PARP-1 AND METHODS OF USE THEREOF

[0001] This application claims priority from U.S. Provisional Application No. 62/469,436 filed on March 09, 2017, the entire contents of which is incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby

incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

GOVERNMENT INTERESTS

[0004] This invention was made with government support under Grant Nos. HL072889 and P20GM103S0I awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION [0005] This invention is directed to roles for PARP-1 in disease.

BACKGROUND OF THE INVENTION

[0006] Colon cancer is complex and involves a large number of genetic and environmental factors such as mutations in specific genes and chronic inflammation. In familial adenomatous polyposis (FAP) syndrome, mutations in both alleles of the APC gene that result in its inactivation are considered one of the initial events in colorectal carcinogenesis. It is not clear whether this type of cancer is driven by chronic inflammation although inflammation manifests itself during the course of the disease. Several studies showed conflicting results on the effect of anti-inflammatory conditions on A -driven tumor burden. In carcinogen/chronic inflammation (AOM/DSS)-driven colon cancer, most of the anti-inflammatory factors prevent or reduce tumor burden in mice.

SUMMARY OF THE INVENTION

[0007] Immunotherapy is increasingly regarded as a critical approach to treat many forms of cancers. Its efficacy is sometimes a limitation. Given the role of PARP-1 in the function of MDSCs and the ability of partial PARP inhibition to reduce the suppressive activity of these cells, it is conceivable to use PARP inhibitors at a dose that can be gaged according to the type of cancer and affected patient to achieve a better clinical outcome with immunotherapy approaches. It is also conceivable to use this approach (i.e. partial PARP inhibition) with therapies whose targets do not include DNA repair/damage.

[0008] Aspects of the invention are directed to wards a method for treating a tumor in a subject.

[0009] Aspects of the invention are further directed towards a method of reducing progression or promoting regression of a tumor in a subject,

[0010] Still further, aspects of the invention are directed towards a method of reducing cellular proliferation of a tumor cell in a subject.

[0011] In embodiments, the tumor comprises a solid tumor or a liquid tumor. A tumor can be benign, premalignant, or malignant. In embodiments, the tumor does not comprise a mutation in a BRCA gene, nor is the tumor considered a "triple negative" cancer based on negative oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-type 2 (HER2) expression A tumor can be one that is influenced by the immune system. A tumor can be a primary tumor, or a metastatic lesion. Non-limiting examples of cancers that are associated with tumor formation comprise brain cancer, head & neck cancer, esophageal cancer, tracheal cancer, lung cancer, liver cancer stomach cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer, uterine cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, rectal cancer, and lymphomas. Non-limiting examples of liquid tumors comprise neoplasia of the reticuloendothelial or haematopoetic system, such as lymphomas, myelomas and leukemias. Non-limiting examples of leukemias include acute and chronic lymphoblastic, myeolblastic and multiple myeloma. Typically, such diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Specific myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Specific malignant lymphomas include, non- Hodgkin lymphoma and variants, peripheral T cell lymphomas, adult T cell

leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[0012] In embodiments, the formation and/or growth of the tumor is exacerbated by chronic inflammation, the amount of which is dependent upon tumor type.

[0013] In embodiments, the method comprises administering to the subject afflicted with the tumor a low-dose, therapeutically effective amount of a PARP inhibitor compound.

[0014] In embodiments, the PARP inhibitor compound inhibits the enzymatic activity of one or more proteins in the PARP family of proteins. For example, the PARP inhibitor compound inhibits the enzymation activity of PARP-1, PARP-2, PARP-3, PARP-4, PARP-5a, PARP- 5b, PARP-6, PARP-7, PARP-8, PARP-9, PARP-10, PARP-1 1, PARP-12, PARP-13, PARP- 14, PARP-15, PARP- 16., or any combination thereof. In specific embodiments, the PARP inhibitor compound inhibits the enzymatic activity of PARP-1, PARP-2, PARP-3, or any combination thereof.

[0015] In embodiments, the enzymatic activity inhibited by a PARP inhibitor compound comprises poly(ADP-ribosylation).

[0016] In embodiments, the PARP inhibitor compound comprises a compound of Formula

(I):

[0017] In embodiments, the PARP inhibitor compound is a compound of Formula (I):

[0018] In embodiments, a subject is administered a PARP inhibitor compound at a low dose, therapeutically effective amount. For example, the low dose of a PARP inhibitor comprises a dose that is about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 10Ox, 1 10x, 120x, 130x, 140x, or 150x lower than what is currently prescribed.

[0019] Embodiments of the invention comprise a dose of about Img per day, about Smg per day, about 10mg per day, about 15mg per day, about 20mg per day, about 2Smg per day, about 30mg per day, about 35 mg per day, about 40mg per day or about 50mg per day.

[0020] In embodiments, a subject is administered a PARP inhibitor compound at a low dose, therapeutically effective amount that reduces the enzymatic activity of a PARP between about 10% reduction in activity and about 50% reduction in activity, but nevertheless does not abolish enzymatic activity. Specifically, the method as described herein pertains to administering to a subject a low dose, therapeutically effective amount of a PARP inhibitor compound that reduces the poly(ADP-ribosyl)ation activity of PARP- 1 by about 10%, about 20%, about 30%, about 40%, or about 50%. In other embodiments, the PARP inhibitor compound reduces the enzymatic activity by about 60%, about 70%, about 80%, about 90%, or about 100%, but does not abolish the enzymatic activity of the PARP. In embodiments, the reduction of the enzymatic activity of PARP is compared to the activity level of PARP when activated by DNA damage (such as by DNA damaging chemotherapeulic agents like cisplatin, etoposide, or gamma radiation).

[0021] In embodiments, the PARP inhibitor compound modulates the tumor

microenvironment. For example the PARP inhibitor compound reduces the activity of myeloid derived suppressor cells (MDSCs). In embodiments, the PARP inhibitor compound reduces the tumor suppressive activity of MDSCs. Modulation of the tumor

microenvironment can be measured by, for example, assessment of immune cells within a biopsy (such as by FACS using markers specific to MDSC, CD8, NK, DC).

[0022] In embodiments, the PARP inhibitor compound is administered as a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises at least one additional anti-cancer agent, such as anti-PDl, and/or an anti-inflammatory agent.

[0023] In embodiments, the PARP inhibitor compound is administered in a single dose.

[0024] In embodiments, the PARP inhibitor compound is administered at intervals of about 4 hours, 12 hours, or 24 hours. In some embodiments, the PARP inhibitor compound is administered to the subject on a regular basis, f or example three times a day, two times a day, once a day, every other day or every 3 days. In other embodiments, the PARP inhibitor compound is administered to the subject on an intermittent basis, for example twice a day followed by once a day followed by three times a day; or the first two days of every week; or the first, second and third day of a week. In some embodiments, intermittent dosing is as effective as regular dosing.

[0025] In embodiments, the PARP inhibitor compound is administered orally,

intraperitoneally, subcutaneously, intravenously, or intramuscularly.

[0026] Other objects and advantages of this invention will become readily apparent from the ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG. 1 shows the partial PARP-1 inhibition is sufficient to block expression of inflammatory genes in LPS-treated colon epithelial cells. Primary colon epithelial cells were isolated from WT, PARP-1+/-, or PARP-1-/- mice. (A) Protein extracts were subjected to immunoblot analysis with antibodies to PARP-1 or actin (Note that PARP-1+/- expressed -50% of PARP-1 shown in WT cells. (B) Cells were treated with 2mg/ml LPS. RNA was extracted then analyzed by real-time PCR, (C) Cells were treated with 10 ng/ml TNF-a, Protein extracts were subjected to immunoblot analysis with antibodies to COX-2, VCAM-1, or actin.

[0028] FIG. 2 shows partial PARP-1 inhibition by gene heterozygosity is more efficient than complete inhibition by gene knockout at reducing chronic inflammation-driven colon tumori genesis in an AOM/DSS mouse model of colon cancer. WT, PARP-1+/- or PARP-1-/- mice received a single injection of the carcinogen azoxymethane (AOM) followed by 4 biweekly cycles of DSS (inducer of chronic inflammation) and sacrificed at 16 wks of age. (A) Tumor numbers along the colon were assessed. (B) H&E staining of tumors and

immunohistochemical analysis with antibodies to PCNA, a marker of cell proliferation. (C) H&E staining showing the protective effect of PARP-1 gene heterozygosity or knockout against AOM/DSS-induced colitis.

