, E-7010 and E 7010. As disclosed for the first time in WO 2021 / 150109 (Stichting het Nederlands Kanker I nstitituut-Antoni van Leeuwenhoek Ziekenhuis) ABT-751 can surprisingly penetrate the brain (cross the blood-brain barrier).

[0043]

[0036] As used herein, the term “modulator of an interphase cell cycle checkpoint” encompasses a compound of any nature with the capability to enhance or to inhibit one or more of the elements involved in an interphase cell cycle checkpoint. The interphase cell cycle encompasses G1-, S and G2-cell phases. Thus, all the phases of the cell cycle with the exception of the mitosis (or M-phase). In this description, the terms “modulator of a G1-phase cell cycle checkpoint”, “modulator of an S-phase cell cycle checkpoint”, and “modulator of a G2-phase cell cycle checkpoint” are used to refer to the compounds that particularly modulate (e.g. inhibit) the network of regulatory proteins which perform as checkpoints, respectively, in G1-phase, S-phase, G2-phase.

[0044]

[0037] In general, cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle. Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage. The cell cannot proceed to the next phase until checkpoint requirements have been met. Checkpoints typically consist of a network of regulatory proteins that monitor and dictate the progression of the cell through the different stages of the cell cycle. There are several checkpoints to ensure that damaged or incomplete DNA is not passed on to daughter cells. Three main checkpoints exist: the G1 / S checkpoint, the G2 / M checkpoint and the metaphase (mitotic) checkpoint (also known as the spindle assembly checkpoint (SAC), or as the metaphase-to-anaphase transition). Some proteins are also involved in the control of the transition from S-phase cell cycle to G2- phase cell cycle (also called S / G2 checkpoint). Besides there is also the intra-S checkpoint. Another checkpoint is the Go checkpoint, in which the cells are checked for maturity. If the cells fail to pass this checkpoint by not being ready yet, they will be discarded from dividing. In this description, the terms “modulator of a G1-phase cell cycle checkpoint”, “modulator of an S-phase cell cycle checkpoint”, “modulator of a G2-phase cell cycle checkpoint”, and “modulator of M-phase cell cycle checkpoint” or “modulator of the metaphase checkpoint” are used to refer to the compounds that particularly modulate (e.g. inhibit) the network of regulatory proteins which perform as checkpoints, respectively, in G1-phase, S-phase, G2-phase and M-phase.

[0045]

[0038] The modulators of the cell cycle checkpoint inhibit or enhance one or more of the regulatory proteins involved in a cell cycle checkpoint. Thus, they mainly interfere in the process of DNA damage verification at that point of the cell cycle, or in other processes, to complete a phase in the cell cycle.

[0046]

[0039] The administration of a modulator of an interphase cell cycle checkpoint according to the invention thus allows the cells to proceed through G1 / S and G2 despite any errors that would normally cause the interphase cell cycle checkpoint to not allow the cell to proceed through G1 / S and G2. By modulating the interphase cell cycle checkpoint in accordance with the invention, the inactivity of the interphase cell cycle checkpoint will now allow the cells to proceed through G1 / S and G2. The modulator of an interphase cell cycle checkpoint according to the invention is a compound that independently of its mechanism of action (inhibitor, enhancer) will ultimately allow the cells to proceed through the interphase checkpoints (i.e., G1 / S and G2). The administration of an interphase cell cycle checkpoint modulator in combination with an M-phase cell arresting compound aims to allow the cells to proceed through G1 / S and G2 despite any errors and to remain the cells arrested in the M-phase.

[0040] Preferred modulators of several of the checkpoints within a phase of the interphase cell cycle (i.e., G1 , S, and G2) are listed below, although they are not to be considered as limitative examples of all the possible ones. Conventional techniques are used and known to determine if a compound is a modulator of an interphase checkpoint, including among others microscopy, flow cytometry and biochemical assays as previously discussed for the M-phase cell arresting compounds.

[0047]

[0041] As used herein, the term “berzosertib” refers to the chemical agent 3-[3-[4- (methylaminomethyl)phenyl]-5-isoxazolyl]-5-(4-propan-2-ylsulfonylphenyl)-2- pyrazinamine and any pharmaceutically acceptable salts, esters or ethers thereof. Berzosertib (also known under the terms VE-822, VX-970, M6620) is disclosed for the first time in the family of the European Patent EP3354650 of Vertex Pharmaceuticals and further licensed for development to Merck KGaA. It acts as a potent inhibitor of the enzyme ataxia telangiectasia and Rad3 related (ATR), and with lower potency as an inhibitor of ATM serine / threonine kinase (ATM). These enzymes are both involved in detecting DNA damage as part of cell cycle checkpoints during cell division. By inhibiting their activity, berzosertib interferes with the ability of rapidly dividing cells to detect damage to DNA, and this makes it useful as a potential treatment for some forms of cancer by causing accumulation of DNA damage in the cancer cells, and thus reducing their viability. In addition, by inhibiting these enzymes, berzosertib promotes the cells to speed through the S- and / or G-phase cell cycle. It has progressed furthest in trials for the treatment of ovarian cancer, though also shows activity against numerous other cancer types. Berzosertib is thus a modulator, namely an inhibitor, of a cell cycle checkpoint. Therefore, berzosertib acts as a modulator (i.e., inhibitor) of a cell cycle checkpoint according to the invention.

[0048]

[0042] As used herein, the term “tuvusertib” refers to the chemical agent 2-Amino-6- fluoro-N-[5-fluoro-4-(1-methyl-1 H-imidazol-5-yl)-3-pyridinyl]pyrazolo[1 ,5-a]pyrimidine- 3-carboxamide, and any pharmaceutically acceptable salts, esters, or ethers thereof. Tuvusertib (also known under the term M-17742) was disclosed for the first time in the family of the European Patent EP2941432 of Vertex Pharmaceuticals. Tuvusertib is an orally available inhibitor ATR kinase, with potential antineoplastic activity. Upon oral administration, tuvusertib selectively inhibits ATR activity and blocks the downstream phosphorylation of the serine / threonine protein kinase checkpoint kinase 1 (CHK1).

[0043] As used herein, the term “adavosertib” refers to the chemical agent

[0049] 1-[6-(2-hydroxypropan-2-yl)-2-pyridinyl]-6-[4-(4-methyl-1-piperazinyl)anilino]-2-prop-

[0050] 2-enyl-3-pyrazolo[3,4-d]pyrimidinone and any pharmaceutically acceptable salts, esters, or ethers thereof. Adavosertib (also known under the terms AZD-1775 and MK- 1775) is disclosed for the first time in the family of the European Patent EP2016080 of Banyu Pharmaceutical Co. Ltd. It is a small molecule inhibitor of the tyrosine kinase Wee1 with potential antineoplastic sensitizing activity. It is being developed by AstraZeneca. It is being investigated as a treatment for several cancer types, such as pancreatic cancer and ovarian cancer. By inhibiting Wee1 , adavosertib does not allow the cells to exit the mitotic phase of the cell cycle. Due to its mode of action to inhibit the G2 checkpoint, addition of adavosertib promotes the cells also enter the M-phase. Therefore, adavosertib acts as a modulator (i.e. , inhibitor) of a cell cycle checkpoint according to the invention, and also is an example of compound that deprive the cell to exit mitosis and progress to the G1-phase cell cycle.

[0051]

[0044] As used herein, the term “zedoresertib” refers to the chemical agent 3-(2,6- Dichlorophenyl)-1-methyl-7-((3-methyl-4-(1-methylpiperidin-4-yl)phenyl)amino)-2,3- dihydropyrimido[4,5-d]pyrimidin-4(1 H)-one, and any pharmaceutically acceptable salts, esters, or ethers thereof. Zedoresertib (also known as Debio123) and with the CAS No. 2243882-74-6 is being used as a Wee1 inhibitor and its biology and mechanism of action is as indicated for adavosertib in this description.

[0052]

[0045] As used herein, the term “rabusertib” refers to the chemical agent (S)-1-(5- bromo-4-methyl-2-(morpholin-2-ylmethoxy)phenyl)-3-(5-methylpyrazin-2-yl)urea, and any pharmaceutically acceptable salts, esters, or ethers thereof. Rabusertib (also known as LY2603618) is disclosed for the first time in the family of the European Patent EP1869020 of ICOS Corporation (compound 28). It is an inhibitor of the cell cycle checkpoint kinase 2 (chk2) with potential chemopotentiating activity. Rabusertib binds to and inhibits the activity of chk2, which may prevent the repair of DNA caused by DNA-damaging agents, thus potentiating the antitumor efficacies of various chemotherapeutic agents. Due to its mode of action to inhibit the G2 checkpoint, addition of rabusertib promotes the cells also enter the M-phase. Therefore, rabusertib acts as a modulator (i.e., inhibitor) of an interphase cell cycle checkpoint according to the invention.

[0046] As used herein, the term “prexasertib” refers to the chemical agent 5-[[5-[2-(3- aminopropoxy)-6-methoxyphenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile, and any pharmaceutically acceptable salts, esters, or ethers thereof. Prexasertib (also known as LY2606368) is disclosed for the first time in the family of the European Patent EP2379532B1 of Lilly Co Eli, as the formic acid, dihydrogen chloride, and methanesulfonic acid salts. Prexasertib is an inhibitor of checkpoint kinase 1 (chk1) with potential antineoplastic activity. Upon administration, prexasertib selectively binds to chk1 , thereby preventing activity of chk1 and abrogating the repair of damaged DNA. Due to its mode of action to inhibit the G2 checkpoint, addition of prexasertib promotes the cells also enter the M-phase. Therefore, prexasertib acts as a modulator (i.e., inhibitor) of an interphase cell cycle checkpoint according to the invention.

[0053]

[0047] As used herein, the term “lunresertib” refers to the chemical agent 2-amino-1- (3-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3- carboxamide, and any pharmaceutically acceptable salts, esters, or ethers thereof. Lunresertib (also known as RP-6306) is disclosed in the family of the International Patent Application WO2021 195781 of REPARE THERAPEUTICS INC. Lunresertib is an orally bioavailable inhibitor of the human membrane-associated tyrosine- and threoninespecific cdc2-inhibitory kinase (PKMYT1), with potential antineoplastic activity. Upon oral administration, lunresertib targets, binds to and inhibits the activity of PKMYT1. This results in the inhibition of CDK1 phosphorylation, which may promote both premature mitosis and a prolonged mitotic arrest. Due to its mode of action to inhibit the G2 checkpoint, addition of lunresertib promotes the cells also enter the M-phase. Therefore, lunresertib acts as a modulator (i.e., inhibitor) of an interphase cell cycle checkpoint according to the invention.

[0054]

[0048] As used herein “exit mitosis” or “to exit the mitotic phase” refers to the final cell cycle transition, and it represents the completion of mitosis and the entry into a new interphase. This process requires a tight coordination of multiple signaling pathways to ensure a faithful distribution of the genomic material and the cellular content between the dividing cells. The key event in mitotic exit is the onset of anaphase, which is driven by the APC / C-dependent ubiquitination. Degradation of APC / C substrates such as cyclin B and securin is important for events during mitotic exit, including sister chromatid separation, spindle disassembly, chromosome decondensation, cytokinesis, and reformation of nuclear envelope (Poon RY. Cell Cycle Control: A System of Interlinking Oscillators. Methods Mol Biol. 2016;1342:3-19. doi: 10.1007 / 978- 1-4939-2957-3_1. PMID: 26254915).

