Therapeutic combinations of PARP1 targeting agents with radiation therapy

Abstract

The present disclosure relates to therapeutic combinations of PARP1 targeting agents with radiation therapy and methods of using the same.

Description

THERAPEUTIC COMBINATIONS OF PARP1 TARGETING AGENTS WITH RADIATION THERAPYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 678,092, filed on August 1, 2024, the contents of which are hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to therapeutic combinations of poly(ADP-ribose) polymerase 1 (PARP1) targeting agents with radiation therapy and methods of using the same.BACKGROUND

[0003] Efforts to enhance tumor control has been an active area of research and approaches to tumor control include the use of combining radiation therapy locally with hypoxic modulators, chemotherapies, DNA damage inhibitors and most recently immunotherapies. Currently, cisplatin and gemcitabine are used as the gold-standard in combinations with radiation therapy for the treatment of cancer.

[0004] Combinations of poly(ADP-ribose) polymerase (PARP) inhibitors with radiation therapy are also being investigated. Zhang et al. investigated the use of niraparib as a radiosensitizer in EGFR-mutated non-small cell lung cancer and demonstrated anti-tumor effects in immunocompetent mice. They reported that the mechanism of action was primarily through the activation of CD8+ lymphocytes and activation of the STING pathways (Zhang et al., Clin. Trans., 2021). Liu et al. tested the combination of Olaparib and radiotherapy on radioresistant FaDu-RR cell lines and xenograft models and proposed that Olaparib increased the percentage of cell lines in the G2 / M cell cycle phase, which is the most radiosensitive compared to other cell cycle phases (S phase is the most resistant) (Liu et al., Oral Oncol.. 2020). Tran Chau et al. have also tested Olaparib in an orthotopic model lung cancer model (LL2) and demonstrated enhanced tumor control; however, the study faced major issues with toxicity (prostration, weakness and w eight loss) when combining Olaparib with radiation (Tran Chau et al.. Br. J. Cancer, 2020).

[0005] Clinically the combination of PARP inhibitors with radiotherapy has also been tested, and as of October 2021, there are 12 registered clinical trials that are comparing PARP-RT combinations (Barcellini et al., Cancers, 2021). Three of these trials have reported outcomes,and demonstrated no statistical difference in Overall Survival or Progression Free Survival compared to standard of care chemotherapies4. The major challenge in combining PARP inhibitors with radiotherapy is managing toxicities. All reviewed trials reported mild to severe (>grade 3) lymphopenia and neutropenia.

[0006] Thus there remains an unmet need for more effective and better tolerable combinations of PARP inhibitors and radiation therapy, as well as new ways of identifying patients who are likely to respond.SUMMARY

[0007] In one aspect, provided herein is a method of treating cancer in a subject thereof, the method comprising administering to the subject an effective amount of radiation therapy and an effective amount of a poly(ADP-ribose) polymerase 1 (PARPl)-selective inhibitor.

[0008] In some embodiments, wherein the PARP 1 -selective inhibitor is administered before the radiation therapy. In some embodiments, the PARP 1 -selective inhibitor is AZD5305, AG- 14361, or A-966492. In some embodiments, the radiation therapy is photon radiation therapy, electron radiation therapy, or proton radiation therapy. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, said fractions having a range of radiation from 1.5 Gy to 80 Gy, collectively. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 0.3 Gy to 16 Gy. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, said fractions having a range of radiation from 8 Gy to 80 Gy, collectively. In some embodiment, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 1.6 Gy to 16 Gy.

[0009] In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, said fractions having a range of radiation from 10 Gy to 80 Gy, collectively. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 2 Gy to 16 Gy. In some embodiments, the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy radiotherapy in 1 to 40 fractions or less at a dose rate of 0. 1 Gy / sec or higher. In some embodiments, the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy of radiation in 1 to 40 fractions or less at a dose rate of 40 Gy / sec or higher.

[0010] In some embodiments, the radiation therapy is ultra-high dose rate (FLASH) radiation therapy. In some embodiments, the radiation therapy is proton FLASH. In some embodiments,the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy proton FLASH in 1 to 40 fractions or less at a dose rate of 40 Gy / sec or higher.

[0011] In some embodiments, the cancer does not have a mutation rendering it deficient in homologous recombination. In some embodiments, the cancer does not have a BRCA1 mutation. In some embodiments, the cancer does not have a BRCA2 mutation. In some embodiments, the cancer does not have a RAD51 mutation. In some embodiments, the cancer does not have an ATM mutation. In some embodiments, the cancer is lung cancer, colon cancer, breast cancer, or melanoma. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the method significantly reduces bone marrow toxicity compared to other PARP inhibitors. In some embodiments, the method significantly reduces hematological toxicity compared to other PARP inhibitors.

[0012] In some embodiments, the method results in a decrease in tumor size that is at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50% greater than the decrease in tumor size achieved by administering the effective amount of radiation therapy in combination with a PARP inhibitor that is not PARP1 selective. In some embodiments, the method results in a decrease in healthy tissue toxicity' of about 10%. at least about 20%, at least about 30%, at least about 40% or at least about 50% compared to the toxicity observed when administering the effective amount of radiation therapy in combination with a PARP inhibitor that is not PARP1 selective. In some embodiments, the healthy tissue toxicity is bone marrow toxicity. In some embodiments, the healthy tissue toxicity is anemia, leukopenia, neutropenia or thrombocytopenia.

[0013] In another aspect, provided herein is a method for identifying a subject for treatment, the method comprising: (a) obtaining a sample from the subject; (b) measuring the expression level of a set of genes comprising NGF, BRMS1L, RAD51AP1. ADCY2, RAD54L. BRCA2, POLD3, and MEI.K'. (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is decreased relative to the expression level of the gene in a control sample; and (d) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP 1 -selective inhibitor if the subject is categorized as likely to respond.

[0014] In some embodiments, the set of genes further comprises NOTCH2 and / or PDK1. In some embodiments, if a subject is not identified as likely to respond, the subject is treated with an alternative therapy. In some embodiments, if a subject is not identified as likely to respond, the subject is monitored.

[0015] In another aspect, provided herein is a method for identifying a subject for treatment, the method comprising: (a) obtaining a sample from the subject; (b) measuring the gene expression level of a set of genes comprising IFITM2, ACKR3, TAGLN, DIO2, GLIPR1, NNT, ADAM12, and IFI27; (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is increased relative to the expression level of the gene in a control sample; and (d) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categorized as likely to respond.

[0016] In some embodiments, the set of genes further comprises BMPR1B, DMTN, LTBP2, STC2, SLC22A4, CD44, IFI44L, COL16A1, CALB2 and / or GJA1. In some embodiments, if a subject is not identified as likely to respond, the subject is treated with an alternative therapy. In some embodiments, if a subject is not identified as likely to respond, the subject is monitored.

[0017] In another aspect, provided herein is a method of treating cancer in a subject, the method comprising: (a) obtaining a sample from the subject; (b) measuring the expression level of a set of genes comprising NGF. BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2. POLD3. and MELK; (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is decreased relative to the expression level of the gene in a control sample; and (d) (i) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categorized as likely to respond or (ii) administering to the subject an alternative therapy or monitoring the subject, if the subject is not identified as likely to respond.

[0018] In some embodiments, the set of genes further comprises NOTCH2 and / or PDK1. In some embodiments, the control sample is a normal tissue control sample.

[0019] In another aspect, provided herein is a method of treating cancer in a subject, the method comprising: (a) obtaining a sample from the subject; (b) measuring the gene expression level of a set of genes comprising IFITM2, ACKR3, TAGLN, DIO2, GLIPR1, NNT, ADAM12, and IFI27; (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is increased relative to the expression level of the gene in a control sample; and (d) (i) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categorized as likely to respond or (ii) administering to the subject an alternative therapy or monitoring the subject, if the subject is not identified as likely to respond.

[0020] In some embodiments, the set of genes further comprises BMPR1B, DMTN, LTBP2, STC2, SLC22A4, CD44. IFI44L, COL16A1, CALB2 and / or GJA1. In some embodiments, the control sample is a normal tissue control sample.

[0021] In another aspect, provided herein is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of radiotherapy and an effective amount of a PARP1 -selective inhibitor, wherein the subject has a decreased expression level of one or more genes relative to a normal tissue control sample, and wherein the one or more genes is selected from the group consisting of: NGF, BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3, MELK, NOTCH2, and PDK1.

[0022] In another aspect, provided herein is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of radiotherapy and an effective amount of a PARP1 -selective inhibitor, wherein the subject has an increased gene expression level of one or more genes relative to a normal tissue control sample, and wherein the one or more genes is selected from the group consisting of: IFITM2, ACKR3, TAGLN, DIO2, GLIPR1, NNT, ADAM12, IFI27. BMPR1B, DMTN, LTBP2, STC2, SLC22A4. CD44, IFI44L, COL16A1, CALB2. and GJA1.DESCRIPTION OF DRAWINGS

[0023] FIG. 1 shows the results of a cell line screen of PARP inhibitors in combination with radiation THERAPY or with chemotherapy.

[0024] FIGs. 2A-2D show the effects of AZD5305 alone or in combination with 2 Gy ionizing radiation on the survival of H650 (FIG. 2A), Hl 975 (FIG. 2B), Hl 573 (FIG. 2C), and H358 (FIG. 2D) cells, respectively.

[0025] FIGs. 3A-3D show the effects of AZD5305 (FIG. 3A). talazoparib (FIG. 3B), olaparib (FIG. 3C) and rucaparib (FIG. 3D), respectively, in combination with varying amounts of ionizing radiation on the proliferation of H650 cells.