[0029] FIG. 3 shows pharmacological inhibition of PARP by olaparib is very effective at reducing COX-2 partially reduces the tumor burden in AOM/DSS-treated WT mice. WT were treated with AOM and DSS as described above . Groups of mice were administered S mg/kg or 25 mg/kg olaparib twice a week. Mice were sacrificed at 16 wks of age. (A) Tumor numbers were counted. (B) H&E staining showing the protective effect of PARP-1 inhibition by olaparib against AOM/DSS-induced colitis. [0030] FIG.4 shows PARP-1 inhibition reduces systemic inflammation in AOM/DSS-treated mice. Sera from the different experimental groups and controls were assessed for IL-6, TNF- a, and MCP-1 by ELISA.

[0031] FIG. 5 shows partial inhibition of PARP-1 (by gene heterozygosity or olaparib) protects against, while complete inhibition (by gene knockout) aggravates, induced tumor burden in mice: No major connection with systemic inflammation. WT, PARP-1 +/- or PARP-1-/- mice were bred into an ApcMin background and sacrificed at 16 weeks of age. (A) Tumor numbers along the intestinal track were assessed. Note the opposing effects of PARP-I gene heterozygosity and knockout (B) Size of tumors was assessed and classified as smalt (<2mm), medium (2-4mm), or large (>4rnm). (C) H&E staining of tumors and immunohistochemical analysis with antibodies to PCNA, a marker of cell proliferation. (D) Five wk old ApcMin/+ mice were administered 5 mg/kg olaparib twice a week. Mice were sacrificed at 16 wks of age. Note the protective effect of olaparib. (E) Sera from the different experimental groups and controls were assessed for IL-6, TNF-a, and MCP-1 by ELISA. Note that all forms of PARP inhibition reduced TNF-a and MCP-1 but not IL-6.

[0032] FIG. 6 shows PARP-1 inhibition provides a tumor-suppressive environment and reduces splenomegaly in MCA-38 cell-based allograft (immunocompetent) model of colon cancer. The colon adenocarcinoma cell line derived from a C57BL/6 mouse were engrailed subcutaneously into WT, PARP- 1+/- or PARP- 1 -/- mice. (A) Tumor sizes were measured at different days (IS and 21 days are shown). (B) actual tumors isolated from the different mouse strains. Note that tumors developed in PARP-1+/- or PARP-1-/- mice were significantly smaller than those of WT mice.

[0033] FIG. 7 shows PARP- 1 inhibition by gene heterozygosity or knockout blocks the suppressive activity of MDSCs. Myeloid-derived suppressor cells (MDSCs) were isolated from tumors developed on WT, PARP-1+/- or PARP-1-/- mice. The MDSCs were then tested for their ability to suppress proliferation of CFSC-labeled WT T cells at a MDSC/T cell ratio of 8/1 or 4/1. Note that both PARP-1+/- and PARP-1-/- MDSCs failed to suppress T cell proliferation albeit PARP-1-/- cells showed the least suppression.

[0034] FIG. 8 shows Olaparib (5 mg/kg) reduces the tumor burden of the APCMin/+ mice. Mice received i.p. injections of AZD2281 twice a week for 1 1 weeks. The treatment was started at 5 weeks of age. Tumor burden was assessed at 16 weeks of age.

[0035] FIG. 9 shows tumor burden in mice treated with Olaparib. Mice received an i.p. injection of 10 mg/kg of AOM at 8 weeks of age. A week later, they were given 1.25% of DSS in drinking water for a week followed by two weeks of regular water. This DSS regimen was repeated 4 times. Two groups of WT received i.p. injections of AZD2281 twice a week at either low dose of 5 mg/kg or high dose at 25 mg/kg. All mice were sacrificed at the end of 21 weeks and were subjected to colon tumor burden count. Note that the most effective protection against the tumor burden was achieved by PARP-1 gene heterozygosity.

[0036] FIG. 10 shows Poly(ADP-ribose) polymerase- 1 (PARP-1 ).

[0037] FIG. 11 shows exemplary processes in which PARP-1 is involved in. See

Cancers 2013, 5(3), 943-958.

[0038] FIG. 12 shows chemical structure of (A) Rubraca and (B) Olaparib. Rubraca comes in tablet form— the starting dose is two 300-mg tablets, taken orally twice a day, with or without food.

[0039] FIG. 13 shows the concept of synthetic lethality. Major issues with the strategy include continuous use of high doses of the drug (such as a minimum of 600 mg/day), and development of resistance.

[0040) FIG. 14 shows partial PARP-1 inhibition is sufficient to block expression of inflammatory genes in primary colon epithelial cells. (A) Phase-contrast microscopy of a typical CEC colony emerging from a pure crypt (black arrow); the right panel is a higher magnification, demonstrating isolation of primary colon epithelial cells. (B) CECs were isolated from WT, mice. Protein extracted were subjected to immunoblot analysis, demonstrating that gene heterozygosity reduces (only partially) expression of PARP-1, while knockout eliminates the protein completely. (C and D) CECs were treated with LPS (2μg/ml) for 6 h or TNF after which RNA was extracted; cDNAs were subjected to real-time PCR using set of primers for mouse TNF, IL-6, ICAM-1, VCAM-1 or β-aclin. Fold changes (ΔΔCT values) were then calculated using β-actin as a normalization control. *, p≤0.05; **, p<0.01; ***, p<0.001 ; ****, p<0.0001, demonstrating that partial inhibition of PARP- 1 by heterozygosity is sufficient to inhibit inflammation as shown by markers of inflammation such as TNF, and others.

[0041] FIG. 15 shows partial inhibition of PARP-1 is more effective at reducing

inflammation-driven colon tumorigenesis. WT, mice received 10 mg/kg οΓΑΟΜ, /'./;. once followed by 4 cycles of 2.5% DSS in drinking water. (A) At 21 weeks of age, mice were sacrificed and colon tumor burden was counted. (B) H&E staining of colon tumor sections. (C) IHC with an anti-PCNA antibody. (D) H&E staining showing the protective effective of against AOM/DSS-induced colitis. (E) Sera were assessed for IL-6, TNF or MCP-1 using sandwich ELISA. *, p<0.05; **, p<0.01 ; ***, p<0.001 ; ****, p<0.0001. [0042] FIG. 16 shows involvement of immune cells in tumor microenvironment and tumorigenesis.

[0043] FIG. 17 shows PARP-1 inhibition by gene heterozygosity or knockout blocks the suppressive activity of MDSCs

[0044] FIG. 18 shows PARP-1 inhibition does not interfere with T or dendritic cell function. Further, see J Immunol June 15, 2006, 176 (12) 7301-7307.

[0045] FIG. 19 shows Low dose of a PARP inhibitor is as effective as a high dose in blocking colon cancer when administered at a very early stage

[0046] FIG. 20 shows Low dose of a PARP inhibitor is much more effective than the high dose in blocking colon cancer when administered after a clear development of tumors

[0047] FIG. 21 shows (A) A sample of genotyping: Bands representing WT APC or (top panel) or WT PARP-1 or KO (bottom panel). Genotype of the mice is displayed below the panels. (B) Tumor numbers were counted at 16-week-old of age in A

mice (per group>10 mice). (C) Tumor burden was analyzed based on size and divided in groups lower than 2mm, 2-4mm, and tumors bigger than 4 mm. Colon tumor sections from were subjected to immunohistochemistry (IHC) with antibodies specific to PCNA (D) or COX-2 (E). *, p<0.05; ·*, p<0.01; ***, p<0.001;

p<0.0001.

[0048] FIG. 22 shows mice were randomized into 3 groups and received, i.p., 5 mg/kg of olaparib (0.005% DMSO in saline), 25 mg/kg of the drug twice a week, or vehicle from 5 weeks up to 16 weeks of age. Mice were then sacrificed and tumor burden was quantified. (B) Weight of mice from the different groups at 16 weeks of age. *, p<0.05; **, p<0.01 ; ***, p<0.001.

[0049] FIG. 23 shows sera from the different mouse groups were assessed for IL-6, TNF or MCP-1 using sandwich ELISA according to manufacturer's instructions.