[0055]

[0049] The term “microtubule-stabilizing agent” or “microtubule stabilizer” (used both as synonyms), are a subclass of microtubule-targeting agents that stimulate the assembly of purified tubulin and increase the density of cellular microtubules by shifting the equilibrium of tubulin polymer from the soluble to the polymerized form. In contrast, “microtubule-destabilizing agents” or a “microtubule depolymerizers” (both used as synonyms) are the substances which interact with tubulin to inhibit the tubulin polymerization to microtubules, either because they depolymerize tubulin or because they do not allow the polymerization to take place. Independently of the mechanism of action, both microtubule-stabilizing agents and microtubule-destabilizing agents make the cell not to be able to perform and / or complete the mitotic phase, and this is why they have been used as anticancer compounds. A mitotic phase that cannot be completed is also herewith termed as an “arrested M-phase”. Determining if a compound is a microtubule-stabilizing or a microtubule-destabilizing agent can be done using conventional techniques widely know by the skilled person. Some of these include Microscopy (Live-Cell Imaging and Immunofluorescence), by direct visualization using fluorescence microscopy with fluorescently labeled tubulin, where destabilizing agents will show rapid microtubule breakdown, while stabilizing agents will show static, dense bundles of microtubules. By microscopy also the spindle morphology can be determined, by staining fixed cells to visualize the nucleus and microtubules which can reveal abnormal, non-functional mitotic spindles or the complete absence of a spindle. Other techniques include Flow Cytometry. Biochemical assays can also be used, such as in vitro Tubulin Polymerization Assays. Turbidity Assays are also used, wherein purified tubulin can be induced to polymerize in a test tube, and the resulting turbidity (cloudiness) of the solution can be measured by light scattering.

[0056]

[0050] The terms “about” and “approximately”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 % and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0057]

[0051] As used herein, the term “amount” is used interchangeably with the term “dose”.

[0052] As used herein the term “dose” refers to a specific individual amount of drug / medication, for example of a pharmaceutical agent, chemotherapeutic agent or ionizing radiation, taken at one time. Typically, a dose is encompassed in, for example, one tablet, one capsule or one other vehicle for administration of medication to a subject.

[0058]

[0053] As used herein, the term “and / or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0059]

[0054] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc.

[0060]

[0055] As used herein, the terms “cancer” and “tumor” (used interchangeably) refer to or describe the physiological condition in a host organism (e.g., a human) that is typically characterized by unregulated cell growth. The terms “cancer” and “tumor” also refer to cells that have undergone a cancerous / malignant transformation that makes them pathological to the host organism. Cancerous cells can be distinguished from non-cancerous cells by techniques known to the skilled person.

[0061]

[0056] The term “chemotherapeutic treatment” or “chemotherapy” (often abbreviated as “chemo”) as used herein refers to (the use of) a pharmaceutical agent or a combination of pharmaceutical agents in a cancer treatment, e.g., which can be part of a standardized chemotherapy regimen. Chemotherapy is given with a curative intent (which almost always involves combinations of chemotherapeutic agents), or it may aim to prolong life or to reduce symptoms (palliative chemotherapy).

[0062]

[0057] The term “chemotherapeutic agent(s)” or “chemotherapeutic drug(s)” (also known as cytotoxic agents) as used herein refers to pharmaceutical agents capable of acting on or targeting rapidly dividing normal (non-cancer cells) and cancerous cells. Several types of chemotherapeutic agents exist, including alkylating agents (e.g. altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxaliplatin, temozolomide, thiotepa, and others), antimetabolite agents (e.g. 5-fluorouracil (5-Fll), 6- mercaptopurine (6-MP), capecitabine (Xeloda), cytarabine (Ara-C), floxuridine, fludarabine, gemcitabine (Gemzar), hydroxycarbamide and others), anti-microtubule agents (e.g. paclitaxel (Taxol), vinorelbine (Navelbine), docetaxel (Taxotere), vinblastine (Velban), and many others), topoisomerase inhibitors type I and II (type I: e.g. irinotecan, topotecan, camptothecin and lamellarin D, and others; and type II: e.g. etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, HU-331 , and others), and cytotoxic antibiotic agents (e.g. dactinomycin, bleomycin, daunorubicin, and others), and many others.

[0058] As used herein the term “glioblastoma”, refers to glioblastoma multiforme and / or all primary and secondary glioblastoma subtypes, wherein the glioblastoma is defined as being a tumor that has originated from glial cells which have undergone tumor transformation that makes them pathological to the host organism. A skilled person is accustomed with glioblastoma and knows how to distinguish the glioblastoma from other malignancies (Louis et al (2016) Acta Neuropathol. 31(6):803- 20).

[0063]

[0059] As used herein, the term “ionizing radiation” refers to a type of energy released by atoms in the form of electromagnetic waves or particles. As used herein the term “ionizing radiation” refers to the ionizing radiation that occurs as electromagnetic rays, for example X-rays, gamma rays or particles, for example alpha and beta particles. Ionizing radiation as used herein can be provided to a subject by, for example, radiation therapy (RT), radiotherapy and the like. Ionizing radiation is well known in the art (Hellman, Essentials of Radiation Therapy, Cancer, Principles & Practice of Oncology, 248-275 (DeVita et al. ed., 10th Ed., V1 , 2014)). Ionizing radiation may herein be abbreviated to “IR”.

[0064]

[0060] The term “metastasis” (plural: “metastases”) or “cancer metastasis” (plural: “cancer metastases”) as used herein refers to a situation where cancer cells break away from where they first formed (primary cancer), travel through the blood or lymph system, and form new tumors (metastatic tumors or lesions; also referred to as secondary tumors) in other (distal) parts of the body. The term is well-known in the art. The terms metastatic tumor and secondary tumor may be used interchangeable.

[0065]

[0061] As used herein the term “mitotic enrichment” refers to a shift in the cell cycle of a cell induced by a pharmaceutical agent, in particular by a M-phase cell cycle arresting compound such ABT-751. The administration of the M-phase cell cycle arresting compound, as ABT-751 , to a subject and / or by adding the said compounds (e.g., ABT-751) in vitro to a cell culture can induce mitotic enrichment in said subject and / or cell culture. The M-phase cell cycle arresting compound, such as ABT-751 , may for example synchronize the cell cycle of cells, preferably tumor cells, to the M- phase, for example by inducing an arrest of the normal cell cycle in M-phase by interfering with the colchicine site on B-tubulin.

[0066]

[0062] As used herein, the term "pharmaceutically acceptable salts, esters or ethers" refers to the relatively non-toxic, inorganic and organic acid or base addition salts, or to the esters or the ethers, of any of an M-phase cell cycle arresting compound (e.g., ABT-751), and / or of a modulator of a cell cycle checkpoint, and / or the chemotherapeutic agents encompassed by the invention. Representative salts are known in the art (See, for example, Berge, et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66: 1-19). The skilled person in the art will also recognize which esters and or ethers of the compounds herewith referred are encompassed, as well as how they are commonly obtained from the acids and or alcohols of the compounds.

[0067]

[0063] The term “primary tumor”, such as a “primary brain tumor” as used herein refers to tumors that are localized in the tissue or organ where they were originated. In the specific case of a primary brain tumor, the term refers to brain tumors (tumors that are localized in the brain) that find their origin in the brain, i.e. , in brain tissue. The primary brain tumor, for example, can be from (neuro)epithelial tissues such as glioma from astrocytes and / or oligodendrocytes, meningioma from meningeal cells and medulloblastoma from primitive neuroepithelial cells.

[0068]

[0064] The term “secondary tumor”, such as a “secondary brain tumor” as used herein refers to tumors formed in another than the tissue where they are located, and which resulted from the spreading of a primary tumor. In the particular case of a secondary brain tumor, it refers to the tumors formed in the brain as a result of metastasis, for example as a result of a situation where cancer cells break away from where they first formed (primary cancer) in other (distal) parts of the body, travel through the blood or lymph system, and form new tumors (metastatic tumors or lesions) in the brain. Secondary brain tumors are tumors arising from cancer cells that have disseminated from another body part to the brain (but are now present in the brain), for example from the lung, breast, genitourinary tract, skin, colon and head and / or neck.

[0069]

[0065] As used herein, the term "subject" refers to any vertebrate animal, but will typically pertain to a mammal, for example a human, a domesticated animal (such as dog or cat), a farm animal (such as horse, cow, or sheep) or a laboratory animal (such as rat, mouse, non-human primate or guinea pig). In preferred examples, the subject is human and includes males, females, adult, elderly, children or infants, for example suffering from or expected to be suffering from a tumor, for example a brain tumor, regardless of the stage or state of the (brain) tumor.

[0070]

[0066] As used herein, the term “therapeutically-effective amount" or “effective amount” refers to the amount either a M-phase cell cycle arresting compound, such as ABT-751 , or a modulator of an interphase cell cycle checkpoint, such as adavosertib or berzosertib, or any other chemotherapeutic agent as disclosed herein, which is effective for producing, e.g. an increase in the fraction of cells in a tumor that are in M-phase of the cell cycle e.g. during a period of at least 6 hours, and therefor effective in producing an desired (therapeutic) effect in a subject at a reasonable benefit / risk ratio applicable and within the context of the treatment of the invention.

[0071]

[0067] As used herein, the terms “treatment” and “treating” refer to therapeutic treatment. The object of the treatment is to at least slow down the disease condition. Those in need of the treatment include those already with the disease condition.

[0072] Detailed description

[0073]

[0068] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0074]

[0069] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0075]

[0070] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

[0076]

[0071] As embodied and broadly described herein, the present invention is directed to the surprising finding that the combination of one or more M-phase cell cycle arresting compounds, such as the microtubule inhibitor ABT-751 , and one or more modulators of an interphase cell cycle checkpoint, preferably inhibitors such as one or more inhibitors of the interphase cell cycle checkpoints (e.g., inhibitors of ATR, CHK1 or Wee1), preferably inhibitors of the S-phase and G2-phase cell cycle checkpoints, results in an efficient synchronization and accumulation of cancer cells in mitosis.

[0077]

[0072] The examples below show that, effectively in cancer cell lines of several kinds of tissues, including models of brain tumor tissue, the combined administration of a M- phase cell cycle arresting compound and a modulator of an interphase cell cycle checkpoint, drove the cells to a superior mitotic enrichment which ultimately translated to a superior radio sensitization.

[0078]

[0073] Thus, herewith are proposed combinations of drugs for use in therapy of cancer, said therapy comprising the delivery of ionizing radiation, as well as the administration of the combination of drugs (compounds) according to the invention.

[0079]

[0074] M-phase cell cycle arresting compounds and modulators of an interphase cell cycle checkpoint for use in the treatment of a tumor.

[0080]

[0075] As previously indicated, the invention provides a M-phase cell cycle arresting compound for use in the treatment of a tumor in a subject, wherein the treatment comprises administering the M-phase cell cycle arresting compound to the subject, administering a modulator of an interphase cell cycle checkpoint to the subject, and delivering ionizing radiation to the subject; and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

[0081]

[0076] Also provided therein is, as indicated, a modulator of an interphase cell cycle checkpoint for use in the treatment of a tumor in a subject, wherein the treatment comprises administering a M-phase cell cycle arresting compound to the subject, administering the modulator of an interphase cell cycle checkpoint to the subject, and delivering ionizing radiation to the subject; and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

[0082]

[0077] This description discloses a method for the treatment of a tumor in a subject, the method comprising administering to said subject a therapeutically effective amount of a mitotic-phase (M-phase) cell cycle arresting compound, and a therapeutically effective amount of a modulator of an interphase cell cycle checkpoint, and delivering ionizing radiation to the subject; and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

[0083]

[0078] In particular embodiments of the invention, the M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use as indicated, is such that the M-phase cell cycle arresting compound is selected from the group consisting of a microtubule-stabilizing agent, a microtubule-destabilizing agent, and combinations thereof.

[0084]

[0079] In yet another particular embodiment, the M-phase cell cycle arresting compound is a microtubule-destabilizing agent selected from the group consisting of a combretastatin, preferably the compound ABT-751 (N-[2-(4-hydroxyanilino)-3- pyridinyl]-4-methoxybenzenesulfonamide), a kinesin inhibitor, and combinations thereof.

[0085]

[0080] In some embodiments the combretastatin is the compound, N-[2-(4- hydroxyanilino)-3-pyridinyl]-4-methoxybenzenesulfonamide (ABT-751).

[0086]

[0081] Other combretastatins known as M-phase cell cycle arresting compounds are Combretastatin A4 phosphate (CA4P), combretastatin A1 phosphate (CA1 P), N- Acetylcolchicinol dihydrogenphosphate (ZD6126), and 2-Methoxy-estradiol.