[0026] FIGs. 4A-4F show the effects of selective PARP1 degradation by Proteolysis Targeting Chimeras (PROTACs) i-Rucaparib-AP6 and SK-575, respectively, in combination with radiotherapy on the survival of H650 (FIG. 4A and 4D), H1573 (FIG. 4B and FIG. 4E) and H1437 (FIG. 4C and FIG. 4F) cells.

[0027] FIGs. 5A and 5B show the effects of AZD5305 alone or administered before or after 2 Gy ionizing radiation on the proliferation of H650 (FIG. 5A) and Hl 975 (FIG. 5B), respectively.

[0028] FIGs. 6A-6D show results of a gene set enrichment analysis in NSCLC cells responding to PARP1 inhibition in combination with radiation therapy. FIGs. 6A and 6B show normalized enrichment scope and the enrichment plot, respectively. FIGs. 6C and 6D show the relative expression of marker genes for sensitivity to combination therapy with a PARP1 inhibitor and radiation therapy, with FIG. 6C showing the expression level of genes whose expression is increased in resistant cell lines and FIG. 6D showing the expression level of genes whose expression is decreased in resistant cell lines relative to control.

[0029] FIGs. 7A-7D show the effect of varying amounts of radiation in combination with talazoparib on the proliferation A549 cells (FIG. 7A) and Hl 299 cells (FIG. 7B), and the effect of varying amounts of radiation in combination with saruparib on the proliferation of A549 cells (FIG. 7C) and Hl 299 cells (FIG. 7D).

[0030] FIGs. 8A-8D show the effect of varying amounts of ionizing radiation on the proliferation of A549 cells (FIG. 8A) and H1299 cells (FIG. SB) treated with 0.06 nM or 0.20 nM of talazoparib, and the effect of vary ing doses of ionizing radiation on the proliferation of A549 cells (FIG. 8C) and H1299 cells (FIG. 8D) treated with 0.06 nM or 0.20 nM of saruparib. The effect of ionizing radiation on untreated cells was assessed as a control.

[0031] FIGs. 9A-9D show a non-treated control normalized for response in comparison to three treatment conditions: 10 pM of AZD5305, 2 Gy ionizing radiation therapy, and a combination therapy of 10 pM of AZD5305 and 2 Gy ionizing radiation. Data are shown for H1975 (FIG. 9A), H1573 (FIG. 9B). H650 (FIG. 9C), and H358 (FIG. 9D) cells.DETAILED DESCRIPTION

[0032] Provided herein are methods of treating cancer comprising administering to the sub] ect an effective amount of radiation therapy and an effective amount of a PARP 1 -selective inhibitor, as w ell as methods of identifying a subject likely to respond to such a combination therapy.Definitions

[0033] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary' skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa unless the content clearly dictates otherwise. In the event that any description of aterm set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0034] The term '‘a” or "an" refers to one or more of that entity, i.e., can refer to plural referents. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0035] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0036] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0037] As used herein, the terms “or” and “and / or” include any, and all, combinations of one or more of the associated listed items.

[0038] As used herein, the terms “include” and “comprise” are used synonymously. In addition, it should be understood that the polypeptides comprising the various combinations of the components (e.g, domains or regions) and substituents described herein, are disclosed by the present application to the same extent as if each polypeptide was set forth individually. Thus, selection of particular components of individual polypeptides is within the scope of the present disclosure.

[0039] As used herein, the terms “treat,” “treating.” or “treatment”, and grammatical vanants thereof, have the same meaning as commonly understood by those of ordinary skill in the art. In aspects, these terms may refer to an approach for obtaining beneficial or desired clinical results. The terms may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above. For thepurposes of this invention, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (e.g, not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treat,’' “treating,” or “treatment” can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject (e.g., a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The terms “treat,” “treating,” or “treatment” includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment and is not necessarily meant to imply complete cessation of the relevant disease or condition.

[0040] As used herein, the terms “prevent.” “preventing,” “prevention” and grammatical variants thereof refer to an approach for preventing the development of, or altering the pathology of, a condition or disease. Accordingly, “prevention” may refer to prophylactic or preventive measures. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable. A subject (e.g.. a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. The term “prevention” includes slowing the onset of disease relative to the absence of treatment and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition. Thus “preventing” or “prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition or preventing or delaying the development of symptoms associated with the condition.

[0041] As used herein, the term “subject” refers to any subject, e.g., a human or anon-human mammal, for whom diagnosis, prognosis, or therapy is desired. The term “subject” may mean a human or non-human mammal affected, likely to be affected, or suspected to be affected with a disease. The terms “subject” and “patient” are used interchangeably herein. In some embodiments, a subject is a mammal. A mammal includes primates, such as humans, monkeys, chimpanzee, and apes, and non-primates such as domestic animals, including laboratory animals (such as rabbits and rodents, e.g., guinea pig, rat, or mouse) and household pets and farm animals (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and nondomestic animals, such as wildlife, birds, reptile, fish, or the like.Methods of Treatment

[0042] In one aspect, provided herein is a method of treating cancer in a subject thereof, the method comprising administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor. The methods disclosed herein are at least in part based on the surprising finding that PARP1 and radiotherapy combinations have an enhanced therapeutic window compared to standard PARP1 / 2 and radiation therapy combinations. Without wishing to be bound by theory, this enhanced therapeutic window is believed to be due to the sparing of PARP2, which is not required of therapeutic efficacy. Toxicities associated with PARP inhibitor combination strategies are believed to results from PARP2 inhibition, but also from non-specific inhibition of other PARP protein family members.Doses and Administration

[0043] As used herein, the term “PARP 1 -selective inhibitor'’ describes a PARP inhibitor that does not substantially inhibit the activity of PARP2, PARP3 or PARP4. For example, a PARP 1 -selective inhibitor may inhibit PARP1 with an IC50 that is at least 5-fold, at least 10- fold, at least 15-fold or at least 20-fold lower than its IC50 for inhibition of PARP2, PARP3 or PARP4.

[0044] Examples of PARP 1 -selective inhibitors include AZD5305, AG-14361, and A- 966492. but any targeted agent that inhibits or degrades PARP1 selectively may be used. In some embodiments, the PARP 1 -selective inhibitor is AZD5305. In some embodiments, the PARP 1 -selective inhibitor is AG- 14361. In some embodiments, the PARP 1 -selective inhibitor is A-966492. In some embodiments, the PARP 1 -selective inhibitors comprise one or more of AZD5305, AG-14361, and A-966492.

[0045] Any suitable type of radiation therapy may be used in the methods described herein. Examples of radiation therapy include but are not limited to, photon radiation, proton radiation, electron radiation, electron radiation, external beam radiation, carbon ion therapy, radioisotopes, stereotactic body radiation, brachytherapy, and intraoperative radiation. In some embodiments, the radiation therapy comprises the administration of electron radiation, proton radiation, or photon radiation, or any combination thereof. In some embodiments, the radiation therapy comprises ionizing radiation. In some embodiments, the radiation therapy comprises non-ionizing radiation. In some embodiments, the radiation therapy comprises administering ultra-high dose (FLASH) radiation therapy, e.g.. electron FLASH, protonFLASH, or photon FLASH. In some embodiments, the radiation therapy comprises hypofractionated and hyperfractionated radiation therapy.

[0046] The radiation therapy of the methods described herein may be administered using any suitable system (or device) known in the art including, for example, an electron linear accelerator, a proton source, or an x-ray source. For example, radiation therapy may be delivered using electrons delivered by a linear electron accelerator is described, for example, in Favaudon et al.. Transl. Med. 6. 245ra93 (2014). In other embodiments, the radiation therapy is administered using high energy charged particles, electrons, protons, heavy ions, high energy photons, x-rays, gamma rays, or neutrons.

[0047] In some embodiments, the radiation therapy comprises proton beam treatment, electron beam treatment, or a combination thereof. In some embodiments, the radiation therapy comprises proton beam treatment. Proton beam treatment has the advantage of being able to penetrate deeper into the tissue than electron beams. Furthermore, proton beams deposit the maximum of their energy at the end of their path, avoiding further penetration into healthy tissue (Liu, Chin J Cancer. 2011 May; 30(5): 315-326). Proton radiation therapy may be administered using a passive beam scattering system (e.g., a single scattering system or double scattering system) or a dynamic spot scanning system. In some embodiments, the radiation therapy or treatment system used to deliver proton radiation therapy is a proton pencil beam scanning system.

[0048] Illustrative devices that may be used to administer radiation therapy are described in, for example, U.S. Patent No. 9,855,445, which is incorporated by reference herein in its entirety for the systems that may be used in the methods described herein. In some embodiments, the system used to administer radiation therapy in accordance with a method described herein comprises a nozzle, an accelerator, and a beam transport system. The nozzle may further comprise a scanning magnet, which guides the beam towards the target, and a beam energy adjuster. The accelerator may be based on radio frequency (e.g., a linear accelerator, a cyclotron, or a synchrotron) or a laser-based accelerator.

[0049] The dose of radiation therapy that is administered to a subject treated in accordance with a method described herein may depend on the characteristics of the subject and the cancer being treated. Without wishing to be bound by theory, it is believed that FLASH radiation therapy may be delivered at substantially higher doses than conventional dose rate radiation therapy due to its decreased normal tissue toxicity. As used herein, the term “conventional dose rate radiation therapy” is used to refer to radiation therapy that is administered at rates of about 2 Gy / sec or less. One of skill in the art will appreciate that the radiation dose ofconventional dose rate therapy that is administered will depend on many variables, including, without limitation, the tumor being treated, the stage and / or progression of the disease, patient co-morbidities, concurrent treatments, the device used to administer the radiation, and prior therapies. “FLASH” radiation therapy is generally administered at dose rates of at least 40 Gy / s.