[0050] FIG. 24 shows (A) Protein extracts from MCA-38 cells, RAW264.7 cells, or colon epithelial cells derived from WT, PARP- 1 -, or mice were subjected to

immunoblot analysis. MCA-38 cells (2.5x10 ⁵ ) were injected subcutaneously into the left flank of WT, (B) Tumor sizes were measured at day 15. (C)

Images of two representative tumors isolated from the different groups. Bar=lcm. (D) Sera from the different mouse groups were assessed for TNF by ELISA as above. Tissue sections from MCA-38-generated tumors were subject to IHC with antibodies to ICAM-l(E) or CD68, a macrophage marker (F). For (B) and (D): *, p<0.05; **, p<0.01; ***, p<0.001. [00511 FIG. 25 shows (A) MDSCs derived from tumors of WT, PARP- mice (n=4 for each group) were assessed for their ability to suppress proliferation of CFSC-labeled WT T cells at a MDSC/T cell ratio of 1 :8. (B) Bone marrow (BM)-derived DCs were incubated with 10ng/ml GM-CSF for 8 days; some WT DCs were cultured in the presence of 1μΜ olaparib (spiked every 2 days). CD1 lc ⁺ cells were co-cultured with CFSE-labeled CD4' T cells from OTII mice. T cell proliferation was assessed by FACS. *, p<0.05; **, p<0.01; ***, p<0.001.

[0052] FIG. 26 shows A549 cells were subjected to knockdown using a lentiviral vector encoding a shRNA or to a CRISPR/Cas9 plasmid system (Santa Cruz.) targeting PARP-1 and their respective controls. After selection, protein extracts were subjected to immunoblot analysis with antibodies to PARP-1 or actin.

[0053] FIG. 27 shows high PCNA immunoreactivity (a marker of cell proliferation) was detected in tumors of AOM/DSS-treated WT and PARP-1 mice, which was much lower in tumors of treated PARP- 1 ' mice (p<0.001 ).

[0054] FIG. 28 shows weight loss observed in the mice was prevented by PARP-1

heterozygosity but not KO (FIG. 28).

[0055] FIG. 29 shows rrepresentative PCR products showing the genotype of the different mouse strains..

[0056] FIG.30 shows (A) primary macrophages were isolated from WT or PARP- mice. Cells were then incubated either for 12 or 24 h in the presence or absence of the indicated concentration of oxLDL. Protein extracts were prepared from then collected cells the subjected to immunoblot analysis with antibodies to CHOP or Aclin. (B) RAW cells (mouse macrophage cell line) were treated with 10 mg/ml oxLDL for 6 h in the presence or absence of 1 mM of the PARP inhibitor TIQ-A. Protein extracts were prepared from the collected cells then subjected to immunoblot analysis with antibodies to CHOP.

[0057] FIG.31 shows that (A) PARP inhibition my also enhance the antitumor effects of low dose of PARP inhibitor compared to higher dose, and that (B) this effect may be due to increased infiltration of cytotoxic CD8+ T- cells in the tumor microenvironmenL

[0058]

DETAILED DESCRIPTION OF THE INVENTION [0059] Detailed descriptions of one or more preferred embodiments of compositions and methods for treating tumors comprising administering PARP inhibitor compounds are provided herein.

[0060] It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0061] The singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0062] Wherever any of the phrases "for example," "such as," "including" and the like are used herein, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. Similarly "an example," "exemplary" and the like are understood to be nonlimiting.

[0063] The term "substantially" allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[006 ] The terms "comprising" and "including" and "having" and "involving" (and similarly "comprises", "includes," "has," and "involves") and the like are used

interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b and c. Wherever the terms "a" or "an" are used, "one or more" is understood, unless such interpretation is nonsensical in context.

[0065] As used herein, the term "about" can refer to approximately, roughly, around, or in the region of. When the term "about" is used i n conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. The term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0066] "Amelioration" can refer to any lessening of severity, delay in onset, slowing of growth, slowing of metastasis, or shortening of duration of a tumor or cancer, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of aPARP inhibitor compound or composition.

[0067] The terms "individual", "patient" and "subject" can be used interchangeably. They refer to a mammal (e.g., a human) which is the object of treatment, or observation. Typical subjects to which PARP inhibitor compounds can be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.

[00681

[0069] Partial Inhibition of Polv(ADP-ribose) polymerase ) for the treatment of disease

[0070) Poly(ADP-ribose polymerases (PARPs) is a family of proteins that play a role not only in DNA repair, but also in fundamental cellular processes such as chromatin remodeling, transcription, and regulation of the cell cycle. PARPs interact with various cellular proteins and transcription factors, including those that aid inflammation. Various studies have shown that DNA damage occurs during inflammatory conditions, and that PARPS (for example, PARP- 1) participate in inflammation through the response to DNA damage. In fact, DNA damage that occurs during inflammation leads to an over activation of PARPs (Deslee G, et al. Chest (2009) 135:965-74; Althaus FR, et al. Mol Cell Biochem (1999) 193:5-1 1 ; Pereira C, et al. Inflamm Bowel Dis (2015) 21 :2403-l 7; Palmai-Pallag T, et al. Microbes Infect (2014) 16:822-32) which can result in an energy crisis due to depletion of their substrate, i.e., nicotinamide adenine dinucleotide (NAD+), thus leading to non-specific cell death (i.e., necrosis) (Ha HC, Snyder SH. Proc Natl Acad Sci U SA (l 999) 96: 13978-82.).

[0071] Mammalian PARP-1 is a 1 16-kDa protein which comprises of an AMerminal DNA- binding domain, a nuclear localization sequence (NLS), a central automodification domain, and a C-terminal catalytic domain (Luo X, et al. Genes De\ (2012) 26:417-32). The C- terminal region is the most conserved part of the PARP family of proteins, and executes its catalytic function. Specifically, the C-terminal region synthesizes poly(ADP)ribose (PAR) using NAD+ as a substrate (35, 36) and transfers the PAR moieties to several proteins, including histones, DNA repair proteins, and transcription factors (Ame JC, et al. Bioessays (2004) 26:882-93; Schreiber V, et al. Nat Rev Mol Cell Biol (2006) 7:517-28), ultimately altering the structure and functions of the acceptor proteins. Target proteins comprise, for example, a PARP protein itself (for example automodification of PARP-l 's BRCT (Breast Cancer Carboxy-Terminal) domain), or modification of other proteins (i.e.,

heteromodification) such as Histone HI, H2B, DNA pol a, topoisomerase I, II, lamin B, XRCC1, SV40 T-Ag, DNAS1L3; DFF40; p65 NF-kB, and/or STAT6. Under genotoxic stress conditions, PARP-1 binds itself to the nucleosomes containing intact (Kim MY, et al. Cell (2004) 1 19:803-14) as well as damaged DNA structures (e.g., nicks and double-strand breaks) which leads to the activation of DNA repair enzymes (D'Amours D, et al. Biochem J (1999) 342(Pt 2): 249-68).

[0072] The covalently attached PAR can be hydroly/ed to free PAR or mono(ADP-ribose) by PAR glycohydrolase (PARG) (Min W, et al. From Biosci (Landmark Ed) (2009)

14: 1619-26). Synthesis and degradation of PAR chains is tightly controlled in vivo and PAR residues have a very short half-life in the cell (few minutes) (Luo X, et al. Genes Dev (2012) 26:417-32). Free or protein-bound PAR polymers also work as signal transducers by binding other proteins.