[0087]

[0082] In yet another embodiment, the M-phase cell cycle arresting compound is the tubulin inhibitor eribulin (CAS 253128-41-5, O'Rourke B,et al. Cell Cycle. 2014;13(20):3218-21. doi: 10.4161 / 15384101.2014.950143. ).

[0088]

[0083] In yet another embodiment, the M-phase cell cycle arresting compound is the tubulin inhibitor lisavanbulin (BAL101553 (CAS 1263384-43-5), prodrug of avanbulin (BAL27862, CAS 798577-91-0) (Holdhoff M, et al. Neurooncol Adv. 2024 Aug 28;6(1):vdae150. doi: 10.1093 / noajnl / vdae150, Burgenske DM et al. Neuro Oncol. 2022 Mar 12;24(3):384-395. doi: 10.1093 / neuonc / noab162.).

[0089]

[0084] In yet another embodiment, the M-phase cell cycle arresting compound is the tubulin inhibitor rigosertib (Jost M et al. Mol Cell. 2020 Jul 2;79(1):191-198.e3. doi: 10.1016 / j.molcel.2020.06.008., CAS 592542-60-4).

[0090]

[0085] Eribulin, lisavanbulin and rigosertib have the same target as the compound ABT-751.

[0086] In an embodiment, the kinesin inhibitors are kinesin inhibitors of the kinesins selected from kinesin-like motor protein kinesin spindle protein (KSP, also knowns as KIF11 or Eg5, and herewith abbreviated as KSP / KIF11 / Eg5), kinesin-like motor protein centromere-associated protein E (CENP-E), kinesin-related protein HSET and Kinesin family member KIF18A (KIF18A).

[0091]

[0087] In some embodiments, the kinesin inhibitors are inhibitors of KSP / KIF11 / Eg5 selected from one or more of the compounds ispinesib (N-(3-Aminopropyl)-N-[(1 R)-1- [7-chloro-3,4-dihydro-4-oxo-3-(phenylmethyl)-2-quinazolinyl]-2-methylpropyl]-4- methylbenzamide), filanesib, litronesib, SB-743921 (CAS No. : 940929-33-9), ARQ621 (CAS No. : 1095253-39-6), MK-0731 (CAS No. : 845256-65-7), and N-[4’- (Trifluoromethyl)-4-biphenylyl]sulfamide, mentioned as compound 20 by Parrish et al. J. Med. Chem. 2007 50: 4939-4952.

[0092]

[0088] In some embodiments, the kinesin inhibitors are inhibitors of CENP-E, selected from one or more of the compounds GSK-923295 (CAS No. : 1088965-37-0), and PF- 2771 (CAS No. : 2070009-55-9).

[0093]

[0089] In some embodiments, the kinesin inhibitors are inhibitors of HSET selected from one or more of the compounds AZ82 (CAS No. : 1449578-65-7), and CW-069 (CAS No. : 1594094-64-0).

[0094]

[0090] In some embodiments, the kinesin inhibitors are inhibitors of KIF18A, selected from one or more of the compounds sovilnesib (CAS No. : 2410796-79-9), and AM- 9022 (CAS No. : 2446872-46-2).

[0095]

[0091] Other known kinesin inhibitors are the compounds S-trityl-L-cysteine, 2-butyl- 5-(3-hydroxyphenyl)-5,6, 11 , 11 a-tetrahydro-1 H-imidazo[1',5': 1 ,6]pyrido[3,4-b]indole- 1 ,3(2H)-dione (HR22C16), and Dihydropyrrole 19 ((1S)-1-Cyclopropyl-2-[(2S)-4-(2,5- difluorophenyl)-2-phenyl-2,5-dihydro-1 H-pyrrol-1-YL]-2-oxoethanamine).

[0096]

[0092] These and other kinesin inhibitors are disclosed in the document of Shahin R, Aljamal S. Kinesin spindle protein inhibitors in cancer: from high throughput screening to novel therapeutic strategies. Future Sci OA. 2022 Feb 21 ;8(3):FSO778. doi: 10.2144 / fsoa-2021-0116. PMID: 35251692; PMCID: PMC8890118. The assays to test that a compound is a kinesin inhibitor form part of the conventional techniques in biochemistry and cell-based assays.

[0097]

[0093] Other than kinesin inhibitors or combrestastatins that are also known as microtubule-destabilizing agents and, thus, as M-phase cell cycle arresting compounds according to this invention, include some colchicines, colchicine site binding compounds, vinca alkaloids (e.g., vinblastine, vinorelbine, vincristine, vindesine, vinflunine and combinations thereof), benzylisoquinoline alkaloids (for example noscapine), separase inhibitors, an anaphase-promoting complex inhibitors, and [1 ,2,4]triazolo[1 ,5-a]pyrimidines.

[0098]

[0094] In embodiments of the invention, the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint, for example any combination of the individual M-phase cell cycle arresting compounds and individual modulators of an interphase cell cycle checkpoint as described herein, are for use in the treatment of a tumor in a subject, as disclosed herein (e.g., in combination with radiation therapy as disclosed herein).

[0099]

[0095] When in this description a list of specific M-phase cell cycle arresting compounds, or a list of modulators of the interphase cell cycle checkpoints are indicated, they encompass the compounds as such, as well as any pharmaceutically acceptable salt or ester or ether thereof, as well as salts of the esters or of the ethers.

[0096] Data in the examples include assays carried out with the M-phase cell cycle arresting compound ABT-751. These assays are the proof of concept that M-phase cell cycle compounds when used in combination with modulators of interphase cell cycle checkpoints, improve the fraction of mitotic cells in tumor cell lines. These cells are more sensitive to ionizing radiation.

[0100]

[0097] In another embodiment of the M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to the invention, the M-phase cell cycle arresting compound is a microtubule-stabilizing agent selected from the group consisting of a taxane, an epothilone, and combinations thereof.

[0101]

[0098] In a more particular embodiment, the M-phase cell cycle arresting compound is a taxane, preferably selected from the group consisting of paclitaxel, docetaxel, 2-(4- morpholinyl)-4H-pyrimido[2,1-a]isoquinolin-4-one (TPI-287), and combinations thereof.

[0102]

[0099] Among the modulators of the interphase cell cycle checkpoint for use according to the invention, are encompassed those compounds or compositions that either enhance or inhibit the action of one or more proteins of the network of regulatory proteins involved in a determined interphase cell cycle checkpoint, preferably one or more proteins of the network of regulatory proteins involved in S-phase or G2-phase cell cycle checkpoints. Extended reviews of the cell cycle checkpoints and of the pathways therein involved are contained in several publications (see, for example, Otto et al. Nat Rev Cancer 2017’17(2):93-115. doi: 10.1038 / nrc.2016.138 and Suski et al. Cancer Cell 2021 ;39(6):759-778. doi: 10.1016 / j .ccell.2021.03.010). Preferred modulators are inhibitors of one or more of the proteins involved in a checkpoint, which by definition and by means of any inhibition mechanism (competitive, uncompetitive, allosteric) will ultimately block / inhibit the checkpoint due to its interaction with one or more of the proteins involved in a checkpoint. These inhibitors will thus impede the checkpoint to perform its actions, and the cells will progress to the next phase in the cell cycle. On the other hand, other preferred modulators of interphase cell cycle checkpoints, are those compounds or compositions that may enhance the activity of one of the proteins in the network of a cell cycle checkpoint. This may be the case when the enhanced protein is indeed a regulatory protein that inhibits or ultimately block / inhibit the checkpoint, thus by enhancing the action of this protein, the result of the modulation of the checkpoint is also the blocking / inhibition of the checkpoint, and the progression the phase of the cell cycle to the subsequent one.

[0103]

[0100] In an embodiment of the invention, the modulator of an interphase cell cycle checkpoint is selected from the group consisting of a modulator of a G1-phase cell cycle checkpoint, preferably an inhibitor of a G1-phase cell cycle checkpoint, a modulator of an S-phase cell cycle checkpoint, preferably an inhibitor of an S-phase cycle cell checkpoint, a modulator of a G2-phase cell cycle checkpoint, preferably an inhibitor of a G2-phase cell cycle checkpoint, and combinations thereof. In even a most preferred embodiment, the modulator of an interphase cell cycle checkpoint is selected from a modulator of the S-phase cell cycle checkpoint, and / or a modulator of the G2- phase cell cycle checkpoint, preferably an inhibitor of the S-phase cell cycle checkpoint, and / or an inhibitor of the G2-phase cell cycle checkpoint.

[0104]

[0101] In an embodiment, the modulator of an interphase cell cycle checkpoint is a modulator of an S-phase cell cycle checkpoint selected from the group consisting of a modulator of Ataxia telangiectasia mutated (ATM) kinase, a modulator of Ataxia Telangiectasia and Rad3 related (ATR) kinase, and combinations thereof. Preferred modulators of either ATM or ATR kinases are selected among inhibitors of any of these enzymes.

[0102] Examples of modulators of ATM or ATR, some of them in clinical studies are disclosed in several documents. All these compounds are to be understood as useful modulators of the interphase cell cycle checkpoints according to the invention (see (1) Bhanu Priya, et al., Targeting ATM and ATR for cancer therapeutics: Inhibitors in clinic, Drug Discovery Today, Volume 28, Issue 8, 2023,103662, https: / / doi.orq / 10.1016 / Ldrudis.2023.103662; (2) Deng, Set al., Targeting the DNA Damage Response and DNA Repair Pathways to Enhance Radiosensitivity in Colorectal Cancer. Cancers 2022, 14, 4874. (3) Vlatkovic, T et al., Targeting Cell Cycle

[0105] Checkpoint Kinases to Overcome Intrinsic Radioresistance in Brain Tumor Cells. Cancers 2022, 14, 701. https: / / doi.org / 10.3390 / cancers1403070, (4) Otto et al. (supra), and (5) Suski et al (supra)) :

[0106]

[0103] In even a more particular embodiment, the modulator of an interphase cell cycle checkpoint, is the modulator of ATR kinase known as berzosertib (i.e., berzosertib and / or any pharmaceutically acceptable salt, ester or ether thereof, and / or any salts of the ester or of the ether).

[0107]

[0104] In even a more particular embodiment, the modulator of an interphase cell cycle checkpoint, is the modulator of ATR kinase known as tuvusertib (i.e., tuvusertib and / or any pharmaceutically acceptable salt, ester or ether thereof, and / or any salts of the ester or of the ether).

[0108]

[0105] In another embodiment of the invention, the modulator of an interphase cell cycle checkpoint is a modulator of a G2-phase cell cycle checkpoint selected from the group consisting of a modulator of Checkpoint kinase 1 , a modulator of Checkpoint kinase 2, a modulator of Wee1-like protein kinase, a modulator of Membrane- associated tyrosine- and threonine-specific cdc2-inhibitory kinase (Myt1), and combinations thereof.

[0109]

[0106] Checkpoint kinase 1 , commonly referred to as Chk1 , is a serine / threonine- specific protein kinase involved in the initiation of cell cycle checkpoints, cell cycle arrest, DNA repair and cell death to prevent damaged cells from progressing through the cell cycle. Chk1 impacts various stages of the cell cycle including the G2-phase, but also, the S-phase, G2 / M transition and M-phase.

[0107] Checkpoint kinase 2, commonly referred as Chk2, is a tumor suppressor gene that a serine-threonine kinase. Chk2 is involved in DNA repair, cell cycle arrest or apoptosis in response to DNA damage.

[0110]

[0108] Wee1-like protein kinase is a nuclear protein, which is a tyrosine kinase belonging to the Ser / Thr family of protein kinases. This protein catalyzes the inhibitory tyrosine phosphorylation of CDC2 / cyclin B kinase and appears to coordinate the transition between DNA replication and mitosis by protecting the nucleus from cytoplasmically activated CDC2 kinase. Wee1-like protein kinase has a key role as an inhibitory regulator of the G2 / M checkpoint that precedes entry into mitosis.