[0050] In some embodiments, the dose rate of radiation therapy administered is at least 0. 1 Gy / s. In some embodiments, the dose rate of radiation therapy administered is at least 1 Gy / s. In some embodiments, the dose rate of radiation therapy administered is between 1 Gy / s and 60 Gy / s. In some embodiments, the dose rate of radiation therapy administered is about 1 Gy / s to about 5 Gy / s, about 5 Gy / s to about 10 Gy / s, about 10 Gy / s to about 15 Gy / s, about 15 Gy / s to about 20 Gy / s, about 20 Gy / s to about 25 Gy / s, about 25 Gy / s to about 30 Gy / s. about 30 Gy / s to about 35 Gy / s, about 35 Gy / s to about 40 Gy / s, about 40 Gy / s to about 45 Gy / s, about 45 Gy / s to about 50 Gy / s, about 50 Gy / s to about 55 Gy / s, about 55 Gy / s to about 60 Gy / s, about 60 Gy / s to about 65 Gy / s, or about 65 Gy / s to about 70 Gy / s. including any values or ranges therebetween.

[0051] In some embodiments, the dose of radiation therapy administered is between 1 Gy and 60 Gy. In some embodiments, the dose of radiation therapy administered is about 1 Gy to about 5 Gy, about 5 Gy to about 10 Gy, about 10 Gy to about 15 Gy, about 15 Gy to about 20 Gy, about 20 Gy to about 25 Gy, about 25 Gy to about 30 Gy, about 30 Gy to about 35 Gy, about 35 Gy to about 40 Gy, about 40 Gy to about 45 Gy, about 45 Gy to about 50 Gy, about 50 Gy to about 55 Gy, about 55 Gy to about 60 Gy, about 60 Gy to about 65 Gy, about 65 Gy to about 70 Gy, about 70 Gy to about 75 Gy, or about 75 Gy to about 80 Gy, including any values or ranges therebetween.

[0052] In some embodiments, the dose rate of FLASH or conventional radiation therapy administered is at least 1 Gy / s. In some embodiments, the dose rate of FLASH or conventional radiation therapy administered is between 1 Gy / s and 60 Gy / s. In some embodiments, the dose rate of FLASH or conventional radiation therapy administered is about 1 Gy / s to about 5 Gy / s, about 5 Gy / s to about 10 Gy / s. about 10 Gy / s to about 15 Gy / s, about 15 Gy / s to about 20 Gy / s, about 20 Gy / s to about 25 Gy / s. about 25 Gy / s to about 30 Gy / s. about 30 Gy / s to about 35 Gy / s, about 35 Gy / s to about 40 Gy / s, about 40 Gy / s to about 45 Gy / s, about 45 Gy / s to about 50 Gy / s, about 50 Gy / s to about 55 Gy / s, about 55 Gy / s to about 60 Gy / s, about 60 Gy / s to about 65 Gy / s, or about 65 Gy / s to about 70 Gy / s, including any values or ranges therebetween.

[0053] In some embodiments, the dose of FLASH or conventional radiation therapy administered is between 1 Gy and 60 Gy. In some embodiments, the dose of FLASH or conventional radiation therapy administered is about 1 Gy to about 5 Gy, about 5 Gy to about 10 Gy, about 10 Gy to about 15 Gy, about 15 Gy to about 20 Gy, about 20 Gy to about 25 Gy, about 25 Gy to about 30 Gy, about 30 Gy to about 35 Gy, about 35 Gy to about 40 Gy, about 40 Gy to about 45 Gy, about 45 Gy to about 50 Gy, about 50 Gy to about 55 Gy. about 55 Gy to about 60 Gy. about 60 Gy to about 65 Gy. about 65 Gy to about 70 Gy, about 70 Gy to about 75 Gy, or about 75 Gy to about 80 Gy, including any values or ranges therebetween.

[0054] The radiation therapy may be delivered in a pulsed manner, a continuous manner, or a quasi-continuous manner. In some embodiments, the radiation therapy is administered in a pulsed manner with pulses at a frequency of about 100Hz. In some embodiments, the dose of radiation therapy is delivered in a single pulse. In some embodiments, the dose of radiation therapy is delivered in a series of two or more pulses. Each pulse can have a duration of less than a second, several seconds, or several minutes. The interval between pulses may also last less than a second, several seconds, or several minutes. In some embodiments, each pulse in a series of pulses has the same duration. In some embodiments, the pulses in a series of pulses have different durations. In some embodiments, the intervals between each pulse in a series of pulses have the same duration. In some embodiments, the intervals between pulses in a series of pulses have different durations.

[0055] The dose and pulse parameters may be varied by a person skilled in the art to optimize the therapeutic effect. In some embodiments, the dose per pulse is at least 1 Gy, at least 2 Gy, at least 3 Gy, at least 4 Gy, or at least 5 Gy.

[0056] In some embodiments, the dose of radiation therapy is administered as fractionated doses, i.e., in a series of small doses over a period of time. Dose fractionation is used with conventional radiation therapy to reduce the incidence of radiation-induced side effects. Generally, a dose of conventional radiation therapy is fractionated into daily doses administered over weeks in order to achieve an acceptable therapeutic index (Dutt et al., Semin Radial Oncol. 2020 April 30(2): 194-200). Commonly, conventional therapy for solid tumors (e.g., standard of care radiation therapy) is fractionated into small doses of 1.8—2 Gy per day. delivered 5 days per week over the course of 6 to 8 weeks, resulting in a total dose of 60-80 Gy (Dutt et al., 2020)). Fractions of radiation may also be administered twice a day (at least 6 hours apart). Such “hyperfractionated’' radiation therapy uses smaller fractions of, for example, about 1.5 Gy. In some embodiments, spatially fractionated radiation therapy (SFRT) is administered.

[0057] In some embodiments of the methods disclosed herein, the radiation therapy is administered in 1 to 100 fractions, 1 to 90 fractions, 1 to 80 fractions, 1 to 70 fractions, 1 to 60 fractions, 1 to 50 fractions, 1 to 40 fractions, 1 to 30 fractions, 1 to 20 fraction, 1 to 10 fractions, 10 to 60 fractions, 10 to 50 fractions, 10 to 100 fractions, 10 to 90 fractions, 10 to 80 fractions, 10 to 70 fractions, 10 to 60 fractions, 10 to 50 fractions, 10 to 40 fractions, 10 to 30 fractions, 10 to 20 fraction, 20 to 100 fractions, 20 to 90 fractions, 20 to 80 fractions, 20 to 70 fractions, 20 to 60 fractions. 20 to 50 fractions, 20 to 40 fractions, 20 to 30 fractions. 30 to 100 fractions, 30 to 90 fractions, 30 to 80 fractions, 30 to 70 fractions, 30 to 60 fractions, 30 to 50 fractions, 30 to 40 fractions, 40 to 100 fractions, 40 to 90 fractions, 40 to 80 fractions, 40 to 70 fractions, 40 to 60 fractions, 40 to 50 fractions, 50 fractions to 100 fractions, 50 to 90 fractions, 50 to 80 fractions. 50 to 70 fractions, 50 to 60 fractions. 60 fractions to 100 fractions, 60 to 90 fractions, 60 to 80 fractions, 60 to 70 fractions, 70 to 100 fractions, 80 to 100 fractions, 80 to 90 fractions, or 90 to 100 fractions, including any values or ranges therebetween. In some embodiments, the radiation therapy is administered in at least 5 fractions, at least 10 fractions, at least 15 fractions, at least 20 fractions, at least 25 fractions or at least 30 fractions. In some embodiments, the radiation therapy is administered in about 1 to 40 fractions. In some embodiments, the radiation therapy is conventional dose rate therapy.

[0058] In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 1 to 10 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 1 to 5 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 5 to fractions. In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 10 to 20 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 20 to 30 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 30 to 40 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 40 to 50 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 50 to 60 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is conventional dose rate therapy administered in 30 fractions.

[0059] Without wishing to be bound by theory, it is believed that FLASH radiation therapy may be administered with fewer fractions than conventional dose rate radiation therapy dueto its decreased healthy tissue toxicity. In some embodiments of the methods disclosed herein, the radiation therapy is FLASH radiation therapy administered in 1 to 5 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is FLASH radiation therapy administered in 5 to 10 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is FLASH radiation therapy administered in 10 to 15 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is FLASH radiation therapy administered in 15 to 20 fractions. In some embodiments of the methods disclosed herein, the radiation therapy is FLASH radiation therapy administered in 1 to 40 fractions. In some embodiments, FLASH radiation therapy is administered in five or more fractions. In some embodiments, five fractions of FLASH radiation therapy is administered. In some embodiments, two fractions of FLASH radiation therapy are administered. In some embodiments, three fractions of FLASH radiation therapy are administered. In some embodiments, four fractions of FLASH radiation therapy are administered. In some embodiments, five fractions of FLASH radiation therapy are administered.

[0060] In some embodiments, the radiation therapy administered in combination with a TITR effector comprises the administration of one or more radionuclides. Examples of radionucleotides include sealed sources (e.g., brachytherapy) as well as unsealed sources (e.g., radiopharmaceuticals).

[0061] In some embodiments, the radiation therapy is administered in an effective amount to treat cancer in a subject thereof. In some embodiments, the effective amount of radiation therapy comprises at least 1 , at least 5, at least 10, at least 15, at least 20, at least 25, at least 30 fractions, at least 35 fractions, or at least 40 fractions of radiation. In some embodiments, the effective amount of radiation therapy comprises at least five fractions of radiation. In some embodiments, the effective amount of radiation therapy comprises five fractions of radiation.