[0073] PARP-1 gets activated in response to DNA damage induced by ROS/RNS under inflammatory conditions (Ba X, el al. Am J Pathol (201 1) 178:946-55, Pacher P, el al. Am J Pathol (2008) 173:2-13.). Although, the primary aim of PARP-1 is to maintain the genome integrity, its over activation under extensive and persistent DNA damaging environment promote inflammatory conditions. Over activation of PARP-1 depletes its substrate, i.e., NAD+, bringing the cell to an energy deficient state, thus leading to necrosis (Islam BU, et al. Indian J Clin Biochem (2015) 30:368-85). Recently, PARP-1 has been reported to cause cell death by suppressing the activity of hexokinase-1 (an essential enzyme of glycolysis) by adding PAR chains (Fouquerel E, et al. Cell Rep (2014) 8: 1819-31). Apart from inducing cellular death, PARP-1 promotes inflammation by influencing chromatin remodeling and expression of several pro-inflammatory factors. Since the DNA is negatively charged, poly(ADP)ribosylalion (also negatively charged) of histones results in relaxing of nucleosomal structures and, hence, aids the transcription of pro-inflammatory genes

(Martinez-Zamudio R, et al. Mol Cell Biol (2012) 32:2490-502, Martinez-Zamudio RI, et al. Brain Behav (2014) 4:552-65). PARP-1 regulates the expression of several NF-KB- dependent cytokines, chemokines, adhesion molecules, inducible nitric-oxide synthase

(iNOS), required for the manifestation of inflammatory cycle (Naura AS, et al. Eur RespirJ (2009) 33:252-62; Chiang j, et al. Eur J Pharmacol (2009) 610: 119-27; von Lukowicz T, et al. Cardiovasc Res (2008) 78:158-66; Park EM, et al. Stroke (2004) 35:2896-901; Zingarelli B, et al. Circ Res (1998) 83:85-94; Sharp C, et al. Inflammation (2001) 25: 157-63; Ullrich O, et al. Nat Cell Biol (2001 ) 3: 1035-42). PARP-1 gene deletion or its pharmacological inhibition results in suppressed migration of leukocytes to the inflammatory sites (Rosado MM, et al. Immunology (2013) 139:428-37). Overall, studies demonstrate that PARP-1 plays a pro-inflammatory role by inducing cellular death and upregulating the expression of various inflammatory genes, via interaction with NF-κΒ (Zerfaoui M, et al. J Immunol (2010) 185:1894-902; Hassa PO, Cell Mol Life Sci (2002) 59:1534-53). Further, see Sethi GS el al. From. Immunol. (2017) 8: 1 172).

[0074] Poly(ADP-ribose) polymerase (PARP-1 ) is a nuclear enzyme that polymerize adenosine diphosphate ribose on substrate proteins to regulate various processes. See FIG. 1 1 , for example. The function of PARP-1 in cancers may be intimately related to its role in providing alternative and efficient pathways to cancer cells to survive especially for those associated with defects in DNA repair (e.g. triple negative breast and ovarian cancers). The role of PARP-1 in DNA repair requires full activity of the enzyme as partial inhibition of PARP-1 has not been associated with obvious defects in DNA repair. Achieving maximum inhibition of PARP-1 to treat cancers with DNA repair deficiency (e.g. breast or ovarian cancers with mutations in BRCAI gene) is critical to induce synthetic lethality of cancer cells. As discussed herein, the focus on achieving maximal or about maximal inhibition of PARP may contribute to the failure of two recent clinical trials using the PARP-1 -inhibitor, olaparib (Lynpar/a ^rM ), on patients with advanced colon cancer as a monotherapy or in combination with irinotecan (Camptosar ^{I M} ), a topoisomerase I inhibitor. Achieving maximum inhibition of PARP to treat cancers with DNA repair deficiency (e.g. breast or ovarian cancers with mutations in BRCAI gene) is critical to induce synthetic lethality of such cancer cells.

However, partial inhibition may be the best approach to blocking inflammation-driven cancer or certain mutation driven cancers, such as APC mutation-driven (e.g. FAP) colon cancer.

[0075] Without wishing to be bound by theory, DNA repair enzymes such as PARP- 1 play important roles in not only cancer-related processes but also in the pathogenesis of many inflammatory diseases. However, it's role in inflammation may be very different than that in cancer. PARP-1 has a critical role during inflammation, in part, through its relationship with NF-kB, and embodiments as described herein demonstrate the roles of PARP-1 in colon inflammation and cancer and their relationship. Examples described herein demonstrate the clinically relevant role of partial inhibition of a PARP enzyme, such as PARP-1 , by using chemical inhibitors of PARP, while also taking advantage of the fact that PARP-1 gene heterozygosity reduces expression and activity of PARP-1 by about 50%. As an example of a clinically relevant PARP inhibitor, examples described herein utilize olaparib (AZD2281)), a potent inhibitor of PARP-1, PARP-2, and PARP-3 that is used in the clinic as a monotherapy for triple-negative ovarian cancer. Triple negative ovarian cancer is defined based on negative oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-type 2 (HER2) expression. Experiments described herein also use the extensively studied mouse model of intestinal cancer, which is a standard model for spontaneous tumorigenesis. In this model, aberrant Wnt/β-catenin signaling following loss of the tumor suppressor gene, adenomatous polyposis coli (APC), is thought to initiate colon adenoma formation. Still further, experiments described herein also use the

carcinogen/inflammation colon cancer model (azoxymethane+DSS-driven). In this model, DSS regimen is given to induce chronic relapsing inflammation after the potentiation by the carcinogen, axoxymethane (AOM), in colon. Finally, a MCA-38 colon carcinoma cell-based allograft model was used in examples described herein, which allows for the investigation of the host environment response to tumor growth.

[0076) Referring to the Examples, partial PARP-1 inhibition (50%) was very effective at reducing or even blocking expression of inflammatory genes in response to LPS or TNF-α treatment and that complete inhibition of the enzyme was not necessary to achieve maximal effects. See FIG. 14, for example. This appears to be due, in part, to a reduction in NF-KB- signal transduction. The remarkable anti-inflammatory effects of PARP-1 inhibition (partial or total) can be protective against chronic inflammation-driven colon carcinogenesis.

Surprisingly, partial PARP- 1 inhibition by gene heterozygosity was more efficient than complete inhibition by gene knockout at reducing chronic inflammation-driven colon tumorigenesis using an azoxymethane (AOM) followed by dextran sulfate sodium (DSS) exposure-based model of the condition although both genotypes provided similar reduction in the levels of systemic and colonic inflammation. See FIG. 15, for example. When a mutation in the p-catenin pathway was the main driver for intestinal tumorigenesis, partial PARP-1 inhibition by gene heterozygosity or olaparib protected against the tumor burden in mice while complete inhibition by gene knockout aggravated the burden. See FIG. 5 and FIG. 22, for example. These differential effects were not mirrored with respective effects on systemic or intestinal inflammation, splenomegaly, or cachexia as all conditions lowered the aforementioned traits. Using a MCA-38 colon carcinoma cell-allograft mouse model, the opposing effects of PARP-1 gene dosages on intestinal tumorigenesis occurred despite that they both provide a tumor suppressive microenvironment through a regulation of the function of Myeloid-Derived Suppressor Cells (MDSCs). These results exemplify the complexity of the role of PARPs in colon tumorigenesis, inflammation, and immunity that could be harnessed to effectively treat not only colon cancer but also other cancers that exist within a tumor microenvironment, such as breast, liver and prostate..

[0077] The results of the inventors' studies exemplify the complexity and the potentially paradoxical roles of PARPs in cancer by using colon carcinogenesis as a model. Achieving maximum inhibition of PARP to treat cancers with DNA repair deficiency (e.g. breast or ovarian cancers with mutations in BRCAI gene) is critical to induce synthetic lethality of cancer cells. However, partial inhibition may be the best approach to blocking inflammation- driven or APC mutation-driven (e.g. FAP) colon cancer. The focus on achieving maximal inhibition of PARP may be the reason for the failure of two recent clinical trials using olaparib (Lynparza™) on patients with advanced colon cancer as a monotherapy or in combination with irinotecan (Camptosar ^{I M} ), a topoisomerase I inhibitor. In some instances, the patient may be developing resistance to the drug, in part due to the high doses administered (such as 600mg/day).

[0078) In cancer, the normal intercellular interactions in tissues are disrupted, and the tumor microenvironment evolves to accommodate the growing tumor. The tumor microenvironment (TME) refers to the cellular environment in which a tumor exists, including components such as surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM). Referring to FIG. 6 which utilizes a MA-38 cell-based allograft

(immunocompetent) model of colon cancer, PARP inhibition provides a tumor-suppressive microenvironment. Specifically, this example demonstrates the effect of inhibiting PARP only in the immune cells of the subject (and not in the cancer cells), However, of clinical relevance, when PARP is inhibited in both immune cells and in cancer cells (as is the case when a PARP inhibitor is administered to a subject), low doses of PARP inhibitors selectively affect the tumor microenvironment (such as MDSCs) but not cancer cells. See FIG. 5, for example.