[0111]

[0109] In a more particular embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint for use according to the invention, the modulator of an interphase cell cycle checkpoint is selected from one or more of: a modulator of Checkpoint kinase 1 , which modulator is a compound preferably selected from the group consisting of prexasertib (LY2606368), rabusertib (LY2603618), and combinations thereof; and / or a modulator of Checkpoint kinase 2; and / or a modulator of Wee1-like protein kinase, which modulator is preferably the compound adavosertib (AZD1775) or zedoresertib (also called Debio123); and / or a modulator of Myt1 (PKMYT1 ; protein kinase, membrane associated tyrosine / threonine 1), preferably the compound lunresertib (see, www.cancer.gov / publications / dictionaries / cancer-drug / def / lunresertib.).

[0112]

[0110] Examples of modulators of either Chk1 , Chk2, Wee1 , and PKMYT1 , some of them in clinical studies are disclosed in several documents. All these compounds are to be understood as useful modulators of the interphase cell cycle checkpoints according to the invention (see (1) Bhanu Priya, et al (supray, (2) Deng, Set al., (supray, (3) Vlatkovic, T et al., (supra) (4) Otto et al. (supra), and (5) Suski et al (supra)).

[0113]

[0111] In another embodiment, the modulator of an interphase cell cycle checkpoint, is the compound adavosertib (i.e. , modulator / inhibitor of the Wee1-like protein kinase).

[0114]

[0112] In another embodiment, the modulator of an interphase cell cycle checkpoint, is the compound zedoresertib (i.e., modulator / inhibitor of the Wee1-like protein kinase)

[0115]

[0113] In another embodiment, the modulator of an interphase cell cycle checkpoint, is the compound berzosertib.

[0114] In another embodiment, the modulator of an interphase cell cycle checkpoint, is the compound prexasertib.

[0116]

[0115] In another embodiment, the modulator of an interphase cell cycle checkpoint, is the compound rabusertib.

[0117]

[0116] In another embodiment, the modulator of an interphase cell cycle checkpoint, is the compound lunresertib.

[0118]

[0117] In another embodiment, the modulator of an interphase cell cycle checkpoint, is the compound tuvusertib.

[0119]

[0118] In another embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the M-phase cell cycle arresting compound is the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint is selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof.

[0120]

[0119] For the schedule of administration of the M-phase cell arresting compound and the modulator of the interphase cell cycle checkpoint prior to the delivery of the ionizing radiation, several options may be considered and that result in the progression of one or more cells in a tumor sample to the M-phase where the cells are then arrested (i.e., the cells preferably do not progress to a G1-phase).

[0121]

[0120] Therefore, in the following paragraphs particular embodiments of the schedule of administration are provided.

[0122]

[0121] In a particular embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint for use in the treatment of a tumor according to the invention, the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, , are, independently, administered to the subject at least 4 hours, preferably at least 6 hours, before delivering ionizing radiation to the subject, and / or preferably at most 48 hours before delivering ionizing radiation to the subject.

[0123]

[0122] In another embodiment of the M-phase cell cycle arresting compound or of the modulator of a cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of a cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, are, independently, administered to the subject 4 - 48 hours, 4 - 24 hours, 6 - 18 hours, 6 - 12 hours, or 6 - 8 hours before delivering ionizing radiation to the subject. It has been found that administering an M-phase cell arresting compound, preferably ABT-751 , and the modulator of the interphase cell cycle checkpoint, preferably adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, at least 4 hours, preferably at least 6 hours, for example 4 hours, 4,5 hours, 5, 5,5, 6, 6,5, 7, 7,5, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 30, 32, 36, 40, 42, 44, 48 hours or more before delivering ionizing radiation to the subject and / or preferably at most 48 hours before delivering ionizing radiation to tumor model cells lines or to a subject results in low percentage (e.g., below 10%) of cell surviving fraction, or in an improved treatment of tumors, such as brain tumors.

[0124]

[0123] In another embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, are, independently, administered simultaneously or sequentially, preferably sequentially, and within the range of hours as indicated for the previous embodiments, before delivering ionizing radiation to the subject. The order of the administering of the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint may be any one, meanwhile with the schedule of administration the treated cells progress to the M-phase by the effect of the modulator of an interphase cell cycle checkpoint and remain then M-phase arrested due to the effect of the M-phase cell cycle arresting compound.

[0125]

[0124] In an embodiment, the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, are administered simultaneously. When administered simultaneously, the doses of each of the compounds are adjusted in order to allow the cells to progress to the M-phase and remain then M-phase arrested. Advantageously, with this schedule an active dose of the M-phase cell cycle arresting compound can be administered at a time point prior to the administering of the ionizing radiation (e.g. 8 hours prior to RT), and an active dose of an interphase cell cycle checkpoint is administered at the same time point, which dose may be one that achieves pharmacologically active levels for a period of time within the time window between the time point prior to the administering of the ionizing radiation and the administering of the ionizing radiation but not necessarily for the whole time window.

[0126]

[0125] In a particular embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the treatment comprises administering the modulator of an interphase cell cycle checkpoint before the administering of M-phase cell cycle arresting compound. In another embodiment, the treatment comprises the administering of the M-phase cell cycle arresting compound before the administering of the modulator of an interphase cell cycle checkpoint.

[0127]

[0126] The skilled person will understand that when the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are sequentially administered, the time between the administering of a dose of each compound may be determined according to conventional techniques from the pharmacokinetic and pharmacodynamics of the compounds in order to allow the cells to progress to the M- phase and remain then M-phase arrested.

[0128]

[0127] Thus, in another embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, are sequentially administered to the subject at different times before delivering ionizing radiation to the subject.

[0129]

[0128] In another particular embodiment of the M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of a cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, are, independently, administered to the subject in two or more separate doses, and wherein there is at least 6 hours between the separate doses, preferably wherein there is 6 - 24 hours, more preferably 6 -12 hours between the separate dose. The separate dose is to be understood in this embodiment as two or more doses of the M-phase cell cycle arresting compound and as two or more doses of the modulator of an interphase cell cycle checkpoint, given apart within the indicated timing intervals, and before the delivery of the ionizing radiation.

[0130]

[0129] The administration of the M-phase cell cycle arresting compound and of the modulator of an interphase cell cycle checkpoint may be done simultaneously or sequentially, preferably sequentially, for example when the mode of administration differs between the compounds that are administered. By providing two or more separate doses, preferably wherein there is 4 - 48 hours, 4 - 24 hours, 6 - 24 hours, 6 -12 hours between the separate doses, the blood plasma level of either the M-phase cell cycle arresting compound, such as ABT-751 , or the blood plasma level of the modulator of the interphase cell cycle checkpoint, such as adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, can be sustained for a longer period at a level sufficient to cause mitotic enrichment prior to delivering the ionizing radiation to the subject.

[0131]

[0130] In another particular embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the treatment comprising administering the M- phase cell cycle arresting compound to the subject, preferably the compound ABT- 751 , administering the modulator of an interphase cell cycle checkpoint to the subject, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, and the delivering of ionizing radiation to the subject, is performed, depending on the tumor type, (in some embodiments at least; in some embodiments at most) once every two weeks, 1 time per week, 2 times per week, preferably 3, 4, or, most preferably, 5 times per week and / or wherein the total treatment is performed for or over a period of at least 1 week, preferably for at least 2 weeks, for 1 - 10 weeks, preferably for 1 - 6 weeks, preferably 6 weeks. In the case of brain tumors is preferably performed at least 2 times per week, preferably 3, 4, or, most preferably, 5 times per week and / or wherein the total treatment is performed for or over a period of at least 1 week, preferably for at least 2 weeks, for 1 - 10 weeks, preferably for 1 - 6 weeks, preferably 6 weeks. For example, in an embodiment of the invention, the treatment comprises a treatment cycle, wherein the treatment round comprises administering the M-phase cell cycle arresting compound and administering the modulator of an interphase cell cycle checkpoint to the subject, followed by delivering ionizing radiation as disclosed herein. In embodiments of the invention, the subject receives 1 , 2, 3, 4, or preferably 5, treatment cycles per week, and, in preferred embodiments, for a period of at least 1 week preferably for at least 2 weeks, for 1 - 10 weeks, preferably for 1 - 6 week. The skilled person will understand that variations in the treatment schedule, for example in the number of treatment cycles per week, or the total weeks of treatment, or the timing or (combination of) used compounds / modulators within a treatment cycle are allowable, for example, within the various embodiments and preferences as disclosed herein and / or, for example, in accordance with the practice in the treatment of a particular tumor or cancer.

[0132]

[0131] In another particular embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, between one or more subsequent treatments, said treatment comprising administering the M-phase cell cycle arresting compound to the subject, preferably the compound ABT-751 , administering the modulator of an interphase cell cycle checkpoint to the subject, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, and the delivering of ionizing radiation to the subject, there is a period of nontreatment, preferably wherein the non-treatment period is for 1 - 30 days, preferably 1 - 14 days, 1 - 7 days or 1 - 3 days.

[0133]

[0132] The treatment, for which the compounds and the ionizing radiation are used in according to the invention, is to be understood as a treatment cycle that comprises the administering of the M-phase cell cycle arresting compound to the subject, the administering of the modulator of an interphase cell cycle checkpoint to the subject, and subsequently the delivering of ionizing radiation to the subject (i.e. , wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject). Therefore, when in this description the times per week of the said treatment is referred to, is to be understood that each time a complete round including administering the M-phase cell cycle arresting compound to the subject, the administering of the modulator of an interphase cell cycle checkpoint to the subject, and the delivery of ionizing radiation is carried out. When in this description a particular period of non-treatment is referred to, it means that within that period no administering of the M-phase cell cycle arresting compound to the subject, no administering of the modulator of an interphase cell cycle checkpoint to the subject, and no delivery of ionizing radiation takes place, and preferably also not other chemotherapy. A period of non-treatment directly follows a period of treatment and precedes another period of treatment. A period of non-treatment is commonly referred to as a “drug holiday”, “medication vacation” or “drug vacation”. Said period is a strategic interruption of the treatment of certain cancers, such as a brain cancer. It is believed that a period of non-treatment allows recovery of the body of the subject. Said recovery enables a subsequent period of treatment of said subject. The skilled person is able to determine the length of the period of non-treatment based on the duration of the period of treatment preceding the period of non-treatment. The skilled person further may consider the subject’s age, weight, lifestyle, severity of disease condition, activity level and the like in determining the duration of the period of non-treatment.

[0134]

[0133] In an embodiment, the period of non-treatment is such that the amount or levels of the administered compounds determined in plasma or in another tissue / fluid sample of the subject, are those allowing the cells in a tumor previously submitted to a treatment cycle, to go through one or more rounds of mitosis. In other words, to allow the cells to naturally progress through the cell cycle from G1-phase to M-phase, and then to intervene in this progress by means of a subsequent treatment cycle according to the invention, increasing the number of cells sensitive to the administered ionizing radiation.

[0135]

[0134] In another particular embodiment of the M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the treatment that comprises administering the M- phase cell cycle arresting compound to the subject, preferably the compound ABT- 751 , administering the modulator of an interphase cell cycle checkpoint to the subject, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, and the delivery of ionizing radiation to the subject, is performed 1 time per week, 2 times per week, preferably 3, 4, or, most preferably, 5 times per week and / or wherein the total treatment is performed for or over a period of at least 1 week, preferably for at least 2 weeks, for 1 - 10 weeks, preferably for 1 - 6 weeks, preferably 6 weeks; and wherein between one or more subsequent treatments there is a period of non-treatment, preferably wherein the nontreatment period is for 1 - 30 days, preferably 1 - 14 days, 1 - 7 days or 1 - 3 days, preferably 2 days.

[0136]

[0135] In another embodiment when the ABT-751 is one of the M-phase cell cycle arresting compounds for use as indicated according to the invention, administering ABT-751 to a subject is in an amount of 50 mg - 400 mg, preferably 50 mg - 100 mg and / or wherein administering ABT-751 to the subject is performed 4 - 24 hours, preferably 6 - 18 hours, more preferably 6 - 8 hours before delivering ionizing radiation to the subject.