[0062] In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, said fractions having a range of radiation from 1.5 Gy to 80 Gy, collectively. In some embodiments, the effective amount of radiation therapy comprises Ito 40 fractions of radiation, said fractions having a range of radiation from 8 Gy to 80 Gy, collectively. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, said fractions having a range of radiation from 10 Gy to 80 Gy, collectively. In some embodiments, the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy proton radiation in 1 to 40 fractions at a dose rate of 0.1 Gy / sec or higher. In some embodiments, the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy proton radiation in 1 to 40 fractions at a dose rate of 1 Gy / sec or higher.In some embodiments, the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy proton radiation in 1 to 40 fractions at a dose rate of 40 Gy / sec or higher.

[0063] In some embodiments, each of the at 1 to 40 fractions of radiation has a range of radiation from 0.3 Gy to 12Gy, 1.6 Gy to 12 Gy, or 2 Gy to 12 Gy, including any values or ranges therebetween. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 0.3 Gy to 16 Gy. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 1.6 Gy to 16 Gy. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 2 Gy to 16 Gy.

[0064] In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of proton FLASH, said fractions having a range of radiation from 1.5 Gy to 80 Gy, collectively. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of proton FLASH, said fractions having a range of radiation from 8 Gy to 80 Gy, collectively. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of proton FLASH, said fractions having a range of radiation from 10 Gy to 80 Gy, collectively. In some embodiments, the effective amount of radiation therapy is a dose of about 10 Gy to about 60 Gy' proton FLASH radiotherapy in 1 to 40 fractions or less at a dose rate of 40 Gy / sec or higher.

[0065] In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of proton FLASH, wherein each fraction has a range of radiation from 0.3 Gy to 12 Gy. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of proton FLASH, wherein each fraction has a range of radiation from 1.6 Gy to 12 Gy. In some embodiments, the effective amount of radiation therapy comprises 1 to 40 fractions of proton FLASH, wherein each fraction has a range of radiation from 2 Gy to 12 Gy.

[0066] In preferred aspects of the methods disclosed herein, the PARP1 -selective inhibitor is administered before the radiation therapy. In some embodiments, the PARP1 -selective inhibitor is administered the day' before the radiation therapy. In some embodiments, the PARP1 -selective inhibitor is administered 12 hours to 36 hours before the radiation therapy. In some embodiments, the PARP1 -selective inhibitor is administered 16 hours to 30 hours before the radiation therapy. In some embodiments, the PARP1 -selective inhibitor is administered 16 hours to 24 hours before the radiation therapy. In some embodiments, thePARP1 -selective inhibitor is administered 12 hours, 16 hours, 18 hours, 24 hours, 30 hours or 36 hours before the radiation therapy.

[0067] In some embodiments, the PARP1 -selective inhibitor is administered 2, 3, 4, 5, 6, or 7 days before the radiation therapy.

[0068] In some embodiments, a patient receives one cycle of treatment comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. The administration of the PARP 1 -selective inhibitor followed by radiation therapy may be repeated as necessary. A person of skill in the art will appreciate that the amounts of repeat treatments will depend on factors such as the patient’s ability to tolerate the treatment, the severity of the disease (e.g., the stage of the cancer) and the patient’s response to the initial treatment. In some embodiments, a patient receives two cycles of treatment, each cycle comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. In some embodiments, a patient receives three cycles of treatment, each cycle comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. In some embodiments, a patient receives four cycles of treatment, each cycle comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. In some embodiments, a patient receives five cycles of treatment, each cycle comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. In some embodiments, a patient receives six cycles of treatment, each cycle comprising administration of a PARP- 1 selective inhibitor followed by radiation therapy. In some embodiments, a patient receives seven cycles of treatment, each cycle comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. In some embodiments, a patient receives eight cycles of treatment, each cycle comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. In some embodiments, a patient receives nine cycles of treatment, each cycle comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. In some embodiments, a patient receives ten cycles of treatment, each cycle comprising administration of a PARP-1 selective inhibitor followed by radiation therapy. The time between cycles may vary’ depending on factors such as the patient’s ability to tolerate the treatment, the severity of the disease (e.g., the stage of the cancer) and the patient’s response to the initial treatment. In some embodiments, the time between cycles is 2 weeks. In some embodiments, the time between cycles is 3 weeks. In some embodiments, the time between cycles is 2 weeks. In some embodiments, the time between cycles is 4 weeks. In some embodiments, the time between cycles is 5 weeks. In some embodiments, the time between cycles is 2 weeks. In some embodiments, the time between cycles is 6 weeks. In someembodiments, the time between cycles is 7 weeks. In some embodiments, the time between cycles is 8 weeks.Indications

[0069] Historically , the cancers indicated for PARP inhibitor therapy were cancers deficient in homologous recombination. Cancers may become deficient in homologous recombination due to mutations in one or more gene(s) encoding DNA repair enzymes, for example, mutations in BRCA1, BRCA2, ATM, PALB2, RAD51, FANCM, FANCD2, ATR, and / or CDK12. In contrast, it is believed that the cancers that can be treated with the methods described herein are not limited to cancers that are deficient in homologous recombination due to such mutations. Without wishing to be bound by theory, it is believed that the efficacy of the methods in treating cancers that are not deficient in homologous recombination may be due to the presence of other, non-genetic drivers of drug susceptibility in these cancers. For example, while there may be no mutational change in DNA-repair enzy mes, a cancer could have lower expression or activity of such enzymes, which may mimic the phenotype observed in the instances driven by mutation status. Thus, in some embodiments, the cancer treated in accordance with the methods described herein does not have a mutation rendering it deficient in homologous recombination. In some embodiments, the cancer treated in accordance with the methods described herein does not have a BRCA1 mutation. In some embodiments, the cancer treated in accordance with the methods described herein does not have a BRCA2 mutation. In some embodiments, the cancer treated in accordance with the methods described herein does not have a RAD51 mutation. In some embodiments, the cancer treated in accordance with the methods described herein does not have an ATM mutation.

[0070] In some embodiments, the cancer treated in accordance with the methods described herein is lung cancer, colon cancer, breast cancer, or melanoma. In some embodiments, the cancer treated in accordance with the methods described herein is non-small cell lung cancer.

[0071] It will be apparent to a person of skill in the art that the anatomical site of the cancer is not necessarily the determining factor in deciding whether or not the cancer is likely to respond to treatment in accordance with a method described herein.Improvements of Toxicities

[0072] Without wishing to be bound by theory, it is believed that the methods of treatment described herein results in less severe off-target toxicity than combination therapy of the samedose of radiation therapy in combination with a PARP inhibitor that is not selective for PARP1, due to the sparing of PARP2 inhibition.

[0073] Thus, in some embodiments, a method of treatment described herein significantly reduces bone marrow toxicity compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1.

[0074] In some embodiments, the method of treatment described herein reduces bone marrow toxicity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%. at least 70%, at least 80%, or at least 90% compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, the method of treatment described herein reduces bone marrow toxicity by about 5-10%, about 10-15%, about 15-20%, about 20-25%. about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-55%, about 55-60%, about 60-65%, about 65-70%, about 70-75%, about 75-80%, about 80-85%, about 85-90%, about 90-95%, or about 95-100% compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. Bone marrow toxicity may be measured using any suitable method known in the art or described herein. For example, bone marrow toxicity may be determined using complete blood counts and / or histological examination of the bone marrow.

[0075] In some embodiments, a method of treatment described herein significantly reduces a hematological toxicity compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1 .

[0076] In some embodiments, the hematological toxicity is anemia (low red blood cell counts). In some embodiments, the hematological toxicity is leukopenia (low- white blood cell counts). In some embodiments, the hematological toxicity is neutropenia (low neutrophil counts). In some embodiments, the hematological toxicity is thrombocytopenia (low platelet counts).

[0077] In some embodiments, the method of treatment described herein reduces hematological by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%. or at least 90% compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, the method of treatment described herein reduces bone marrow toxicity by about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-55%, about 55-60%, about 60-65%, about 65-70%, about 70-75%, about 75-80%, about 80-85%, about 85-90%,about 90-95%, or about 95-100% compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1.

[0078] Hematological toxicities can be classified by grade (Grade 1 being the mildest and Grade 4 being the most severe).

[0079] In some embodiments, the method of treatment described herein reduces hematological by 1 grade compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, the method of treatment described herein reduces hematological by 2 grades compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, the method of treatment described herein reduces hematological by 3 grades compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, the method of treatment described herein reduces hematological by 4 grades compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1.Improvement of Efficacy

[0080] As described above and without wishing to be bound by theory, it is hypothesized that the methods of treatment described herein result in decreased toxicities compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. A person of skill in the art will appreciate that decreases in toxicity may allow for a larger dose of radiation therapy to be administered and may thus result in better efficacy compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1.Decrease in Tumor Size

[0081] In some embodiments, a method of treatment described herein results in a decrease in tumor size compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. The decrease in tumor size may be measured using any suitable method known in the art or described herein, for example, using calipers or bioluminescence imaging in animal models, or using imaging suchas computer tomography (CT) scanning, magnetic resonance imagining (MRI), positron emission tomography (PET), x-ray. or physical examination in human subjects.