[0079] Tumor microenvironment is complex and is heavily influenced by immune system. Emerging immune cells that influence and/or drive tumorigenesis are known as Myeloid- derived suppressor cells (MDSCs). MDSCs are a heterogeneous population of cells that are defined by their myeloid origin, immature state and ability to potently suppress T cell responses. MDSCs migrate from the basement membrane (BM) and recruit to the site of tumor by tumor-associated macrophages (TAM). MDSCs are potent immune suppressors (i.e., immunosuppressive), which as a result contributes to tumor progression. Specifically, MDSCs can infiltrate a developing tumor and promote vascularization, inhibit major pathways of immunosurveillance, inhibit natural killer (NK) cell-dependent cytotoxicity, inhibit T and B cell proliferation, inhibit antigen presentation by dendritic cells (DC), and drive Ml macrophage polarization.

[0080] MDSCs are increasingly being viewed as important players in promoting progression or even resistance of most cancers. Referring to the Examples, PARP-1 plays a role in the function of MDSCs. Again, partial inhibition of PARP-1 is sufficient to interfere with the suppressive capacity of these cells (see FIG. 7 and FIG. 17, for example). The role of PARP-1 in the function of MDSCs may be harnessed as an added therapy to block many forms of cancers including colon cancer.

[00811 Importantly, PARP- 1 inhibition interferes with the suppressive capacity of MDSCs, while having little to no effect on T cell or dendritic cell function (see FIG. 18, for example).

[0082)

[0083) Therapeutic Methods

[0084] Described herein are methods of treating a subject afflicted with a tumor and/or cancer comprising administering to a subject a low dose of a PARP inhibitor compound. The term "low dose" refers to a very small quantity of the PARP inhibitor compound relative to the well-established/conventional larger quantities (such as 600mg/day) that are known to produce an effect in certain cancers with DNA repair deficiency (e.g. breast or ovarian cancers with mutations in BRCA1 gene). As described herein, a low dose of a PARP inhibitor compound produces a different effect than the well-established higher dose. Referring to FIG. 20, for example, low dose of a PARP inhibitor compound is much more effective than the high dose in blocking colon cancer when administered to a subject after the clear

development of tumors.

[0085] A low dose of a PARP inhibitor compound is a quantity that is effective for partial inhibition of PARP-1 en/ymatic activity. In another embodiment, a low dose of a PARP inhibitor compound is a dose that is below that which represents the threshold tor maximal and/or complete inhibition of en/ymatic activity. "Partial inhibition" can refer to any measurable reduction in the en/ymatic activity of a PARP that is less than maximal (i.e., complete) inhibition. For example, partial inhibition of a PARP can refer to reducing the enzymes activity to about 50%, enzymatic activity, about 40% enzymatic activity, about 30% enzymatic activity, about 10% enzymatic activity, or about 1% enzymatic activity. Referring to FIG. 5, for example, partial inhibition of PARP-1 enzymatic activity (such as by about 50%) protects against, while complete inhibition of PARP-1 activity aggravates, tumor burden. Referring to FIG. 15, for example, partial inhibition of PARP-1 activity (such as by about 50%) is more effective at reducing inflammation-driven colon tumorigenesis than complete inhibition of PARP-1 activity. Mechanistically, partial inhibition of PARP-1 activity is sufficient to block expression of inflammatory genes in primary colon epithelial cells (see FIG. 14, for example), indicating that it is not necessary to inhibit all enzymatic activity (as is the goal of large doses of PARP inhibitors) to reduce inflammation.

[0086] A low dose of a PARP inhibitor compound can refer to a dose that is about 1 Ox to 150x lower than what is currently prescribed (e.g. see description herein). For example the low dose of a PARP inhibitor compound comprises a dose that is about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1 10x, 120x, 130x, 140x, or 150x lower than what is currently prescribed.

[0087] For example, certain studies currently administer to a subject 5mg/kg per day of a PARP inhibitor compound to achieve an effect, wherein an embodiment of the invention can comprise a low dose of about 0.2 mg/kg per day of a PARP inhibitor compound. Thus, a 60kg individual may be administered approximately 300mg per day of a PARP inhibitor to achieve an effect, whereas embodiments of the invention may comprise administering to a subject about 12mg per day of a PARP inhibitor compound. In other embodiments of the invention, a subject can be administered a PARP inhibitor at a dose of between about Img per day to about 30mg per day. For example, embodiments of the invention comprise a dose of about Img per day, about 5mg per day, about 10mg per day, about 15mg per day, about 20mg per day, about 25mg per day, about 30mg per day, about 35 mg per day, about 40mg per day or about 50mg per day. The terms "treat," "treating" or "treatment" can refer to the lessening of severity of a tumor or cancer, delay in onset of a tumor or cancer, slowing the growth of a tumor or cancer, slowing metastasis of cells of a tumor or cancer, shortening of duration of a tumor or cancer, arresting the development of a tumor or cancer, causing regression of a tumor or cancer, relieving a condition caused by a tumor or cancer, or stopping symptoms which result from a tumor or cancer. The terms "treat," "treating" or "treatment", can include, but are not limited to, prophylactic and/or therapeutic treatments. Referring to FIG. 5, for example, partial inhibition of PARP-1 activity protects against tumor burden in vivo (e.g., reduces tumor number/mouse and tumor size), while complete inhibition of PARP-1 activity aggravates tumor burden in vivo.

[0088) As used herein, the terms "rumor" and "cancer" can be used interchangeably, and generally refer to a physiological condition characterized by the abnormal and/or unregulated growth, proliferation or multiplication of cells. [0089] In embodiments, a "tumor" or "solid tumor" can refer to a solid mass of tissue that is of sufficient si/e such that an immune response can be detected in the tissue. A tumor may be benign, premalignant, or malignant. A tumor may be a primary tumor, or a metastatic lesion. Examples of cancers that are associated with tumor formation include brain cancer, head & neck cancer, esophageal cancer, tracheal cancer, lung cancer, liver cancer stomach cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer, uterine cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, rectal cancer, and lymphomas. One of ordinary skill in the art would be familiar with the many disease entities that can be associated with tumor formation.

[0090] A "liquid tumor" can refer to neoplasia that is diffuse in nature as they do not typically form a solid mass. Examples include neoplasia of the reticuloendothelial or haematopoetic system, such as lymphomas, myelomas and leukemias. Non-limiting examples of leukemias include acute and chronic lymphoblastic, myeolblastic and multiple myeloma. Typically, such diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Specific myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Specific malignant lymphomas include, non-Hodgkin lymphoma and variants, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[0091] The approach as described herein (i.e., administration of a low dose of a PARP inhibitor) will provide clinical benefit, defined broadly as any of the following: inhibiting an increase in cell volume, slowing or inhibiting worsening or progression of cancer cell proliferation, reducing primary tumor size, reducing occurrence or size of metastasis, reducing or stopping tumor growth, inhibiting tumor cell division, killing a tumor cell, inducing apoptosis in a tumor cell, reducing or eliminating tumor recurrence. Referring to FIG. 5 and FIG. 21, for example, partial inhibition of PARP- 1 activity protects against (e.g., reduces tumor number/mouse and tumor size) tumor burden in vivo, while complete inhibition of PARP- 1 activity aggravates tumor burden in vivo. Notably, ail large tumors are absent in the subjects with partial inhibition of PARP- 1.

[0092] [0093] Administration

[0094] Described herein are methods of treating a subject afflicted with a tumor and/or cancer comprising administering to a subject a low dose of a PARP inhibitor compound. The term "administration" can refer to introducing a PARP inhibitor compound or composition comprising the same into a subject. In general, any route of administration can be utilized. Non-limiting examples of routes of administration comprise parenteral (e.g., intravenous), intraperitoneal, oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments. In some embodiments, administration is intraperitoneal. Additionally or alternatively, in some embodiments, administration is parenteral. In some embodiments, administration is intravenous. In other embodiments, administration is orally.

[0095] In embodiments, the PARP inhibitor compound can be administered to a subject before, during or after the development of a tumor or cancer. See, for example, FIG. 1 and 20. In some embodiments, the PARP inhibitor compound is used as a prophylactic and is administered continuously to subjects with a propensity to develop a tumor. In some embodiments, the PARP inhibitor compound is administered to a subject during or as soon as possible after the development of a tumor. In some embodiments, the administration of the PARP inhibitor compound is initiated within the first 48 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. In some embodiments, the initial administration of the PARP inhibitor compound is via any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, intraperitoneally and the like, or combination thereof. The PARP inhibitor compound should be administered as soon as is practicable after the onset of a cancer is detected or suspected, and for a length of time necessary for the treatment of the cancer, such as, for example, from about 1 month to about 3 months. The length of treatment can vary for each subject, and the length can be determined using the known criteria. In some

embodiments, the PARP inhibitor compound is administered for at least 2 weeks, between about 1 month to about 5 years, or from about 1 month to about 3 years.