[0137]

[0136] In another particular embodiment of the M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the delivering of ionizing radiation (IR) to the subject is in an amount of 0.5 Gy - 20.0 Gy, 0.5 Gy - 15.0 Gy, 0.5 G-y - 10 Gy, or 1.5Gy - 2.8 Gy

[0138]

[0137] Commonly, the dose of ionizing radiation, for example used in radiation therapy is expressed in the unit “gray” (Gy). The skilled person knows that Gy stands for the absorption of one joule of radiation energy per kilogram of matter. For the treatment as provided herein ionizing radiation can be delivered by means of external radiation therapy, for example by external beam radiation therapy (EBRT), Intensity Modulated Radiation Therapy (IMRT), Image Guided Radiation Therapy (IGRT) and the like, or by means of internal RT, for example by brachytherapy. IR may be delivered in fractions, for example in at least 1 daily fraction, at least 2 daily fractions, at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more fractions, to the subject. A typical used schedule for glioblastoma is for example 2 Gy for 30 fractions, resulting in a (cumulative) dose of 60 Gy. IR may also be provided, for example, as a fraction once every other day, or once every two, three, or four days, and so on. The skilled person is aware that delivering ionizing radiation in multiple fractions per time period, for example per hour, per day or per week, relates to the term “hyperfractionation”. Hyperfractionation is known to the person skilled in the art as the delivery of a high number of low-dose fractions of ionizing radiation over a period of time, cumulating over time to a high dose of ionizing radiation, for example of at least 50 Gy, 60 Gy, 70 Gy, 80 Gy, 90 Gy. Present invention does not in particular relate to the method used for delivering IR. The method for delivering ionizing radiation may be determined by, for example, the attending physician.

[0139]

[0138] In one preferred embodiment when the ABT-751 is one of the M-phase cell cycle arresting compounds for use as indicated according to the invention, administering ABT-751 to a subject is in an amount of 50 mg - 400 mg, preferably 50 mg - 100 mg and / or wherein delivering ionizing radiation to said subject is in an amount of 0.5 Gy - 20.0 Gy, 0.5 Gy - 15.0 Gy, 0.5 Gy - 10 Gy, 1 Gy - 4 Gy, or 1.5 - 2.8 Gy, and / or wherein administering ABT-751 to the subject is performed 4 - 48 hours, 4 - 24 hours, 6 - 18 hours, or 6 - 8 hours before delivering ionizing radiation to the subject. For example, in such embodiment, the subject is first provided with, for example, 100 mg ABT-751 , and, for example, 7 hours after the patient was provided with 100 mg ABT-751 , the patient is provided with, for example, 2.0 Gy ionizing radiation. After the subject has been treated with ionizing radiation, for example, 2, 4, 6, 10, 12, 16, 24, 48, or 72 hours after the subject has been treated with ionizing radiation, a next dose of ABT-751 (e.g., 100 mg) may be provided to the subject. In other words, between two treatments with ionizing radiation, the subject is treated with at least one dose ABT-751. The treatment (for example, ABT-751 and a modulator of a cell cycle checkpoint followed by ionizing radiation, as described herein), may, in preferred embodiments, be repeated, for example daily, for example every other day, or, for example for 4, 5 or 6 days in a week during a period of 4, 5, 6, 7 or more weeks.

[0140]

[0139] As provided herein mg refers to milligrams of active pharmaceutical agent. The skilled person is able to calculate the exact amount of pharmaceutical active agent comprised in a pharmaceutical composition that can be administered to said subject as is provided herein. The amount of ABT-751 , as disclosed herein, is a dose of ABT-751 , as defined herein, that is administered to the subject having a tumor, preferably a brain tumor. A dose of a compound, such as ABT-751 , comprises any suitable pharmaceutical composition of the compound, such as ABT-751 , for administration to a subject. The amount to be administered to a subject should in the end be determined by the skilled person treating said subject.

[0141]

[0140] In a further embodiment the present invention provides ABT-751 for use in the treatment as disclosed, optionally together with one or more M-phase cell cycle arresting compounds, and together with the one or more modulators of the interphase cell cycle checkpoint, wherein administering ABT-751 to the subject is performed by administering ABT-751 in two or more separate (sub)doses in an amount of 50 mg - 400 mg, preferably 50 mg - 100 mg; and / or delivering ionizing radiation to the subject is in an amount of 0.5 Gy - 20.0 Gy, 0.5 Gy - 15.0 Gy, 0.5 Gy - 10 Gy, 1 Gy - 4 Gy, or 1.5 - 2.8 Gy, wherein administering the final dose, for example the second dose, third, fourth, fifth, nthetc., of ABT-751 to the subject is performed 0 - 48 hours, 2 - 48 hours, 4 - 48 hours, preferably 4 - 24 hours, 6 - 18 hours, or 6 - 8 hours before delivering ionizing radiation to the subject.

[0142]

[0141] With the term “final dose”, referred to any compound for use as indicated according to the invention, is meant to indicate the dose of that compound, as for example ABT-751 , that is the last dose provided before the ionizing radiation is delivered to the subject. For example, if two doses of a compound are administered prior to the delivery of the ionizing radiation, a first dose of 50 mg and a second dose of 75 mg, the final dose prior to the administration of the ionizing radiation is the dose of 75 mg.

[0143]

[0142] The combination of the previously indicated compounds and the radiation with other (bio)pharmaceutical agent is also encompassed in the invention. Thus, in another particular embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, the treatment further comprises administering a further (bio)pharmaceutical agent to the subject.

[0144]

[0143] In a preferred embodiment, the further (bio)pharmaceutical agent is selected from one or more of the previously listed “chemotherapeutic agents” or “chemotherapeutic drugs”, namely alkylating agents, preferably selected from one or more of altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxaliplatin, temozolomide, and thiotepa; antimetabolite agents, preferably selected from one or more of 5- fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda), cytarabine (Ara- C), floxuridine, fludarabine, gemcitabine (Gemzar), and hydroxycarbamide; topoisomerase inhibitors type I and II, preferably of type I one or more of irinotecan, topotecan, camptothecin and lamellarin D, and of type II one or more of etoposide (VP- 16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, and HU-331 ; and cytotoxic antibiotic agents, preferably selected from one or more of dactinomycin, bleomycin, and daunorubicin.

[0144] In another preferred embodiment, the further (bio)pharmaceutical agent is selected from a compound or composition for use in immunotherapy, such as in cell immunotherapy, comprising preferably one or more of chimeric antigen receptor T-cell therapy, tumor-infiltrating Lymphocyte (TIL) Therapy, Engineered T Cell Receptor (TCR) Therapy, natural killer cell therapy, and dendritic cell therapy.

[0145]

[0145] In another embodiment, the further (bio)pharmaceutical agent is an antibody, wherein said antibody is preferably a monoclonal antibody, for example bevacizumab. Bevacizumab, known under tradename Avastin®, is a widely used monoclonal antibody that binds to circulating VEGF-A and inhibits its biological activity by preventing the interaction with the VEGF receptor.

[0146]

[0146] The M-phase cell cycle arresting compound and the modulator of a cell cycle checkpoint for use according to the invention, are for use in the treatment of any kinds of tumors.

[0147]

[0147] In a particular embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, preferably a modulator of the S- phase or G2-phase cell cycle checkpoint, for use according to the invention, the tumor is selected from the group consisting of: a brain tumor, a pulmonary tumor, a breast tumor, a genitourinary tract tumor, a bone tumor, a skin tumor, a head tumor, a neck tumor, a meroblastic tumor, a gastrointestinal tumor, a colorectal tumor, a pancreatic tumor, an hematopoietic tumor and lymphoid tissue tumor, a soft tissue sarcoma, preferably a fat tissue sarcoma and / or a muscle tissue sarcoma. In a more preferred embodiment is a brain tumor.

[0148]

[0148] Within the brain tumors, the M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, preferably a modulator of the S- phase or G2-phase cell cycle checkpoint, are for use in the treatment of a primary brain tumor selected from the group consisting of astrocytomas, meningiomas, oligodendrogliomas, medulloblastomas, ependymomas, craniopharyngiomas, gliomas, central nervous system lymphomas, chordomas, pineoblastomas and schwannomas, preferably wherein the primary brain tumor is an astrocytoma, even more preferably wherein the astrocytoma is a glioblastoma selected from the group consisting of primary glioblastoma, recurrent glioblastoma, glioblastoma with hypermethylation of the promoter of the gene O6-Methylguanin-Methyltransferase (MGMT), glioblastoma without hypermethylation of the promoter of MGM

[0149] Alternatively, the M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, preferably a modulator of the S-phase or G2-phase cell cycle checkpoint, are for use in the treatment of a secondary brain tumor, which secondary brain tumor is a tumor that has formed in the brain as a result of metastasis, preferably metastasis from a different area in the subject’s body selected from the group consisting of pulmonary tumor, breast tumor, genitourinary tract tumor, bone tumor, skin tumor, head tumor, neck tumor, meroblastic tumor, gastrointestinal tumor, colorectal tumor, pancreatic tumor, hematopoietic tumor and lymphoid tissue tumor, preferably wherein the secondary brain tumor is a pulmonary tumor, breast tumor or skin tumor.

[0149]

[0150] In another embodiment of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, preferably a modulator of the S- phase or G2-phase cell cycle checkpoint, for use according to the invention, the tumor is, independently of its etiology, selected from a tumor located in the brain, a tumor located in the lungs, a tumor located in the breast, a tumor located in the genitourinary tract, a tumor located in bone system, a tumor located in the skin, a tumor located in the head, a tumor located in the neck, a tumor located in the gastrointestinal system, a tumor located in the colon and / or rectum, a tumor located in the pancreas, a tumor located in the hematopoietic and / or lymphoid tissue, preferably is a tumor located in the brain, either primary or secondary. In some embodiments, the tumor is a tumor that is located (localized) behind a blood-brain-barrier, i.e. , located within a region of the (human) body (normally) surrounded by the blood-brain-barrier (Daneman et al. Cold Spring Harb Perspect Biol. 2015 Jan; 7(1): a020412 doi:

[0150] 10.1101 / cshperspect.a020412.).

[0151]

[0151] Because radiotherapy constitutes the backbone of sarcoma care without many alternative options, soft tissue sarcomas, and preferably fat tissue sarcomas and / or muscle tissue sarcomas, are also of interest and a particular target of the invention. Current standard of care radiotherapy regimens, as the ones indicated in this description, are compatible with this kind of tumor. Often sarcomas are localized in the extremities or in easily accessible zones, which allows the targeting of only the tumor (not the surrounding tissues). This accessibility provides also the advantage of an easy taken of biopsies pre and / or post administration of the M-phase cell cycle arresting compound, the modulator of an interphase cell cycle checkpoint, and the ionizing radiation according to the invention. Hence the determining or analysis of the therapy efficacy is also easy, as well as the performance of any companion diagnostics.

[0152]

[0152] Drug combinations

[0153]

[0153] According to an aspect of the invention there is also provided for a combination, namely a drug combination, comprising:

[0154] (a) a M-phase cell arresting compound, preferably a pharmaceutical acceptable M- phase cell arresting compound, preferably the compound ABT-751 ; and (b) a modulator of an interphase cell cycle checkpoint, preferably a pharmaceutical acceptable modulator of an interphase cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof; wherein (a) and (b) are comprised in a single pharmaceutical composition or, alternatively, (a) and (b) are comprised in separate pharmaceutical compositions.

[0155]

[0154] All the embodiments previously disclosed for the M-phase cell cycle arresting compound or for the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention, are to be considered as possible encompassed embodiments of this combination (i.e., drug combination). Thus, for example, the particular M-phase cell cycle arresting compounds, as well as the particular modulators of an interphase cell cycle checkpoint listed above do also apply to the combination of the invention.

[0156]

[0155] In a preferred embodiment of the combination of the invention, optionally in combination with any of the embodiments above or below, the combination comprises: (a) a M-phase cell arresting compound which is the ABT-751 compound, and / or a pharmaceutically acceptable salt, ester or ether thereof; and (b) a modulator of an interphase cell cycle checkpoint, which modulator is selected from a modulator of Wee1-like protein kinase, preferably the inhibitor of Wee1-like protein kinase known as adavosertib (AZD1775) or a pharmaceutically acceptable salt, ester or ether thereof or the inhibitor of Wee1-like protein kinase known as Debio123 or as zedoresertib, or a pharmaceutically acceptable salt, ester or ether thereof; and / or a modulator of ATR kinase, preferably the inhibitor of ATR kinase selected from berzosertib, tuvusertib, and combinations thereof, or a pharmaceutically acceptable salt, ester or ether thereof; and / or a modulator of CHK1 selected from rabusertib, prexasertib, and combinations thereof or pharmaceutically acceptable salts, esters or ethers thereof; and / or a modulator of a of Myt1 (PKMYT1) kinase, preferably the inhibitor of Myt1 kinase known as lunresertib, or a pharmaceutically acceptable salt, ester or ether thereof.