[0082] In some embodiments, a method of treatment described herein results in a decrease in tumor size that is at least 10% greater than the decrease in tumor size that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, a method of treatment described herein results in a decrease in tumor size that is at least 20% greater than the decrease in tumor size that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, a method of treatment described herein results in a decrease in tumor size that is at least 30% greater than the decrease in tumor size that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, a method of treatment described herein results in a decrease in tumor size that is at least 40% greater than the decrease in tumor size that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, a method of treatment described herein results in a decrease in tumor size that is at least 50% greater than the decrease in tumor size that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1.Duration of Response

[0083] In some embodiments, a method of treatment described herein results in a prolongation of the duration of response compared to a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. The duration of response is generally defined as the interval from response initiation (when the treated subject first shows a complete or partial response to therapy) to the earlier of disease progression or death. Disease relapse is generally measured as the recurrence of tumor cells after the subject has achieved a complete response.

[0084] In some embodiments, a method of treatment described herein results in a prolongation of the duration of response that is at least 10% greater than the duration of response that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, a method of treatment described herein results in a prolongation of the durationof response that is at least 20% greater than the duration of response that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, a method of treatment described herein results in a prolongation of the duration of response that is at least 30% greater than the duration of response that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, a method of treatment described herein results in a prolongation of the duration of response that is at least 40% greater than the duration of response that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1. In some embodiments, a method of treatment described herein results in a prolongation of the duration of response that is at least 50% greater than the duration of response that is achieved with a method of treatment comprising administering the same dose of radiation therapy and a PARP inhibitor that is not selective to PARP1.Delayed Tumor Recurrence

[0085] In some embodiments, a method of treatment described herein result in delayed tumor recurrence. Generally, tumor recurrence refers to a tumor becoming detectable again after being undetectable for a prolonged period of time.

[0086] In some embodiments, a tumor treated according to a method described herein does not recur for about 2 month to about 6 month, about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, or about 5 years to about 10 years. In some embodiments, a tumor treated according to a method described herein does not recur for at least about 2 month, at least about 3 month, at least about 6 month, at least about 9 months, at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or at least about 10 years, including any values or rages therebetween.

[0087] Tumors may be detected and measured using computer tomography (CT) scanning, magnetic resonance imagining (MRI), positron emission tomography (PET), x-ray, or physical examination, or any other suitable method known in the art.Decrease in Metastases

[0088] A method of treatment described herein may also reduce metastases of a cancer. Metastasis is the spreading of a cancer to other sites in the body from the primary site.

[0089] In some embodiments, a method of treatment described herein results in a decrease in number and / or size of tumor metastases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%. about 45%, about 50%, about 55%, about 60%, about 65%, about 70%. about 75%, about 80%, about 85%. about 90%, or about 95% compared to the number and / or size of tumor metastases before the administration of the therapeutic combination. In some embodiments, a method of treatment described herein results in a decrease in number and / or size of tumor metastases of about 5-10%, about 10- 15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40- 45%, about 45-50%, about 50-55%, about 55-60%, about 60-65%, about 65-70%, about 70- 75%, about 75-80%, about 80-85%, about 85-90%, about 90-95%, or about 95-100% compared to the number and / or size of tumor metastases before the administration of the therapeutic combination. The number and / or size tumor metastasis may be determined at any suitable time point after treatment, for example. 1 month. 2 months, 3 months. 4 months. 5 months, or 6 months after the administration of the therapeutic combination. In some embodiments, a method of treatment described herein prevents the occurrence of metastases for a least 6 months, at least 9 months, at least 12 months, at least 2 years, or at least 3 years.

[0090] Metastases may be detectable in blood or bone samples, or they can be detected and measured using computer tomography (CT) scanning, magnetic resonance imagining (MRI), positron emission tomography (PET), x-ray, or physical examination, or any other suitable method known in the art.Biomarkers of Response

[0091] In another aspect, provided herein are methods of identifying subjects that are likely to respond to the methods of treatment described herein.

[0092] Thus, in one aspect, provided herein is a method for identifying a subject for treatment, the method comprising (a) obtaining a sample from the subject; (b) measuring the expression level of a set of genes comprising NGF, BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3, and MELK; (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is decreased relative to the expression level of the gene in a control sample and (d) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitorif the subject is categorized as likely to respond. In some embodiments, the set of genes further comprises N0TCH2 and / or PDK1. In some embodiments, the subject is identified as likely to respond if the expression level of two genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of three genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of four genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of five genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of six genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of seven genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of eight genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of nine genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of ten genes in the set of genes in the set of genes is decreased relative to the expression level of the genes in a control sample.

[0093] In another aspect, provided herein is a method for identifying a subject for treatment, the method comprising (a) obtaining a sample from the subject; (b) measuring the gene expression level of a set of genes comprising IFITM2, ACKR3, TAGLN, DIO2, GLIPR1, NNT, ADAMI 2, and IFI27 (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is increased relative to the expression level of the gene in a control sample and (d) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categorized as likely to respond. In some embodiments, the set of genes further comprises BMPR1B, DMTN, LTBP2, STC2, SLC22A4, CD44, IFI44L, COL16A1, CALB2 and / or GJA1. In some embodiments, the subject is identified as likely to respond if the expression level of two genes in the set of genes in the set of genes is increased relative to the expression level ofthe genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of three genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of four genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of five genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of six genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of seven genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of eight genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of nine genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of ten genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of eleven genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of twelve genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample. In some embodiments, the subject is identified as likely to respond if the expression level of 13 genes in the set of genes in the set of genes is increased relative to the expression level of the genes in a control sample.

[0094] If a subject is not identified as likely to respond by a method disclosed herein, the subject may receive an alternative therapy. An alternative therapy may be any other therapy that is indicated for the cancer being treated. For example, the patient may be administered chemotherapy. Alternatively, a patient may be monitored without administration of a therapy for a suitable amount of time and the test repeated.

[0095] In another aspect, provided herein is a method of treating cancer in a subject, the method comprising (a) obtaining a sample from the subject; (b) measuring the expression levelof a set of genes compnsing NGF, BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3. and MELK; (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is decreased relative to the expression level of the gene in a control sample and (d) (i) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categorized as likely to respond or (ii) administering to the subject an alternative therapy or monitoring the subject, if the subject is not identified as likely to respond. In some embodiments, the set of genes further comprises N0TCH2 and / or PDK1.

[0096] In another aspect, provided herein is a method of treating cancer in a subject, the method comprising (a) obtaining a sample from the subject: (b) measuring the gene expression level of a set of genes comprising IFITM2, ACKR3. TAGLN, DIO2, GLIPR1. NNT, ADAMI 2, and IFI27; (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is increased relative to the expression level of the gene in a control sample and (d) (i) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categorized as likely to respond or (ii) administenng to the subject an alternative therapy or monitoring the subject, if the subject is not identified as likely to respond. In some embodiments, the set of genes further comprises BMPR1B, DMTN, LTBP2, STC2, SLC22A4, CD44, IFI44L, COL16A1, CALB2 and / or GJA1

[0097] Methods of determining gene expression are well known in the art. Any suitable method may be used to determine the expression of genes for the methods described herein, including, for example, RNA sequencing.

[0098] Provided herein is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of radiotherapy and an effective amount of a PARP1 -selective inhibitor, wherein the subject has a decreased expression level of one or more genes relative to a normal tissue control sample, and wherein the one or more genes is selected from the group consisting of: NGF, BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3, MELK, NOTCH2, and PDK1.

[0099] Provided herein is also a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of radiotherapy and an effective amount of a PARP1 -selective inhibitor, wherein the subject has an increased gene expression level of one or more genes relative to a normal tissue control sample, and wherein the one or more genes is selected from the group consisting of: IFITM2, ACKR3, TAGLN. DIO2,GLIPR1, NNT, ADAM12, IFI27, BMPR1B, DMTN, LTBP2, STC2, SLC22A4, CD44, IFI44L, C0L16A1, CALB2, and GJA1.

[0100] The methods provided herein may require the comparison of gene expression in a sample from the cancer and a control sample. The samples may be obtained by any suitable method. In some embodiments, the sample is obtained by biopsy, for example, needle biopsy, open biopsy, punch biopsy, lymph node biopsy or bone marrow aspiration.

[0101] In some embodiments, the control sample is a normal tissue control sample. Such a sample may be taken from the same subject as the cancer sample. In some embodiments, the control sample is taken from an unaffected part of the same organ as the cancer, for example, if the subject is afflicted with lung cancer, the control sample is taken from an unaffected part of the lung. In other instances, the control sample can be taken from a patient who has responded to the treatment with a PARP-1 selective inhibitor and radiation therapy. In some embodiments, the control sample is derived from genetically engineered cells that are sensitive to the combination therapy.

[0102] The expression level of the genes in a set may also be compared to a house keeping gene from the same sample.NUMBERED EMBODIMENTS

[0103] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

[0104] Embodiment 1. A method of treating cancer in a subject thereof, the method comprising administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor.

[0105] Embodiment 2. The method of embodiment 1 , wherein the PARP 1 -selective inhibitor is administered before the radiation therapy.

[0106] Embodiment 3. The method of embodiment 1, w herein the PARP 1 -selective inhibitor is AZD5305, AG-14361, or A-966492.

[0107] Embodiment. 4. The method of any one of embodiments 1-3, wherein the radiation therapy is ultra-high dose rate (FLASH) radiation therapy.

[0108] Embodiment 5. The method of any one of embodiments 1-3, wherein the radiation therapy is photon radiation therapy, electron radiation therapy, or proton radiation therapy.

[0109] Embodiment 6. The method of any one of embodiments 1-5, wherein the effective amount of radiation therapy comprises five fractions of proton FLASH, said fractions having a range of radiation from 1.5 Gy to 60 Gy, collectively.