[0096] The terms "co-administration" or the like, as used herein, can refer to the administration of a PARP inhibitor compound and at least one additional compound, such as a second PARP inhibitor compound or an anti-cancer agent, such as anti-PDl, to a single subject, and is intended to include treatment regimens in which the compounds and/or agents are administered by the same or different route of administration, in the same or a different dosage form, and at the same or different time.

[0097] Therapeutically effective amounts can depend on the severity and course of the cancer, previous therapy, the subject's health status, weight, and response to the drugs, and the judgment of the treating physician. Prophylactically effective amounts depend on the subjects state of health, weight, the severity and course of the disease, previous therapy, response to the drugs, and the judgment of the treating physician.

[0098] In some embodiments, the PARP inhibitor compound is administered to the subject on a regular basis, e.g., three times a day, two times a day, once a day, every other day or every 3 days. In other embodiments, the PARP inhibitor compound is administered to the subject on an intermittent basis, e.g., twice a day followed by once a day followed by three times a day; or the first two days of every week; or the first, second and third day of a week. In some embodiments, intermittent dosing is as effective as regular dosing. In further or alternative embodiments, the PARP inhibitor compound is administered only when the patient exhibits a particular symptom, e.g., the onset of pain, or the onset of a fever, or the onset of an inflammation, or the onset of a skin disorder. If two or more compounds are administered, dosing schedules of each compound can depend on the other or can be independent of the other.

[0099] In an embodiment, the administration of the PARP inhibitor compound can be administered chronically, that is, for an extended period of time, including throughout the duration of the subject's life in order to ameliorate or otherwise control or limit the symptoms of the subject's disorder.

[00100] In another embodiment, the administration of the PARP inhibitor compound can be given continuously; alternatively, the dose of drug being administered can be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday can vary between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday may be from 10%- 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[00101 ] In embodiments, a maintenance regimen can be administered if necessary, such as once a subject's condition has improved. Subsequently, the dosage or the frequency of administration of the PARP inhibitor compound can be reduced, for example as a function of the symptoms or tumor si/e, to a level at which the individual's improved condition is retained. Individuals can, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.

[00102] The amount of the PARP inhibitor compound administered to a subject can vary depending upon factors such as the particular compound, cancer and its severity, the identity (e.g., weight) of the subject or host in need of treatment, and is determined according to the particular circumstances surrounding the case, including, e.g., the specific agents being administered, the routes of administration, the tumor being treated, and the subject or host being treated.. In general, however, doses employed for adult human treatment will typically be in the range of about 0.02mg per day to about 50mg/day, or from about 1 mg per day to about 30 mg per day. For example, embodiments of the invention comprise a dose of about 0.1 mg per day, about lmg per day, about 5mg per day, about 10mg per day, about 15mg per day, about 20mg per day, about 2Smg per day, about 30mg per day, about 35 mg per day, about 40mg per day or about SOmg per day. The desired dose of each compound can be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[00103] The PARP inhibitor compound can be provided in a unit dosage form suitable for single administration of precise dosages. The unit dosage may be in the form of a package containing discrete quantities of the compound. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers can be used, in which case it is typical to include a preservative in the

composition. By way of example only, formulations for parenteral injection may be presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative.

[00104] It is understood that a medical professional will typically determine the dosage regimen in accordance with a variety of factors. These factors include the cancer and/or tumor from which the subject suffers, the degree of metastasis, as well as the age, weight, sex, diet, and medical condition of the subject.

[00105]

[00106] Compounds. Pharmaceutical Formulations, and Compositions

[00107] Described herein are methods of treating a subject afflicted with a tumor and/or cancer comprising administering to a subject a low dose of a PARP inhibitor compound. [00108] Definition of standard chemistry terms are found in reference works, including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4 ^th ED." Vols. A (2000) and B (2001), Plenum Press, New York, the entire contents of which are incorporated herein by reference. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art can be employed. Unless specific definitions are provided, the nomenclature employed in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known in the art. Standard techniques are optionally used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[00109] The PARP inhibitor compounds described herein can be selective for PARP- 1 , but can also be selective for PARP-2, PARP-3, or any combination of PARP- 1, PARP-2, and/or PARP- 3 (such as rucaparib). In embodiments, the PARP inhibitor compound is a PARP-1 inhibitor compound. In embodiments, the PARP inhibitor compound is a PARP-1 /PARP-2 inhibitor compound. In still other embodiments, the PARP inhibitor compound is a PARP- 1/PARP-2/PARP-3 inhibitor compound.

[00110] In an embodiment, the PARP inhibitor compound is a compound of Formula (I).

[00111] In some embodiments, the PARP inhibitor is Olaparib (i.e., AZD, Lynparza). Non- limiting examples of other PARP inhibitors comprise nicotinamide analogues (such as 3- Aminobenzamide), TIQ-A, NU 1025, PJ-34, AIQ, PD12873, ABT-888, AGO 14699, among others.

[00112] Pharmaceutical compositions comprising a PARP inhibitor compound can be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), the entire contents of which are incorporated by reference herein in their entireties.

[00113] PARP inhibitor compounds can be incorporated into pharmaceutical compositions suitable for administration to a subject. A pharmaceutical composition can refer to a mixture of a PARP inhibitor compound with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

[00114] For example, such compositions can comprise a compound of formula (I) and a pharmaceutically acceptable carrier. In embodiments, the composition comprises a PARP inhibitor and a pharmaceutically acceptable carrier. For example, non-limiting examples of pharmaceutically acceptable carriers comprise solid or liquid fillers, diluents, and

encapsulating substances, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral oil.

[00115] Pharmaceutical compositions can be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[00116] The pharmaceutical compositions described herein can be administered by any suitable administration route, including but not limited to, oral, inlerparenteral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intraperitoneal, intranasal, buccal, topical, rectal, or transdermal administration routes.

[00117] The pharmaceutical compositions described herein are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by an individual to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In some embodiments, the compositions are formulated into capsules. In some embodiments, the compositions are formulated into solutions (for example, for IV

administration).

[00118] For example, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, for example, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required panicle si/e in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00119J Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients, such as those described herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional ingredient from a previously sterile-filtered solution thereof.

[00120] The pharmaceutical solid dosage forms described herein optionally include one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubili/er, moistening agent, plastici/er, stabili/er, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof.

[00121] In still other aspects, using standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the compositions. In some embodiments, the compositions are formulated into particles (for example for administration by capsule) and some or all of the particles are coated. In some embodiments, the compositions are formulated into particles (for example for administration by capsule) and some or all of the particles are microencapsulated. In some embodiments, the compositions are formulated into particles (for example for administration by capsule) and some or all of the panicles are not microencapsulated and are uncoated.

[00122]

[00123] ATA;

[00124] Described herein are methods of treating tumors and/or cancers comprising administering to a subject a low dose of a PARP inhibitor compound.

[00125] For use in therapeutic methods described herein, kits and articles of manufacture are also described herein. In some embodiments, such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) including one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

[00126] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any disorder that benefit by inhibition of PARP-1, or in which PARP-1 is a mediator or contributor to the symptoms or cause.

[00127] For example, a container may include a compound of Formula (I) and one or more additional compounds. The containers) optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprising a compound with an identifying description or label or instructions relating to its use in the methods described herein.

[00128] A kit will typically include one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint Tor use of a compound described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[00129] In some embodiments, a label is on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. A label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein.

[00130] In certain embodiments, a pharmaceutical composition comprising a PARP inhibitor is presented in a pack or dispenser device which can contain one or more unit dosage forms. The pack can for example contain metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. The pack or dispenser can also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, can be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. EXAMPLES

[00131 ] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

EXAMPLE 1

[001321 Complex roles for PARP-1 in colon cancer, inflammation, and tumor immunity: harnessing these roles to modulate cancer

[00133] One of the critical objectives of our laboratory is to test the hypothesis that DNA repair en/ymes such as PARP-1 play important roles in not only cancer-related processes but also in the pathogenesis of many inflammatory diseases. Without wishing to be bound by theory, the function of PARP-1 in cancers is intimately related to its role in providing alternative and efficient pathways to cancer cells to survive especially for those associated with defects in DNA repair (e.g. triple negative breast and ovarian cancers), however, its role in inflammation may be very different. The role of PARP-1 in DNA repair requires full activity of the enzyme as partial inhibition of PARP-1 has not been associated with defects in DNA repair. The purpose of the present studies is to clarify the roles of PARP-1 in colon inflammation and cancer and determine whether they are related.