[0157]

[0156] In an embodiment of the combination of the invention, (a) and (b) are for use in the treatment of a tumor in a subject, and wherein the treatment comprises the administering of the active compounds (a) and (b) simultaneously or sequentially, preferably sequentially.

[0158]

[0157] Sequential administration is in particular preferred when the mode of administration differs between the compounds that are administered, and / or when the timing of administration of (a) and (b) in relation to each other, or in relation to the further delivery of the ionizing radiation is to be schedule at different points of time to allow the cells to progress to the M-phase and remain M-phase arrested.

[0159]

[0158] In an embodiment of the combination of the invention, the combination is a kit of parts that comprises as separate pharmaceutical compositions, preferably contained or embedded in the same packaging (e.g., package): (a) a M-phase cell arresting compound, preferably the compound ABT-751 ; and (b) a modulator of an interphase cell cycle checkpoint, preferably selected from one or more of adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib; and (c) optionally instructions for the use of the kit, preferably instructions for the administering of the separate pharmaceutical compositions that comprise (a) and (b).

[0160]

[0159] The kit of parts that comprises the compounds (a) and (b) as separate pharmaceutical compositions implies the advantage of providing all the compounds required for the treatment cycle that will also comprise the subsequent delivery of ionizing radiation.

[0161]

[0160] Moreover, the kit of parts provides for the compounds for use according to the invention (i.e. , for the treatment of a tumor), in such a way that the administering of the active compounds (a) and (b) may be done simultaneously or sequentially, preferably sequentially, as previously disclosed.

[0162]

[0161] As the skilled person will deduce, the pharmaceutical compositions comprise the therapeutically effective amounts of the compounds (a or b), and they may comprise one or more pharmaceutically acceptable carriers or excipients. Examples of excipients or carriers comprise, for example, accelerators, anti-adherents, binders, coatings, colors, diluents, disintegrates, emulsifiers, flavors, humectants, glidants, lubricants, preservatives, sorbents, sterilizing agents, sweeteners, solubilizers, vehicles, mixtures thereof and the like. Administration by the oral route of administration is preferred by subjects due to the low to absent invasiveness of the therapy compared to for example intravenous administration.

[0163]

[0162] It is contemplated that a therapeutically-effective amount of either the M-phase cell cycle arresting compound, preferably ABT-751 , or of the modulator of an interphase cell cycle checkpoint, preferably selected from one or more of adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, in the present invention depends on the recipient of the treatment, the type of cancer being treated, and the severity of the same. Further it is contemplated that said therapeutically-effective amount may depend on compositions containing the therapeutically-effective amounts, the route of administration, the duration of the treatment, the potency of therapeutically-effective amounts, the rate of clearance and / or whether the M-phase cell cycle arresting compound, preferably ABT-751 , or the modulator of an interphase cell cycle checkpoint, preferably selected from one or more of adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and a combination thereof, are co-administered with another (bio)pharmaceutical agent as the ones listed in the embodiments of the M-phase cell cycle arresting compound or of the modulator of an interphase cell cycle checkpoint, for use in the treatment of a tumor according to the invention.

[0164]

[0163] The pharmaceutical compositions in the combination, as the skilled person will also deduce may be for per enteral administration, for example oral administration, or for parenteral administration, for example intravenous (into a vein) administration. Preferably, when ABT-751 is used as M-phase cell cycle arresting compound, it is administered per oral administration.

[0165]

[0164] The pharmaceutical compositions in the combination of the invention are, in an embodiment, formulations selected from the group consisting of solid formulations, such as a pill, a tablet, granules, capsules, or liquid formulations, such as solutions or suspensions. Liquid formulations are for oral administration or for parenteral (e.g., injectable).

[0166]

[0165] In another embodiment of the combination of the invention, (a) and (b) are the active ingredients of a single pharmaceutical composition. This embodiment is applicable when both active principles are to be administered by the same route of administration, and when the simultaneous administration of the active ingredients will assure the cells will be drove to and arrested in the M-phase cell cycle for the ionizing radiation to be the most efficient when delivered.

[0167]

[0166] The skilled person in the art, will know how to provide in a single composition two or more ingredients that are delivered sequentially. For example, one of the ingredients may be provided encapsulated in a matrix that will release the same in a retarded mode, in relation to the other ingredient which may not be encapsulated.

[0168]

[0167] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0169]

[0168] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[0170]

[0169] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0171]

[0170] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect or embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspects separately.

[0172]

[0171] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art. EXAMPLES

[0173] Example 1

[0174] M-phase cell cycle arresting compounds and modulators of an interphase cell cycle checkpoint for use in the treatment of a tumor. Drug combinations.

[0175]

[0172] The materials and methods and other experimental settings for the performance of the assays in the examples are listed below.

[0176]

[0173] The methods and results of the examples are also disclosed in the section of the Figures.

[0177]

[0174] Next-generation Mitotic Enrichment using ABT-751 in combination with the Wee1 inhibition (adavosertib). (Figure 2).

[0178]

[0175] Using ABT-751 in combination with the Wee1 inhibition (adavosertib) achieved superior mitotic enrichment and radiosensitization, compared to the already effective administering of ABT-751 alone, in colorectal (HCT-116) and brain cancer cells (U251 , glioblastoma (serum-cultured); U3086MG classical glioblastoma; VUmc-ATRT-o1 SHH subtype AT / RT; VUmc-HGG-15, BRAF mutant pHGG) (See Figure 2). The Wee1 inhibitor adavosertib dose-dependently increased the percentage of mitotic cells (DNA 4N, pHH3+, four column in the sets showed for each dose in the bar graphics) in combination with ABT-751. G1 cells (DNA 2N, pHH3-, first column in the sets showed for each dose in the bar graphics) were already severely depleted by ABT-751 alone and S phase cells (DNA 2N-4N, pHH3-, second column in the sets showed for each dose in the bar graphics) stayed unaffected. However, in line with its mode of action to inhibit the G2 checkpoint, addition of adavosertib to ABT-751 also reduced the amount of G2 cells (DNA 4N, pHH3-, third column in the sets showed for each dose in the bar graphics). These effects synergistically co-operated and resulted in a superior mitotic enrichment compared to using ABT-751 alone. ***p < 0.001 , ****p < 0.0001 (Extra sum-of-squares F test)

[0179]

[0176] Next-generation Mitotic Enrichment using ABT-751 in combination with the ATR inhibition achieves superior mitotic enrichment and radiosensitization (Figure 3).

[0180]

[0177] Using ABT-751 in combination with the ATR inhibition achieves superior mitotic enrichment and radiosensitization, compared to the already effective administering of ABT-751 alone, in colorectal (HCT-116) and brain cancer cells (U251 , glioblastoma (serum-cultured); U3086MG classical glioblastoma; VUmc-ATRT-o1 SHH subtype AT / RT; VUmc-HGG-15, BRAF mutant pHGG). The ATR inhibitor berzosertib dose- dependently increased the percentage of mitotic cells (DNA 4N, pHH3+; four column in the sets showed for each dose in the bar graphics) in combination with ABT-751. G1 cells (DNA 2N, pHH3-, first column in the sets showed for each dose in the bar graphics) were already severely depleted by ABT-751 alone and were unaffected by berzosertib. However, in line with its mode of action to inhibit the intra-S and S-G2 checkpoints, addition of berzosertib to ABT-751 also reduces the combined amount of S phase cells (DNA 2N-4N, pHH3-, second column in the sets showed for each dose in the bar graphics) and G2 cells (DNA 4N, pHH3-, third column in the sets showed for each dose in the bar graphics). These effects synergistically co-operated and resulted in superior mitotic enrichment compared to using ABT-751 alone. ***p < 0.001 , ****p < 0.0001 (Extra sum-of-squares F test).

[0181]

[0178] Superior in vitro radiosensitization by next-generation Mitotic Enrichment using ABT-751 in combination with the Wee1 inhibitor adavosertib compared to first- generation Mitotic Enrichment using ABT-751 alone. (Figure 5).

[0182]

[0179] First-generation Mitotic Enrichment using ABT-751 alone radiosensitized 11251 glioblastoma cells (left panel; see also Figure 2 data with 11251 glioblastoma cells). However, next-generation Mitotic Enrichment using addition of 1 pM adavosertib resulted in a synergic superior radiosensitization of 11251 glioblastoma cells compared to ABT-751 alone, in line with the superior mitotic enrichment achieved by this combination (right panel; see also see also Figure 2 data with 11251 glioblastoma cells). Importantly, adavosertib did not radiosensitize 11251 cells by itself in a Mitotic Enrichment setup. ***p < 0.001 , ****p < 0.0001 (Extra sum-of-squares F test).

[0183]

[0180] Next-generation Mitotic Enrichment using, ABT-751 , in combination with CHK1 inhibition, by rabusertib or prexasertib achieves superior mitotic enrichment (Figure 4)

[0184]

[0181] Flow cytometric cell cycle analysis using propidium iodide (PI; DNA staining) and phospho-histone H3 staining (pHH3; mitotic marker) demonstrates superior induction of mitotic enrichment by combining ABT-751 with a CHK1 inhibitor compared to ABT-751 alone in brain cancer cells (11251 , glioblastoma (serum-cultured)). The CHK1 inhibitors rabusertib and prexasertib dose-dependently increase the percentage of mitotic cells (DNA 4N, pHH3+; four column in the sets showed for each dose in the bar graphics) in combination with ABT-751. G1 cells (DNA 2N, pHH3-, first column in the sets showed for each dose in the bar graphics) are already severely depleted by ABT-751 alone and are unaffected by rabusertib or prexasertib. However, in line with its mode of action to inhibit the G2-M checkpoint, addition of rabusertib or prexasertib to ABT-751 also reduces the amount of G2 phase cells (DNA 4N, pHH3-, third column in the sets showed for each dose in the bar graphics). These combined effects result in superior mitotic enrichment compared to using ABT-751 alone.

[0185]

[0182] Enforcing a G2 checkpoint arrest by CDK1 inhibition by RO-3306 (CAS No. : 872573-93-8), prevents mitotic enrichment by ABT-751 (Figure 6).

[0186]

[0183] Flow cytometric cell cycle analysis using propidium iodide (PI; DNA staining) and phospho-histone H3 staining (pHH3; mitotic marker) demonstrates loss of mitotic enrichment by combining ABT-751 with a CDK1 inhibitor compared to ABT-751 alone in brain cancer (11251 , glioblastoma (serum-cultured), melanoma (Mel57) and cervical cancer cells (HeLa). The CDK1 inhibitor RO-3306 dose-dependently decreases the percentage of mitotic cells (DNA 4N, pHH3+; lines with solid capsized triangle points) in combination with ABT-751. G1 cells (DNA 2N, pHH3-, lines with solid circle points) are depleted by ABT-751 alone but recover by combined inhibition of CDK1. The amount of S phase cells (DNA 2N-4N, pHH3-, lines with solid square points) and G2 phase cells (DNA 4N, pHH3-, lines with solid upside triangles) remained largely unaffected. These combined effects result in loss of mitotic enrichment compared to using ABT-751 alone.

[0187]

[0184] Loss of in vitro radiosensitization by using ABT-751 in combination with the CDK1 inhibitor RO-3306 compared to first-generation Mitotic Enrichment using ABT- 751 alone (Figure 7).