[0110] Embodiment 7. The method of any one of embodiments 1-5, wherein the effective amount of radiation therapy comprises five fractions of proton FLASH, said fractions having a range of radiation from 8 Gy to 60 Gy, collectively.

[0111] Embodiment 8. The method of any one of embodiments 1-5, wherein the effective amount of radiation therapy comprises five fractions of proton FLASH, said fractions having a range of radiation from 10 Gy to 60 Gy, collectively.

[0112] Embodiment 9. The method of any one of embodiments 1-5, wherein the effective amount of radiation therapy is a dose of about 10 Gy to about 60 Gy proton FLASH radiotherapy in five fractions or less at a dose rate of 40 Gy / sec or higher.

[0113] Embodiment 10. The method of any one of embodiments 1 -9, wherein the cancer does not have a mutation rendering it deficient in homologous recombination.

[0114] Embodiment 11. The method of any one of embodiments 1-10, wherein the cancer does not have a BRCA1 mutation.

[0115] Embodiment 12. The method of any one of embodiments 1-10, wherein the cancer does not have a BRCA2 mutation.

[0116] Embodiment 13. The method of any one of embodiments 1-10, wherein the cancer does not have a RAD51 mutation.

[0117] Embodiment 14. The method of any one of embodiments 1-10, wherein the cancer does not have an ATM mutation.

[0118] Embodiment 15. The method of any one of embodiments 1-14, wherein the cancer is lung cancer, colon cancer, breast cancer, or melanoma.

[0119] Embodiment 16. The method of any one of embodiments 1-15, wherein the cancer is non-small cell lung cancer.

[0120] Embodiment 17. The method of any one of embodiments 1-16. wherein the method significantly reduces bone marrow toxicity compared to other PARP inhibitors.

[0121] Embodiment 18. The method of any one of embodiments 1-16, wherein the method significantly reduces hematological toxicity compared to other PARP inhibitors.

[0122] Embodiment 19. The method of any one of embodiments 1-18. wherein the method results in a decrease in tumor size that is at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50% greater than the decrease in tumor size achieved by administering the effective amount of radiation therapy in combination with a PARP inhibitor that is not PARP1 selective.

[0123] Embodiment 20. The method of any one of embodiments 1-19. wherein the method results in a decrease in healthy tissue toxicity of about 10%, at least about 20%, at least about30%, at least about 40% or at least about 50% compared to the toxicity observed when administering the effective amount of radiation therapy in combination with a PARP inhibitor that is not PARP1 selective.

[0124] Embodiment 21. The method of embodiment 20, wherein the healthy tissue toxicity is bone marrow toxicity.

[0125] Embodiment 22. The method of embodiment 20, wherein the healthy tissue toxicity is anemia, leukopenia, neutropenia or thrombocytopenia.

[0126] Embodiment 23. A method for identifying a subject for treatment, the method comprising: (a) obtaining a sample from the subject; (b) measuring the expression level of a set of genes comprising NGF, BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3, and MELK (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is decreased relative to the expression level of the gene in a control sample; and (d) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP 1 -selective inhibitor if the subject is categorized as likely to respond.

[0127] Embodiment 24. The method of embodiment 23. wherein the set of genes further comprises NOTCH2 and / or PDK1.

[0128] Embodiment 25. A method for identifying a subject for treatment, the method comprising: (a) obtaining a sample from the subject; (b) measuring the gene expression level of a set of genes comprising IFITM2. ACKR3, TAGLN, DIO2, GLIPR1, NNT. ADAM 12, and IFI27,- (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is increased relative to the expression level of the gene in a control sample; and (d) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP 1 -selective inhibitor if the subject is categorized as likely to respond.

[0129] Embodiment 26. The method of embodiment 25, wherein the set of genes further comprises BMPR1B, DMTN, LTBP2, STC2, SLC22A4, CD44, IFI44L, COL16A1, CALB2 and / or GJA1.

[0130] Embodiment 27. The method of any one of embodiments 23-26, wherein if a subject is not identified as likely to respond, the subject is treated with an alternative therapy.

[0131] Embodiment 28. The method of any one of embodiments 23-26, wherein if a subject is not identified as likely to respond, the subject is monitored.

[0132] Embodiment 29. A method of treating cancer in a subject, the method comprising: (a) obtaining a sample from the subject; (b) measuring the expression level of a set of genescomprising NGF, BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3, and MEEK' (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is decreased relative to the expression level of the gene in a control sample; and (d) (i) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categorized as likely to respond or (ii) administering to the subject an alternative therapy or monitoring the subject, if the subject is not identified as likely to respond.

[0133] Embodiment 30. The method of embodiment 29, wherein, the set of genes further comprises NOTCH2 and / or PDK1.

[0134] Embodiment 31. A method of treating cancer in a subj ect, the method comprising: (a) obtaining a sample from the subject; (b) measuring the gene expression level of a set of genes comprising IFITM2. ACKR3. TAGLN. DIO2, GLIPR1. NNT, ADAM12, and IFI27 (c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is increased relative to the expression level of the gene in a control sample; and (d) (i) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categonzed as likely to respond or (ii) administering to the subject an alternative therapy or monitoring the subject, if the subject is not identified as likely to respond.

[0135] Embodiment 32. The method of embodiment 31, wherein the set of genes further comprises BMPR1B, DMTN. LTBP2, STC2. SLC22A4. CD44, IFI44L, COL16A1, CALB2 and / or GJA1.

[0136] Embodiment 33. The method of any one of embodiments 23-31, wherein the control sample is a normal tissue control sample.EXAMPLES

[0137] The examples described in this section are for illustrative purposes only and are not intended to limit the invention.Example 1: PARP1 Inhibition in Combination with Radiation Therapy for the Treatment of CancerMethodsCell Lines and Tissue Culture

[0138] All cancer cell lines were obtained from ATCC. Cells were grown in RPMI, 10% FBS. MRC1 was obtained from Horizon and was grown in EMEM, 10% FBS.Compound-Radi other apy Screening

[0139] Cells were plated at optimized cell densities in triplicate in 50 pL / well; 24 hours after plating, cells were treated using the Echo 650 dose-response module to generate a 5-point dose response profile. 30 hours after plating, cells were irradiated using MultiRad225 (Faxitron) at 2 Gy (1 Gy / minute). Once cells reached 85-90% confluency, plates were fixed with 3% paraformaldehyde (PF A) and permeabilized with 0.5% Triton-X. Hoechst 33342 Solution (Thermo 62249) was added at a final 4 ug / mL concentration. Plates were washed with DPBS before imaging on Molecular Devices HT.ai.Quantification of Compound and Radiotherapy Interactions

[0140] Cell counts were normalized to each plate DMSO-treated condition, and the Area Under the Curve (AUC) was calculated for each drug condition with (AUC_RT) and without Radiation (AUC). Delta AUC was calculated by the difference betw een AUC and AUC_RT (DeltaAUC = AUC_RT - AUC). Delta AUC scores were Z-scored to identify hits. Negative Z-scores indicate radiosensitization and positive scores correspond with radioprotectant effects.IC50 P ARP -Radiotherapy (RT) Characterization

[0141] Cells were plated in triplicate at optimized densities; 24 hours after plating, cells w ere treated with drugs using the Echo 650 dose-response module to generate a 12-point dose response profile. Cells were quantified and normalized to DMSO treatment. Curves w ere fit, and ICso was determined using GraphPad Prism nonlinear regression analysis (four parameters curve fitting).PARP-RT Treatment Sequencing

[0142] Cells were plated in triplicate at optimized densities. The following three conditions were used for testing the sequencing of treatments: Drug-RT = Drug treatment 24 hours post-plating + RT treatment 30 hours after plating. RT-Drug = RT Treatment 24 hours post-plating + Drug Treatment 30 hours post-plating. Cells were quantified and normalized to DMSO treatment. Curves were fit, and IC50 was determined using GraphPad Prism nonlinear regression analysis (four parameters).RNA-seq Analysis of RT Response and Gene Set Enrichment Analysis (GSEA)

[0143] The fresh frozen cell lysates of irradiated and un-irradiated cancer cell lines were shipped to Admera Health (South Plainfield, NJ) for RNA extraction and sequencing. Total RNA of each sample was extracted using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Only high-quality RNA samples with RNA Integrity Number (RIN) greater than 9 were used for library preparation by the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). Each sample library was then sequenced on an Illumina NovaSeq 6000 platform, generating approximately 50 million 2xl50bp paired-end reads. The quality of raw sequencing reads was evaluated using FastQC (v0.11.9) and summarized by MultiQC (vl.l 1) (Andrews, 2010; Ewels et al., 2016). High-quality reads were aligned and quantified at the gene level using kallisto software (vO.46.1) against the GRCh38 human reference genome.

[0144] Gene set enrichment analysis (GSEA) was applied to evaluate the enrichment of gene expression from a pre-ranked list of all available genes with functionally related gene sets using GSEA software (v4.3.0) package (Subramanian et al. 2005). The Hallmark gene set collection (h. all. v7.0. symbols) obtained from the Molecular Signatures Database (MSigDB) website (http: / / software.broadinstitute.org / gsea / rnsigdb) were chosen as the reference gene sets. The Benj amini-Hochberg false discovery rate (FDR) method was used for p-value adjustment. The relevant gene set with normalized enrichment score |NES| >1. P-value < 0.05 and FDR < 0.25 was regarded as statistically significant. Multiple GSEA results were visualized using the R software.ResultsNon-Small Cell Lung Cancer Oncology and Radiotherapy Screen identify PARR inhibitors as radiosensitizers.