[00134] Referring to FIG. 1 , for example, we show that partial PARP-1 inhibition (50%) was very effective at reducing or even blocking expression of inflammatory genes in response to LPS or TNF-a treatment and that complete inhibition of the enzyme was not necessary to achieve maximal effects. This appears to be due, in part, to a reduction in NF- κΒ-signal transduction. The remarkable anti-inflammatory effects of PARP- 1 inhibition (partial or total) suggested that such inhibition would be protective against chronic inflammation-driven colon carcinogenesis.

[00135] Referring to FIG. 2, for example, surprisingly, partial PARP-1 inhibition by gene heterozygosity was more efficient than complete inhibition by gene knockout at reducing chronic inflammation-driven colon tumorigenesis using an azoxymethane (AOM) followed by dextran sulfate sodium (DSS) exposure-based mode! of the condition although both genotypes provided similar reduction in the levels of systemic and colonic inflammation. [001361 Referring to FIG. 5, for example, when a mutation in the β-catenin pathway {Apc ^Min ) was the main driver for intestinal tumorigenesis, partial PARP-1 inhibition by gene heterozygosity or olaparib protected against the tumor burden in mice while complete inhibition by gene knockout aggravated the burden. These differential effects were not mirrored with respective effects on systemic or intestinal inflammation, splenomegaly, or cachexia as all conditions lowered the aforementioned traits.

[00137] Referring to FIG. 20, for example, using an MCA-38 colon carcinoma cell- allograft mouse model, we show that the opposing effects of PARP-1 gene dosages on intestinal tumorigenesis occurred despite that they both provide a tumor suppressive microenvironment through a regulation of the function of Myeloid-Derived Suppressor Cells (MDSCs). These results exemplify the complexity of the role of PARP-1 in colon tumorigenesis, inflammation, and immunity that could be harnessed to effectively treat not only colon cancer but also others,

EXAMPLE 2

[00138] PARP-1 heierozygositv noticeably protects against tumorigenesis in mice. 1001391 Without wishine to be bound bv theorv. this orotection can be due. in oart. to the anti-inflammatory effects of PARP-1 inhibition at the level of the colon as well as systemically.

[00140] Although PARP-1 nulli/igosity promotes an anti-inflammatory effect, it actually aggravates tumorigenesis in mice. Preliminary studies using CGH analysis suggests that such effects may be associated with an increase in genomic instability, another driving force in colon carcinogenesis.

[00141] Using a low and a high doses of the PARP inhibitor olaparib, we were able to reproduce the protective effect of PARP-1 gene heterozygosity but not nullizigositv in mice . Of note, using a high dose of olaparib was unreasonably expected to inhibit PARP-1 in a manner similar to that achieved by PARP-1 knockout.

[00142] It is noteworthy, however, that the pro-tumorigenic effect of PARP-1 nulli/igosity may actually be associated with changes within the transformed colon epithelial cells rather the overall host response. Indeed, PARP-1 nulli/igosity promotes a tumor-suppressive rather than a tumor-promoting host response as shown by our results using the MCA-38-allograft model [00143] When inflammation is the major driver, PARP-1 inhibition, pharmacologically or by gene knockout, protects against colon tumorigenesis. However, PARP-1 heterozygosity was more protective against AOM/DSS-indueed tumor burden than gene knockout. This discrepancy may also be associated with the effect of PARP-1 knockout (not heterozygosity) in genomic instability.

[00144] Overall these studies call for a serious caution in the current approach to use PARP inhibitors including olaparib as a therapeutic strategy against colon cancer.

[00145] Referring to FIG. 1 , for example, colon epithelial cells were treated with TNF-o for the indicated time intervals in the presence or absence 5 μΜ of the PARP inhibitor AZD2281. Cells were collected and protein extracts were subjected to immunoblot analysis with antibodies to VCAM-1 and COX2. AZD2281 inhibits the expression of inflammatory markers.

[00146] PARP-1 heterozygosity decreased tumorigenesis of the APCMin /+ mice, not PARP- 1 nullizigosity. (A) Tumor numbers of small and large intestine were counted at 16- week -old of age in APCMin/+mice (n = 13) ApcMin/+ PARP+/- mice (n = !4) and

APCMin/+ PARP-/- (n = 12). (B) Tumors were separated based on their size.

[00147]

EXAMPLE 3

[00148] Low doses of PARP inhibitors as a novel straiesv to enhance anti-tumor immunity

[00149] The objective is to establish a better understanding of the different aspects of the contribution of poly(ADP-ribose) polymerase- 1 (PARP-1) in tumorigenesis in such a way that it can be targeted in cancers other than those with BRCA mutations. Cancers including that of the colon are rank among the most common diseases in the US as well as worldwide with total national expenditure for care of affected individuals exceeding $125 billion. These studies will provide important insights on the utility of PARP inhibitors in targeting cancer cells indirectly by promoting an advantage to the host to use its immune system to attack cancer cells using the drugs at low doses, which reduces the potential for resistance and manifestation of side effects.

[00150]

[ 00151] Summar y

[00152] Targeting poly(ADP-ribose) polymerase (PARP) is a new strategy in cancer therapy. For example, this therapy is primarily effective in BRCA-defective cancers (approved for advanced ovarian cancer). The ultimate goal of this current therapy is maximal inhibition of PARP to achieve "synthetic lethality" taking advantage of the inability of BRCA-deficient cancer cells to repair DNA. See FIG. 13, for example. This strategy is limited by the requirement for a constant administration of high doses of the drugs (e.g. olaparib/Lynparza ^TM ) and the alarming rise of resistance to the drugs. Past studies on the utility of PARP- 1 as a therapeutic target were based on the use of PARP- 1 mice, very high doses of PARP inhibitors (such as 300mg/day or more of PARP inhibitors), and immunocompromised mice. Clinical trials exploring the efficacy of PARP inhibitors on several cancers lead to disappointing results. Because of such focus, many important aspects of PARP inhibition could not be harnessed for full clinical benefits.

[00153] Results herein show that partial PARP-1 inhibition (by gene heterozygosity or a low dose of olaparib) provides excellent protection against intestinal tumor burden in mice while extensive inhibition of the enzyme (by gene knockout or a high dose of olaparib) was ineffective or aggravated such burden. These divergent effects occurred despite a blockade in intra-tumoral and systemic inflammation and a promotion of tumor suppressive microenvironment.

[00154] Myeloid-Derived Suppressor Cells (MDSCs) recruitment and expansion are major determinants of cancer progression and resistance to therapies by modulating the tumor- killing capacity of the immune system. Results show that PARP-1 plays a key role in MDSC function and that partial inhibition of the enzyme is sufficient to block the suppressive function of these suppressor cells in a MCA-38 cell-based allograft model. Interestingly, antitumor immune cells (e.g. T, NK, and dendritic cells) are not affected even with maximal inhibition of PARP- 1.

[00155] Without wishing to be bound by theory, high doses of PARP inhibitors suppress MDSC function but with a concomitant enhancement of the tumorigenic effects of cancer cells by increasing genomic instability. However, low doses of PARP inhibitors selectively reduce the suppressive activity of MDSCs, decrease inflammation, and provide an advantage to tumor-killing immune cell at reducing/blocking tumor progression without enhancing the tumorigenic traits of cancer cells.

[00156] This paradigm-shift will be tested by examining the relationship between PARP-1, its inhibition by decreasing doses of olaparib, and MDSCs differentiation, function, and recruitment to tumors using the allograft model and an ex vivo system. We will then examine whether myeloid-specific or colon epithelial-specific deletion of PARP-1 influences tumorigenesis and MDSC trafficking in a mouse model of Apc ^Min -driven colon cancer. The results of the studies will allow us to establish a foundation on which we can demonstrate that PARP-I inhibition with low doses of drugs constitutes an extraordinary opportunity to modulate the progression of colon cancer as well as many others by blocking MDSC recruitment and function, which potentially enhances the efficacy of many existing and future immunotherapeutic strategies.