[0188]

[0185] First-generation Mitotic Enrichment using ABT-751 alone radiosensitized 11251 glioblastoma cells (line with solid square points vs. line with solid circle points). However, combining ABT-751 with RO-3306 abrogated radiosensitization of 11251 glioblastoma cells compared to ABT-751 alone (line with empty circle points), in line with the loss of mitotic enrichment achieved by this combination (see also Figure 6). Importantly, RO-3306 did not radiosensitize 11251 cells by itself in a Mitotic Enrichment setup (line with solid triangles). Together, these data underline the importance of inhibiting interphase checkpoints and not enforcing them to achieve radiosensitization through Mitotic Enrichment. ***p < 0.001 , ****p < 0.0001 (Extra sum-of-squares F test).

[0189]

[0186] Materials and method for the Examples

[0190]

[0187] Drugs

[0191]

[0188] ABT-751 , berzosertib and prexasertib were purchased from MedKoo Biosciences, Inc (Research Triangle Park, NC), adavosertib and rabusertib from MedChemExpress (Monmouth Junction, NJ) and RO-3306 from Selleck Chemicals (Houston, TX). All drugs were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO).

[0192]

[0189] Cell culture

[0193]

[0190] The human glioma cell line 11251 was purchased from ATCC (Manassas, VA). The human colon cancer cell line HCT116 and the human cervical epithelial adenocarcinoma cell line HeLa were both kindly provided by Prof. Rene Medema (Netherlands Cancer Institute, Amsterdam, the Netherlands). The human melanoma cell line Mel57 was kindly provided by Dr. William Leenders (Radboud lime, Nijmegen, the Netherlands). All these lines were cultured in medium composed of MEM supplemented with 10% FBS, 1% L-Glutamine, 1% MEM-Vitamins, 1 % Non-Essential Amino Acids , 1 % Sodium pyruvate, and 1 % penicillin G / streptomycin (all obtained from Life Technologies, Carlsbad, CA) using standard conditions (37°C and 5% CO2).

[0194]

[0191] The human glioma stem cell line U3086MG was obtained from the Human Glioma Cell Culture repository (Xie et al., EBiomedicine, 2015). It was cultured in Neurobasal-A medium and DMEM / F-12 GlutaMAX (1 :1) supplemented with 1 * B27 (w / o vitamin A) supplement and 1 % penicillin / streptomycin (All from Life Technologies, Carlsbad, CA), 10 ng / mL recombinant human EGF, 10 ng / mL recombinant human FGF2 (both from Peprotech, Rocky Hill, NJ).

[0195]

[0192] The BRAF mutant pediatric high-grade glioma stem cell line VUmc-HGG-14 and the SHH subtype AT / RT line VUmc-ATRT-01 were both kindly provided by Dr. Esther Hulleman (Prinses Maxima Center, Utrecht, The Netherlands). These cells were cultured in Neurobasal-A medium and DMEM / F-12 GlutaMAX (1 :1), 1 % HEPES (1M), 1% Non-Essential Amino Acids ,1 % sodium pyruvate, and 1 % penicillin / streptomycin. Culture medium was freshly supplemented with B27 (w / o vitamin A) (1 :50) (all from Life Technologies, Carlsbad, CA), 20 ng / mL recombinant human EGF, 20 ng / mL recombinant human FGF2, 10 ng / mL recombinant human platelet-derived growth factor-AA, 10 ng / mL recombinant human platelet-derived growth factor-BB (all from Peprotech, Rocky Hill, NJ), and Heparin (5 lE / mL).

[0196]

[0193] Flow cytometric cell cycle analysis assay.

[0197]

[0194] 5 x 104cells / well were seeded in 6 well-plates and left to recover and cycle for 48 hours, after which drug treatment was started for 6 hours. Cells were then washed twice, collected using Trypsin-EDTA (Life Technologies; for serum-cultured lines) or TrypLE Express (Gibco, Thermo Fisher Scientific, Waltham, MA; for neurobasal- cultured cells), centrifuged, resuspended in ice-cold phosphate-buffered saline (PBS; Life Technologies) and fixed using ice-cold 70% ethanol.

[0198]

[0195] The immuno-staining started by blocking with 2% bovine serum albumin fraction V (BSA; Sigma-Aldrich), 0.1 % NaN3 and 0.1% Triton X100 in PBS followed by incubation with primary antibody anti-pH3Ser10 (1 : 10,000 in blocking buffer) (06-570, Merck Millipore, Burlington, MA for 2h on a shaker (400 rpm) at room temperature. After incubation the excessive antibody was diluted and removed. Next, the swine- anti-rabbit-FITC (1 :200) (Dako, Glostrup, Denmark) was used as secondary antibody. The samples were incubated for 1 h on the shaker in the dark at 4°C. Finally, cells were washed twice and pelleted before being re-suspended in PBS containing RNAse (100 pg / mL) and propidium iodide (10 pg / mL). Samples were analyzed on the Attune NxT Flow Cytometer system (Thermo Fisher Scientific). The data was analyzed by FlowJo software (v10.6.2, TreeStar Inc., Ashland, OR). Differences in G1 , S and G2 / M cells were made by gating in the histogram plot. A dot-plot with the FITC (pH3) signal vs. the PI signal was made to discriminate the mitotic cells, which are pH3 positive, from the G2 cells (pH3 negative).

[0199]

[0196] Determination of the radiation-sensitivity using clonogenic assays.

[0200]

[0197] Clonogenic assays were carried out by seeding 200 - 500 cells / well in 6 wellplates. After 24h, the cells were treated with various drugs. After cells were exposed for 6h, they were irradiated using a Cesium source (Gammacell 40 EXACTOR, Best Theratronics, Ontario, Canada), followed by removing the drug and leaving the cells in drug-free medium for 18h. This 24h cycle was repeated for 4 subsequent days, after which colonies were left to grow out. When colonies were formed in the non-treated wells, cells were fixed, and colonies were analyzed using the ‘analyze particles’ feature of FIJI. Using plating efficiency, these results were normalized towards the number of seeded cells and the untreated controls.

[0201]

[0198] Statistics

[0202]

[0199] Curve fitting of the dose-dependent changes in cell cycle distribution was done using non-linear regression on semi-log data using GraphPad Prism 7 (Graphpad Software, Inc., San Diego, CA). Assessment of radiosensitization was determined using the extra sum-of-squares F test with ‘HO = one curve accurately fits all data’ and ‘Ha = two separate curves more accurately fit all data’. In this case rejection of HO led us to conclude that radiosensitization had occurred.

[0203] Example 2

[0204] Other M-phase cell cycle arresting compounds and modulators of an interphase cell cycle checkpoint for use in the treatment of a tumor. Drug combinations.

[0205]

[0200] Following the same methodology disclosed in Example 1 , other experimental set-ups were conducted by the inventors in which several M-phase cell cycle arresting compounds, some in combination with modulators of an interphase cell cycle checkpoint, were tested.

[0206]

[0201] The set-ups and results are as summarized in the legends of the corresponding Figures 8 to 13, and are detailed also in the following.

[0207]

[0202] In Figure 8 it is shown that ABT-751 , Vincristine, Compound-20, and SB- 743921 were able to significantly increase the mitotic fraction of U251 cells without inducing cytotoxicity. (A) Effect of ABT-751 (5 pM), Vincristine (4 nM), Compound-20 (10 pM) and SB-743921 (1 nM) on cell survival after various exposure times. The dotted line indicates the treatment duration in (B). No cytotoxic effects were observed for any of these drugs until after 8 hours of exposure. (B) FACS cell cycle analysis using propidium iodide and phospho-histone H3 staining demonstrates effective induction of mitotic enrichment at non-cytotoxic conditions by all compounds tested.

[0208]

[0203] Although data not shown, short-term exposure to compounds ABT-751 , Vincristine, Compound-20 and SB-743921 , that induce mitotic enrichment doesn’t induce permanent chromosomal aberrations. After 8 hours of exposure, removal of ABT-751 , Vincristine, Compound-20 and SB-743921 from 11251 cells normalized the cell cycle distribution as observed from an assay with by propidium iodide (PI) profiles and mitotic fraction as indicated by the relative number of phospho-histone H3 positive cells to baseline levels within 16 hours after washout. After normalization, both cell cycle indicators remained stable for at least 24 more hours.

[0209]

[0204] Further it was observed in a by-side assay (data not shown), that the mitotic enrichment of 11251 cells by Compound-20 and SB-743921 was attenuated by prior ionizing radiation, since ionizing radiation caused a G2 cell cycle arrest that attenuated and delayed induction of mitotic enrichment by Compound-20 or SB-743921.

[0210]

[0205] Figure 9 is an assay to show that mitotic enrichment radiosensitizes 11251 cells to fractionated radiation. In (A) Schematic depiction of the radiosensitization schedule used. (B) Compound-20 and ABT-751 considerably radiosensitized 11251 cells to the schedule depicted in (A).

[0211]

[0206] In Figure 10 is shown that irradiation prior to exposure to mitotic enrichment inducers (M-phase cell cycle arresting compounds) prevents radiosensitization. From (A) Schematic depiction of the radiosensitization schedule used. From (B) 11251 cells could not be radiosensitized by ABT-751 or Compound-20 when ionizing radiation was given prior to exposure to the compounds.

[0212]

[0207] Figure 11 shows for the M-phase cell cycle arresting compound SB-743921 , brain penetration and plasma pharmacokinetics in WT and ABC transporter knockout mice. In (A) Brain penetration and plasma concentrations of wildtype and transporter knockout mice at 8 hours after receiving i.p. SB-743921. SB-743921 reaches concentrations in the brain of WT mice up to 8 hours after i.p. administration that are sufficient to induce mitotic enrichment in vitro, although it appears to be a substrate for Abcbl as indicated by higher brain penetration in Abcb1a / b- / - mice that did not increase further in Abcg2;Abcb1a / b- / - mice. In (B) Plasma pharmacokinetics curves of the mice from (A). These plasma concentration time curves are in line with those in patients receiving clinically acceptable dose levels (Holen KD et al. Cancer Chemother Pharmacol. 2011. 67(2):447-54. doi: 10.1007 / s00280-010-1346-5).

[0213]

[0208] Mitotic enrichment of E98 tumors was observed using phospho-histone H3 (pHH3) and haematoxylin and eosin (H&E) immunohistochemistry stainings of brain tissue of SB-743921 treated E98 tumors grafted in wildtype athymic FVB nude (FVB / nu) or Abcg2;Abcb1a / b- / - nude (Bab / nu) mice, in relation to controls (mice not treated with SB-743921) (Data dot shown).

[0214]

[0209] Mitotic enrichment of GBM652457 tumors was also observed using phosphohistone H3 (pHH3) and haematoxylin and eosin (H&E) immunohistochemistry stainings of brain tissue of SB-743921 treated GBM652457 tumor grafted in a wildtype athymic nude mouse in relation to controls (non-treated mice) (Data dot shown).

[0215]

[0210] The tests in vivo demonstrate that the M-phase cell cycle arresting compound SB-743921 , intraperitoneally administered, was able to penetrate the brain at amounts to induce M-phase cell cycle arrest.

[0216]

[0211] In Figure 12 the results of an assay with the Eg5 / KIF11 / KSP inhibitors filanesib and litronesib and the CENP-E inhibitors GSK923295 and PF-2771 are shown. These compounds were able to significantly increase the mitotic fraction of cancer cells without inducing cytotoxicity. In (A) effect of filanesib (10 nM), litronesib (10 nM), and GSK-923295 (100 nM) on 11251 cell survival after various exposure times. No cytotoxic effects were observed for any of these drugs until after 8 hours of exposure. In (B) FACS cell cycle analysis using propidium iodide and phospho-histone H3 staining demonstrates effective induction of mitotic enrichment of various cancer cells after 6 hours at non-cytotoxic conditions by all compounds tested.

[0217]

[0212] Short-term exposure to compounds filanesib, litronesib or GSK-923295 that induced mitotic enrichment did not induce permanent chromosomal aberrations, after 8 hours of exposure, and removal of the compounds (filanesib, litronesib or GSK- 923295) from U251 , the cells normalized the cell cycle distribution as indicated by propidium iodide (PI) profiles and mitotic fraction as indicated by the relative number of phospho-histone H3 positive cells to baseline levels within 16 (litronesib, GSK- 923295) or 40 hours (filanesib) after washout (data not shown).