[0145] To assess the landscape of radiotherapy (RT) and drug interactions, a panel of 13 NonSmall Cell Lung Cancer cell lines and one non-tumorigenic cell line (MRC5) was screened. A total of 140 drugs was screened across all 14 cell lines, resulting in 1,960 drug-RT interactions. Clustering drugs and cell lines highlighted distinct response profiles across drugclasses. The response to chemotherapies and cytotoxic agents demonstrated the heterogeneity (Average Interaction Score of -1.72) of response to this class of drugs (FIG. 1). Interestingly, PARP1 inhibitors demonstrated the most potent sensitization to RT across the drug panel (Average interaction Score of 0.36). Within the PARP inhibitor panel, AZD5305 demonstrated the most potent sensitization to RT (FIG. 1), which was the only PARP1 selective inhibitor in the panel.

[0146] H650 and H1975 were the most sensitive cell lines to the combination (FIGs. 2A and 2B). In addition, cell lines were sensitive across different oncogenic drivers, such as KRAS and EGFR mutation (FIGs. 2A - 2D). Our approach demonstrates that our screening approach can effectively map the landscape of RT-Drug combinations and identify classes of drugs, which may improve RT response.PARP1 inhibition is a potent radiosensitizer.

[0147] To test the hypothesis that specific PARP1 inhibition is a more potent radiosensitizer, AZD5305 (PARP1) and Talazoparib, Olapraib, Rucaparib (PARP 1 / 2 inhibitors) were tested in H650. There was a shift in IC50s in H650 from >20 nM to 3. 13 nM (2 Gy) and less than 1 nM at 4 Gy (1.007e-5 pM). Notably, the IC50 shift amount of other PARP inhibitors was not as potent (Figures 3B-3D). Olaparib and Rucaparib showed no significant IC50 shift at 2 Gy, and only a modest shift at 4 Gy.

[0148] Additionally. Proteolysis Targeting Chimeras (PROTACs) were tested to assess the impacts of PARP 1 degradation in combination with radiotherapy. i-Rucaparib-AP6 and SK- 575 (PARP1 Selective PROTACT) were tested across the panel of non-small cell lung cancer cell lines. SK-575 was a more potent radiosensitizer compared I Rucaparib- AP6 (FIGs 4A- 4F).Treatment sequencing impacts the outcome of the PARP 1 -Radiotherapy combination.

[0149] To further characterize the interaction between PARP1 inhibition and Radiotherapy (i.e.. PARP1-RT) the sequence of treatments (see methods) was tested. Pre-treatment with PARP1 inhibitor was the most effective radiosensitizer in H650 and H1975 cell lines (FIGs. 5 A and 5B). In contrast pretreatment with Radiotherapy then, PARP1 inhibition did not have a radiosensitizing effect on the cells.Gene Expression Analysis identifies gene signatures associated with PARP1-RT response.

[0150] Gene expression profiles were obtained from the panel of NSCLC cell lines to assess genomic features associated with response to PARP1-RT combination therapy. Gene SetEnrichment Analysis of the top responders and non-responders to the combination therapy revealed a total of 16 differentially enriched pathways (adj. p-value < 0.05. FDR < 25%). Among the top upregulated pathways in responders' cell lines were Interferon Alpha, Epithelial-Mesenchymal Transition, and Xenobiotic metabolism (Figure 6A). Most interestingly, responder cells had suppression of E2F Targets and G2M Checkpoints, pathways that typically regulate normal progression through the cell cycle (FIG 6A). The core ennchment subset of genes of E2F targets revealed several key regulators of DNA repair, such as WEE1, RAD51 API, BRCA1, and BRCA2. Genomic analysis demonstrated that cell lines responding to the combination therapy have distinct expression profiles that may render them sensitive to PARP1-RT combination.

[0151] The expression values of the genes studied are shown in

[0152] Table 1. NGF. BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3, MELK, NOTCH2, PDK1, NUMA1, MAP2K6, MKNK2, MCM3. RFC3, LUC7L3, POLE. HK2, CCND1, SLC7A1, EZH2, PAICS, NASP, MAP3K6, CCNT1, EXOSC8, CIT, TOPI, PDS5B, BRCA1, PNN. ATRX, RPS6KA1, CUL4A, LIG1, MCM7, POLQ, CDKN1B. KIF4A, TRAF2, TFDP1. RADI. TNFRSF1A. SLC29A2, CENPE. UNG, SLBP, SMC4, SPAG5, MRTO4, PTPN11. USP1, TBK1, NUP205, SSI 8. TK1. PPP1R8, AK2, WEE1, KIF22, RPA1. MCM5, CDCA8, SRSF10, ARPC3, HNRNPD, MCM6, PPRC1, NCK1, NEK2, GRB2, POLDI, HELLS, SMC3, EWSR1, RAD51C and NPM1 were more highly expressed in cells that are resistant to the treatment, while TIMM9, ECU, ARF4, AC 0X1, RHOA, NDUFB4, ADH5, COX8A, DDT, ERCC2, ELMOD3, P4HA1, AC ATI. CNDP2, CHUK. TSPO. UQCRB, TIMM10, VDAC2, GUSB, ACP2, RETSAT, PPIA. MGST3, ATG4A, MOCS2. COX5A, CDR2, CD151, HELZ2, GPL, ANXA2, PSMB10, GHITM, RDH11, MINPP1, PKM, COX4I1, NAGK, ATP6APL SLC10A3. GMPPA, FH, RNASEH2A, PSEN1, OGDH ATP6V0E1, PSEN2, NDUFA6, NUDT12, TRIM21. ETFB, PDHA1, TIMM8B, UQCRC1, IDH3A, IDE, QDPR, NFIB, UQCR10, FOSL2, SCHIP1, ESYT1, GSTO1, SLC25A38. CD59, RHOG, LAMP2, MRPS11, SLC25A1, UROD, YWHAH, MARKS, RRP12, TPM4, BMPR1A, EGFR, HADHA, LRP1, CYB5R3, PTGR1, ARID5B, NDUFS6, XRCC3, KDELR3, FLNA, NBN, HSPA4L, MIF, PMM2, ABLIM1, PGM2, ENTPD5, DHRS7, MMP11, GOT2, GLRX5, SPTBN1, GMPPB, ABHD2, SAP 30, SIPA1L1, PEX11G, MX1, ATP6V1E1, MLYCD, F8, BCL2L13, PHLDB1, CHST12, SOWAHC, MAPRE3, LAMC1, CNN3, UPP1. COQ9. DYNLT3, FSTL3, ATP6V1D, PROC, APOBEC3F, ASB13, BASPL SMS, CBR1, SMTN.BCAR1, ARL4A, ANXA9, RIPK2, SKIL, EXT2. TIMP1, PRDX4, ABHD6, RASGRP1, CPQ, MSRB1. MAPK12, CALM1. APOCI, NDST1, RHOD, PVR. GNB4. MAGEE1, NOG. PTGS2, ACADS, PTPN21, FLNB, VLDLR,ABCA3, ENPEP, INHA, SLC27A5, MATN3. HTATIP2, SNX10, LGALS1, P4HA2. ,SRXN1RBPMS, HSP90AA1. MRAS, SLC22A5, C2. SDC2. DECR1, CTSL, SLC25A6, PAOX,DOCKIO, TMCC2, EL0VL6, ITPK1, ESR2, DLC1, BLVRA, .RHOB ,EMP3 GBP2, MPP1,PRPS1, MGLL, G6PD, C1R, SOX9, FZD2, PRDX2, AOX1, TGFB2, IFITM3, PTHLH, IFI44,THBS1, TGFB3, DGKG, CIS, BDNF, EIF4E3, FABP5, OAS2, MAP IB, OSBP2, HMOX1,SMOX, BMPR1B, DMTN, LTBP2, STC2, SLC22A4CD44, IFI44L COL16A1, CALB2, GJA1,IFITM2. ACKR3. TAGLN, DIO2, GLIPR1, NNT. ADAM12 and IFI27 were more highly expressed in cells that are sensitive to the treatment.Table 1: Relative Gene Expression of Genes significantly associated with Response toPARP1-RT

[0153] FIGs. 6C and 6D show the relative expression levels of genes that are decreased and increased, respectively, in cell lines that were resistant and sensitive to the combination therapy.

[0154] To validate the gene signature, the cell lines linked to PARP1-RT response (e.g., A549 cells) or resistance (e.g.. Hl 299 cells) were further tested. The basal expression of the A549 cell profde matched PARP1-RT responsiveness and showed dose-dependent sensitivity to talazoparib-RT (FIG. 7A) and AZD5305-RT (also referred to as saruparib-RT; FIG. 7C)combination therapy. In contrast, H1299 cells, which correlated with non-responsiveness, exhibited no response to AZD5305-RT (FIG. 7D) and minimal activity with talazoparib-RT (Fig. 7B).

[0155] Notably, A549 and Hl 299 cell lines were both resistant to RT doses. For example, A549 cells exhibited a 5% decrease in proliferation at 2 Gy ionizing radiation (FIGs. 8A and 8C), and H1299 cells exhibited a 15% decrease in proliferation (FIGs. 8B and 8D). Once cell lines were treated with PARP inhibitors, the A549 cells were sensitized to RT with a 75% reduction in proliferation at 6 Gy ionizing radiation for both the 0.06 nM and 0.20 nM doses of AZD5305 (FIGs. 8C). Conversely, H1299 cells, which were predicted to be resistant to the PARP1-RT combination therapies, achieved 40% to 50% reduction in proliferation at 6 Gy ionizing radiation for the 0.06 nM and 0.20 nM doses of AZD5305 (FIG. 8D).

[0156] The cell proliferation among non-treated controls was also assessed in comparison to 10 pM of AZD5305 alone, 2 Gy ionizing radiation alone, and a combination of 10 pM of AZD5305 alone and 2 Gy ionizing radiation, among the various cell lines tested above (FIGs. 9A-9D). These control experiments support that the combination therapy results in an unexpected and significant synergistic effect that would not have been predictable based on the effect of RT or PARP1 inhibition alone.Discussion

[0157] Radiotherapy is a comer stone for the treatment of localized and locally advanced tumors. In the clinic chemo-radiotherapy is one of the most used combination therapies for the treatment of modalities. With the advent of genomic technologies, there is an opportunity to leverage these omic scale datasets and rationally design combination therapies. Described herein is a radiotherapy screening platform that systematically screens drug-RT combinations across genetically defined tumors. This approach allows for the leveraging of genomic data to uncover a subset of tumors that may respond to specific drug-RT combinations and identify the omic drivers of response.

[0158] This approach also demonstrated that most non-specific DNA Damaging agents (Helicase inhibitors, DNA intercalators) exhibited heterogeneous responses across the panel (FIG. 1). This observation highlights the challenges of using chemotherapies in combination with radiotherapy. Additionally, it was found that PARP1-RT inhibition is a novel approach for treating non-small cell lung cancers. This example provides experimental evidence that PARP1 inhibition in combination with Radiotherapy is a potent strategy for targeting non-small cell lung cancer (FIGs. 5 A and 5B). Furthermore, PARP1 inhibition specifically appeared more potent than PARP 1 / 2 inhibition. Finally, by integrating this screening data with gene expression profiling, several pathways associated with the response to PARP1-RT combination therapy were uncovered. Among these pathways, G2M checkpoints and E2F targets are of great interest since these pathways govern several key genes, which may make cell lines vulnerable to PARP1-RT combination therapy.

[0159] These signatures could be used to identify tumors that may be responsive to PARP1- RT inhibition and aid in implementing a precision medicine approach for Radiotherapy. Overall, these signatures correlated with response to PARP1 inhibition as observed when testing AZD5305 (saruparib) and talazoparib, both of which are potent PARP1 inhibitors.

[0160] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

[0161] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Claims

CL IMS1. A method of treating cancer in a subject thereof, the method comprising administering to the subject an effective amount of radiation therapy and an effective amount of a poly(ADP-ribose) polymerase 1 (PARPl)-selective inhibitor.

2. The method of claim 1, wherein the PARP1 -selective inhibitor is administered before the radiation therapy.

3. The method of claim 1, wherein the PARP1 -selective inhibitor is AZD5305. AG- 14361, or A-966492.

4. The method of claim 1, wherein the radiation therapy is photon radiation therapy, electron radiation therapy, or proton radiation therapy.

5. The method of claim 1, wherein the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, said fractions having a range of radiation from 1.5 Gy to 80 Gy, collectively.

6. The method of claim 1, wherein the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 0.3 Gy to 16 Gy.

7. The method of claim 1, wherein the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, said fractions having a range of radiation from 8 Gy to 80 Gy, collectively.

8. The method of claim 1, wherein the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 1.6 Gy to 16 Gy.

9. The method of claim 1, wherein the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, said fractions having a range of radiation from 10 Gy to 80 Gy, collectively.

10. The method of claim 1, wherein the effective amount of radiation therapy comprises 1 to 40 fractions of radiation, wherein each fraction has a range of radiation from 2 Gy to 16 Gy.

11. The method of claim 1, wherein the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy of radiation in 1 to 40 fractions at a dose rate of 0.1 Gy / sec or higher.

12. The method of claim 1, wherein the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy of radiation in 1 to 40 fractions at a dose rate of 40 Gy / sec or higher.

13. The method of claim 1, wherein the radiation therapy is ultra-high dose rate (FLASH) radiation therapy.

14. The method of claim 13, wherein the radiation therapy is proton FLASH.

15. The method of claim 14, wherein the effective amount of radiation therapy is a dose of about 10 Gy to about 80 Gy proton FLASH in 1 to 40 fractions at a dose rate of 40 Gy / sec or higher.

16. The method of claim 1, wherein the cancer does not have a mutation rendering it deficient in homologous recombination.

17. The method of claim 1, wherein the cancer does not have a BRCA1 mutation.

18. The method of claim 1, wherein the cancer does not have a BRCA2 mutation.

19. The method of claim 1, wherein the cancer does not have a RAD51 mutation.

20. The method of claim 1, wherein the cancer does not have an ATM mutation.

21. The method of claim 1, wherein the cancer is lung cancer, colon cancer, breast cancer, or melanoma.

22. The method of claim 1, wherein the cancer is non-small cell lung cancer.

23. The method of claim 1, wherein the method significantly reduces bone marrow toxicity compared to other PARP inhibitors.

24. The method of claim 1, wherein the method significantly reduces hematological toxicity compared to other PARP inhibitors.

25. The method of claim 1, wherein the method results in a decrease in tumor size that is at least about 10%, at least about 20%, at least about 30%. at least about 40% or at least about 50% greater than the decrease in tumor size achieved by administering the effective amount of radiation therapy in combination with a PARP inhibitor that is not PARP1 selective.

26. The method of claim 1, wherein the method results in a decrease in healthy tissue toxicity of about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50% compared to the toxicity observed when administering the effective amount of radiation therapy in combination with a PARP inhibitor that is not PARP1 selective.

27. The method of claim 26, wherein the healthy tissue toxicity is bone marrow toxicity7.

28. The method of claim 26, wherein the healthy tissue toxicity7is anemia, leukopenia, neutropenia or thrombocytopenia.

29. A method for identifying a subject for treatment, the method comprising:(a) obtaining a sample from the subject;(b) measuring the expression level of a set of genes comprising NGF, BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3, andMELK(c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is decreased relative to the expression level of the gene in a control sample; and(d) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP 1 -selective inhibitor if the subject is categorized as likely to respond.

30. The method of claim 29, wherein the set of genes further comprises NOTCH2 and / or PDK1.

31. The method of claim 29, wherein if a subject is not identified as likely to respond, the subject is treated with an alternative therapy.

32. The method of claim 29, wherein if a subject is not identified as likely to respond, the subject is monitored.

33. A method for identifying a subject for treatment, the method comprising:(a) obtaining a sample from the subject;(b) measuring the gene expression level of a set of genes comprising IFITM2, ACKR3, TAGLN, DIO2, GLIPR1, NNT, ADAMI 2, and IFI27;(c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is increased relative to the expression level of the gene in a control sample; and(d) administering to the subject an effective amount of radiation therapy and an effective amount of a P ARP 1 -selective inhibitor if the subject is categorized as likely to respond.

34. The method of claim 33, wherein the set of genes further comprises BMPR1B. DMTN, LTBP2, STC2, SLC22A4, CD44, IFI44L. COL16A1, CALB2 and / or GJAL35. The method of claim 33, wherein if a subject is not identified as likely to respond, the subject is treated with an alternative therapy.

36. The method of claim 33, wherein if a subject is not identified as likely to respond, the subject is monitored.

37. A method of treating cancer in a subject, the method comprising:(a) obtaining a sample from the subject;(b) measuring the expression level of a set of genes comprising NGF, BRMS1L, RAD51AP1, ADCY2. RAD54L, BRCA2, POLD3, and MELK,'(c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is decreased relative to the expression level of the gene in a control sample; and(d) (i) administering to the subject an effective amount of radiation therapy and an effective amount of a P ARP 1 -selective inhibitor if the subject is categorized as likely to respond or (ii) administering to the subject an alternative therapy or monitoring the subject, if the subject is not identified as likely to respond.

38. The method of claim 37, wherein, the set of genes further comprises NOTCH2 and / or PDK1.

39. The method of claim 37, wherein the control sample is a normal tissue control sample.

40. A method of treating cancer in a subject, the method comprising:(a) obtaining a sample from the subject;(b) measuring the gene expression level of a set of genes comprising IFITM2, ACKR3, TAGLN, DIO2, GLIPR1, NNT, ADAMI 2, and IFI27(c) categorizing the subject as a likely to respond if the expression level of one or more of the genes in the set of genes is increased relative to the expression level of the gene in a control sample; and(d) (i) administering to the subject an effective amount of radiation therapy and an effective amount of a PARP1 -selective inhibitor if the subject is categorized as likely to respond or (ii) administering to the subject an alternative therapy or monitoring the subject, if the subject is not identified as likely to respond.

41. The method of claim 40, wherein the set of genes further comprises BMPR1B, DMTN. LTBP2, STC2, SLC22A4, CD44, IFI44L, COL16A1, CALB2 and / or GJA142. The method of claim 40, wherein the control sample is a normal tissue control sample.

43. A method for treating cancer in a subject, the method comprising administering to the subject an effective amount of radiotherapy and an effective amount of a PARP1 -selective inhibitor,wherein the subject has a decreased expression level of one or more genes relative to a normal tissue control sample, and wherein the one or more genes is selected from the group consisting of: NGF BRMS1L, RAD51AP1, ADCY2, RAD54L, BRCA2, POLD3,MELK, NOTCH2, an PDKl.

44. A method for treating cancer in a subject, the method comprising administering to the subject an effective amount of radiotherapy and an effective amount of a PARP1 -selective inhibitor, wherein the subject has an increased gene expression level of one or more genes relative to a normal tissue control sample, and wherein the one or more genes is selected from the group consisting of: IFITM2. ACKR3, TAGLN, DIO2. GLIPR1, NNT. ADAM 12, IFI27, BMPR1B, DMTN, LTBP2, STC2, SLC22A4, CD44, IFI44L, COL16A1, CALB2, and GJA1.

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