[0218]

[0213] The data from all these set-ups provides evidence that several M-phase cell cycle arresting compounds, other than the compound ABT-751 tested in Example 1 do also enrich mitotic phase (Figures 8 and 12), and improve radiosensitization of the arrested cells in several tumor cell lines (Figures 9 and 10).

[0219]

[0214] The inventors also conducted assays to test ABT-751 in combination with Wee1 / Myt1 inhibitors. In Figure 13 these combinations are illustrated. The mitotic enrichment using ABT-751 in combination with Wee1 / Myt1 inhibitors achieves superior Mitotic Enrichment and radiosensitization. Flow cytometric cell cycle analysis using propidium iodide (PI; DNA staining) and phospho-histone H3 staining (pHH3; mitotic marker) demonstrates superior induction of mitotic enrichment by combining ABT-751 with a Wee1 inhibitor or Myt1 inhibitor compared to ABT-751 alone in (A) 11251 and (B) LN-751 brain cancer cells.; *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 (Two- way ANOVA with Dunnett’s multiple comparisons post-hoc test compared to ABT-751 alone). In (C) is depicted the superior in vitro radiosensitization using ABT-751 in combination with the Wee1 inhibitor Debio123 (i.e. , zedoresertib) compared to using ABT-751 alone. First-generation Mitotic Enrichment using ABT-751 alone radiosensitized 11251 glioblastoma cells. However, next-generation Mitotic Enrichment using addition of 1 pM Debio123 resulted in superior radiosensitization of 11251 glioblastoma cells compared to ABT-751 alone, in line with the superior mitotic enrichment achieved by this combination (see (A-B)). Importantly, Debio123 did not radiosensitize 11251 cells by itself in a Mitotic Enrichment setup. **p < 0.01 , ****p < 0.0001 (Extra sum-of-squares F test)

[0220]

[0215] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[0221]

[0216] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

Claims

CLAIMS1. A mitotic-phase (M-phase) cell cycle arresting compound for use in the treatment of a tumor in a subject, wherein the treatment comprises administering the M-phase cell cycle arresting compound to the subject, administering a modulator of an interphase cell cycle checkpoint to the subject, and delivering ionizing radiation to the subject; and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

2. A modulator of an interphase cell cycle checkpoint for use in the treatment of a tumor in a subject, wherein the treatment comprises administering an M- phase cell cycle arresting compound to the subject, administering the modulator of an interphase cell cycle checkpoint to the subject and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

3. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound is selected from the group consisting of a microtubule-stabilizing agent, a microtubule-destabilizing agent, and combinations thereof.

4. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound is a microtubule-destabilizing agent selected from the group consisting of a combretastatin, preferably the compound ABT-751 (N-[2-(4-hydroxyanilino)-3-pyridinyl]-4- methoxybenzenesulfonamide) or the tubulin inhibitors eribulin, lisavanbulin and rigosertib; and kinesin inhibitors.

5. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound is a microtubule-destabilizing agent selected from the group consisting of a combretastatin, preferably the compound ABT-751 (N-[2-(4-hydroxyanilino)-3-pyridinyl]-4- methoxybenzenesulfonamide), and kinesin inhibitors.

6. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound is ABT-751.

7. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound is a kinesin inhibitor selected from N-[4’-(T rifluoromethyl)-4-biphenylyl]sulfamide, ispinesib (N-(3-Aminopropyl)-N- [(1 R)-1-[7-chloro-3,4-dihydro-4-oxo-3-(phenylmethyl)-2-quinazolinyl]-2- methylpropyl]-4-methylbenzamide), filanesib, litronesib, SB-743921 (CAS No. : 940929-33-9), ARQ621 (CAS No. : 1095253-39-6), MK-0731 (CAS No. : 845256-65-7), and N-[4’-(Trifluoromethyl)-4-biphenylyl]sulfamide8. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound is a microtubule-stabilizing agent selected from the group consisting of a taxane, an epothilone, and combinations thereof.

9. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the modulator of an interphase cell cycle checkpoint is selected from the group consisting of a modulator of a G1-phase cell cycle checkpoint, a modulator of an S-phase cell cycle checkpoint, a modulator of a G2-phase cell cycle checkpoint, and combinations thereof, preferably is a modulator of an S-phase cell cycle checkpoint, and / or a modulator of a G2-phase cell cycle checkpoint.

10. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the modulator of an interphase cell cycle checkpoint is a modulator of an S- phase cycle cell checkpoint selected from the group consisting of a modulator of Ataxia telangiectasia mutated (ATM) kinase, a modulator of Ataxia Telangiectasia and Rad3 related (ATR) kinase, and combinations thereof.

11. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the modulator of an interphase cell cycle checkpoint is a modulator of Ataxia Telangiectasia and Rad3 related (ATR) kinase, preferably selected from the compound berzosertib, the compound tuvusertib, and combinations thereof.

12. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the modulator of an interphase cell cycle checkpoint is a modulator of a G2- phase cycle cell checkpoint selected from the group consisting of a modulator of Checkpoint kinase 1 , a modulator of Checkpoint kinase 2, a modulator of Wee1-like protein kinase, a modulator of MYT1 (PKMYT1), preferably an inhibitor.

13. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein: the modulator of Checkpoint kinase 1 is a compound preferably selected from the group consisting of prexasertib (LY2606368), rabusertib (LY2603618), and combinations thereof; and / or the modulator of Wee1-like protein kinase is preferably the compound 1-[6- (2-hydroxypropan-2-yl)-2-pyridinyl]-6-[4-(4-methyl-1-piperazinyl)anilino]-2- prop-2-enyl-3-pyrazolo[3,4-d]pyrimidinone (adavosertib, AZD1775) or the compound 3-(2,6-Dichlorophenyl)-1-methyl-7-((3-methyl-4-(1- methylpiperidin-4-yl)phenyl)amino)-2,3-dihydropyrimido[4,5-d]pyrimidin- 4(1 H)-one (zedorosertib, Debio0123); and / or the modulator of MYT1 is preferably the compound lunresertib.

14. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound is the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint is selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and combinations thereof.

15. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and combinations thereof, are, independently, administered to the subject at least 4 hours, preferably at least 6 hours, before delivering ionizing radiation to the subject, and / or preferably at most 48 hours before delivering ionizing radiation to the subject.

16. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and combinations thereof, are, independently, administered to the subject 4 - 48 hours, preferably 4 - 24 hours, more preferably 6 - 18 hours, or even more preferably 6 - 8 hours before delivering ionizing radiation to the subject.

17. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the M-phase cell cycle arresting compound, preferably the compound ABT-751 , and the modulator of an interphase cell cycle checkpoint, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and combinations thereof, are, independently, administered to the subject in two or more separate doses, and wherein there is at least 6 hours between theseparate doses, preferably wherein there is 6 - 24 hours, more preferably 6 - 12 hours between the separate doses.

18. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the treatment comprising administering the M-phase cell cycle arresting compound to the subject, preferably the compound ABT-751 , administering the modulator of an interphase cell cycle checkpoint to the subject, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and combinations thereof, and the delivering of ionizing radiation to the subject, is performed at least or at most once every two weeks, at least or at most 1 time per week, at least or at most 2 times per week, preferably 5 times per week and / or wherein the treatment is performed for or over a period of at least 1 week, preferably for at least 2 weeks, for 1 - 10 weeks, preferably for 1 - 6 weeks, preferably 6 weeks.

19. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein between one or more subsequent treatments, said treatment comprising administering the M-phase cell cycle arresting compound to the subject, preferably the compound ABT-751 , administering the modulator of an interphase cell cycle checkpoint to the subject, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib, and combinations thereof, and the delivering of ionizing radiation to the subject, there is a period of non-treatment, preferably wherein the non-treatment period is for 1 - 30 days, preferably 1 - 14 days, 1 - 7 days or 1 - 3 days.

20. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the treatment that comprises administering the M-phase cell cycle arresting compound to the subject, preferably the compound ABT-751 , administering the modulator of an interphase cell cycle checkpoint to the subject, preferably selected from adavosertib, berzosertib, prexasertib, rabusertib, lunresertib,tuvusertib, and combinations thereof, and the delivery of ionizing radiation to the subject, is performed 5 times per week, and / or wherein the treatment is performed for or over a period of at least 1 week, preferably for at least 2 weeks, for 1 - 10 weeks, preferably for 1 - 6 weeks, preferably 6 weeks; and wherein between one or more subsequent treatments there is a period of non-treatment, preferably wherein the non-treatment period is for 1 - 30 days, preferably 1 - 14 days, 1 - 7 days or 1 - 3 days, preferably 2 days.

21. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the delivering of ionizing radiation to the subject is in an amount of 0.5 Gy - 20.0 Gy, 0.5 Gy - 15.0 Gy, 0.5 G-y - 10 Gy, or 1.5Gy - 2.8 Gy, preferably 1.5Gy - 2.8 Gy.

22. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the treatment further comprises administering a further (bio)pharmaceutical agent to the subject.

23. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the tumor is selected from a brain tumor, pulmonary tumor, breast tumor, genitourinary tract tumor, bone tumor, skin tumor, head tumor, neck tumor, meroblastic tumor, gastrointestinal tumor, colorectal tumor, pancreatic tumor, hematopoietic tumor and lymphoid tissue tumor, soft tissue sarcoma, preferably is a brain tumor.

24. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the tumor is selected from a tumor located in the brain, a tumor located in the lungs, a tumor located in the breast, a tumor located in the genitourinary tract, a tumor located in bone system, a tumor located in the skin, a tumor located in the head, a tumor located in the neck, a tumor located in the gastrointestinalsystem, a tumor located in the colon and / or rectum, a tumor located in the pancreas, a tumor located in the hematopoietic and / or lymphoid tissue, preferably is a tumor located in the brain.

25. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the tumor is a primary brain tumor selected from the group consisting of astrocytomas, meningiomas, oligodendrogliomas, medulloblastomas, ependymomas, craniopharyngiomas, gliomas, central nervous system lymphomas, chordomas, pineoblastomas and schwannomas, preferably wherein the primary brain tumor is an astrocytoma, even more preferably wherein the astrocytoma is a glioblastoma selected from the group consisting of primary glioblastoma, recurrent glioblastoma, glioblastoma with hypermethylation of the promoter of the gene O6-Methylguanin- Methyltransferase (MGMT), glioblastoma without hypermethylation of the promoter of MGMT.

26. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the tumor is a secondary brain tumor, which secondary brain tumor is a tumor that has formed in the brain as a result of metastasis, preferably metastasis from a different area in the subject’s body selected from the group consisting of pulmonary tumor, breast tumor, genitourinary tract tumor, bone tumor, skin tumor, head tumor, neck tumor, meroblastic tumor, gastrointestinal tumor, colorectal tumor, pancreatic tumor, hematopoietic tumor and lymphoid tissue tumor, preferably wherein the secondary brain tumor is a pulmonary tumor, breast tumor or skin tumor.

27. The M-phase cell cycle arresting compound or the modulator of an interphase cell cycle checkpoint, for use according to any of the previous claims, wherein the tumor is a soft tissue sarcoma, preferably a fat tissue sarcoma and / or a muscle tissue sarcoma.

28. A drug combination, preferably a pharmaceutical drug combination, comprising: (a) a M-phase cell arresting compound, preferably a pharmaceutical acceptable M-phase cell arresting compound, preferably the compound ABT-751 ; and (b) a modulator of an interphase cell cycle checkpoint, preferably a pharmaceutical acceptable modulator of an interphase cell cycle checkpoint, preferably selected from one or more of adavosertib, berzosertib, prexasertib, rabusertib, lunresertib, tuvusertib; wherein (a) and (b) are comprised in a single pharmaceutical composition or, alternatively, (a) and (b) are comprised in separate pharmaceutical compositions.

29. A method for the treatment of a tumor in a subject, the method comprising administering to said subject a therapeutically effective amount of a mitotic- phase (M-phase) cell cycle arresting compound, and a therapeutically effective amount of a modulator of an interphase cell cycle checkpoint, and delivering ionizing radiation to the subject; and wherein the M-phase cell cycle arresting compound and the modulator of an interphase cell cycle checkpoint are, independently, administered to the subject before the ionizing radiation is delivered to the subject.

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