[001571

[00158] Description of Work

[00159] One of the hallmarks of tumor progression and resistance to therapy is the recruitment of myeloid-derived cells that suppress cancer cell-killing immune cells. Myeloid- Derived Suppressor Cells (MDSCs) recruitment and expansion represent critical events during cancer progression. These cells can influence as well as they can be influenced by the tumor microenvironment. Selective interference with the recruitment and/or function of these cells represents an ideal approach to prevent tumor progression, enhance potency of existing therapies, and promote tumor regression.

[00160] Targeting poly(ADP-ribose) polymerase (PARP) is a new strategy in cancer therapy. However, this therapy is primarily effective in BRCA-defective cancers (approved for advanced ovarian cancer). The ultimate goal of this therapy is maximal inhibition of PARP to achieve "synthetic lethality" taking advantage of the inability of BRCA-deficient cancer cells to repair DNA. This strategy is limited by the requirement for a constant administration of high doses of the drugs (e.g. olaparib/Lynpar/aTM) and the premature emergence of resistance to the drugs. Past studies on the utility of PARP-1 as a therapeutic target were based on the use of mice, unreasonably high doses of PARP inhibitors, and immuno-compromised mice. Clinical trials exploring the efficacy of PARP inhibitors on several cancers lead to disappointing results. Because of such focus, many important aspects of PARP inhibition could not be harnessed for full clinical benefits.

[00161] Results herein show that partial PARP-1 inhibition (by gene heterozygosity or a low dose of olaparib) provides excellent protection against intestinal tumor burden in APC ^Min 'mice while extensive inhibition of the en/yme (by gene knockout (KO) or a high dose of olaparib) was ineffective or aggravated such burden. These divergent effects occurred despite a blockade in intra-tumoral and systemic inflammation and a promotion of a tumor suppressive microenvironment. Results show that PARP-1 plays a key role in MDSC function and that partial inhibition of the enzyme is sufficient to block the suppressive capacity of these cells in a MCA-38 cell-based allograft model. Interestingly, anti-tumor immune cells such as T, NK, and dendritic cells are not affected by PARP-1 inhibition. More importantly, low doses of olaparib are more effective than high doses in blocking MCA-38- based tumors in WT mice.

[00162] Without wishing to be bound by theory, high doses of PARP inhibitors may suppress MDSC function but with a concomitant enhancement of the tumorigenic effects of cancer cells by increasing genomic instability. However, low doses of PARP inhibitors selectively reduce the suppressive activity of MDSCs, decrease inflammation, and provide an advantage to tumor-killing immune cell at reducing/blocking tumor progression without enhancing the tumorigenic traits of cancer cells.

[00163] To demonstrate this, we will perform studies using an integrated approach including a MCA-38 colon adenocarcinoma cell-based allograft model and the APC ^Min -based spontaneous intestinal tumorigenesis model. We will take advantage of our newly generated C57BL/6 PARP-1 conditional PARP mice under the control of hematopoietic cell- or colon epithelial-specific Cre strain. We will also use an ex vivo cell culture model to address some aspects of this invention. Additionally, we will use PARP-1 depletion approaches primarily shRNA-based partial knockdown to mimic PARP-1 gene heterozygosity and CRISPR/Cas9 to mimic PARP-1 knockout on MCA-38 cells.

[00164] We will test this paradigm-shift as follows:

[00165) 1 : Examine the relationship between PARP-1, its inhibition by decreasing doses of olaparib, and MDSCs differentiation, intra-tumoral recruitment and function using a MCA-38 colon adenocarcinoma cell-based allograft model and an ex vivo system.

[00166] 2: Examine whether myeloid-speciflc or colon epithelial-specific deletion of PARP- 1 influences tumorigenesis and MDSC trafficking in a mouse model of ApcMin- driven colon cancer.

[00167] The studies will demonstrate that PARP-1 inhibition with low doses of drugs constitutes an extraordinary opportunity to modulate the progression of colon cancer as well as many others by enhancing the efficacy of many existing and future immunotherapeutic strategies.

[00168]

[00169] Research Strategy

[00170] (a) Significance

[00171] A disappointing aspect about the old and new generation cancer therapies is the emergence of drug resistance and the ability of the cancer cell to evade the immune system. PARP inhibitors are emerging as a promising therapy for several human cancers especially those driven by deficiencies in non-PARP-associated DNA repair ¹ . Indeed, olaparib (Lynparza ^TM ) is now approved as a monotherapy for patients with advanced BRCA1 -mutated ovarian cancer. Interestingly, several preclinical and clinical studies offered some hope for PARP inhibitors in the treatment of other cancers with no obvious mutations in BRCA ² . Unfortunately, many of the clinical trials failed. Recently, olaparib was tested in a phase V and a phase II ⁴ clinical trials on patients with advanced colon cancer as a monotherapy or in combination with irinotecan (Camptosar™), with disappointing results but encouraging more detailed studies. Without wishing to be bound by theory, the reason for the failure of PARP inhibitors in treating non-BRCAl -mutated cancers is the fact that we still do not understand the intricacies of the role of the enzyme in the pathogenesis of cancers including that of the colon.

[00172] One of our critical objectives is to unravel the unobvious functions of DNA repair enzymes such as PARP-1 and test the hypothesis that these enzymes play important roles not only in cancer cell-related processes (DNA repair, cell death, genomic integrity, etc.) but also in inflammatory and immune responses. The function of PARP-1 in cancers may be intimately related to its ability to provide an alternative pathway for cancer cells to survive especially for those associated with defects in DNA repair ⁵ ; however, the mechanism by which it contributes to inflammation may be very different. The role of PARP-1 in DNA repair requires the full activity of the enzyme as partial inhibition of PARP-1 has not been associated with major defects in DNA repair ¹ . Indeed, PARP-1 heterozygous cells or mice behave similarly to DNA damaging agents as the WT counterparts ⁶ . Interestingly, we reported that PARP- 1 heterozygosity (which reduces PARP- 1 by ~50%) or low doses of a PARP inhibitor substantially blocks atherosclerosis in high fat diet-fed ApoE mice ⁷ . We have also shown that low doses of PARP inhibitors, including olaparib, protect against asthma ⁷ ' ¹¹ .

[00173] MDSCs play a critical role in providing an advantage to cancer cells to evade the immune system ¹² . These cells are specialized in blocking T cell function by promoting the expansion of TR _C8 cells, depriving T cells of essential amino-acids, producing oxidizing molecules (e.g. H ₂ O ₂ and ONOO ), and blocking T cell recruitment to tumors ¹³ As most cancers, colon cancer is also characterized by the infiltration with immune cell types (e.g. T cells) ¹⁴ . However, because of MDSCs, these cells are dysfunctional. Therefore, interfering with the function and/or recruitment of MDSCs might provide tremendous benefit to strategies targeting cancer cells, representing an attractive strategy to treat not only colon cancer but also many others. Our data suggest that MDSCs are highly sensitive to PARP-1 inhibition and thus can be targeted with low doses of PARP inhibitors. These findings are highly significant because they provide a potential solution that can benefit not only patients with colon cancer but also those with other types of cancers using FDA-approved PARP inhibitors at low doses. This approach would certainly eliminate the possibility of drug resistance and enhance the efficacy of many existing and future immunotherapies.

[001741 (b) Innovation

[00175] Current therapeutic strategies aim at maximally inhibiting PARP, such as with high doses of PARP inhibitor compounds. Embodiment as described herein, which use low doses of the drugs to target MDSCs while preserving the function of cancer-killing immune cells, are novel and represent a paradigm-shifting concept.

[00176] (c) Results

[00177] Partial PARP-1 inhibition is sufficient to block expression of inflammatory genes in primary colon epithelial cells (CECs) upon LPS or TNF exposure. We developed a highly reproducible method to isolate CECs from mice ^{15- 16} and humans. FIG. 14(A) shows the square/hexagonal-shaped cells indicative of the epithelial nature of the cells (additional characterization of cells is described in ¹⁶ ). FIG. 14(B) shows that PARP-1 heterozygosity reduces PARP-1 expression by -50% compared to that of WT cells ⁷ PARP-1 heterozygosity was as effective as KO in reducing TNF, IL-6, and ICAM-1 in response to LPS (FIG. 14(C)) or TNF (FIG. 14(D)) as assessed by qRT-PCR.

[00178]

[00179] Partial PARP- 1 inhibition by gene heterozygosity is more efficient than KO at reducing chronic inflammation-driven colon tumorigenesis in mice despite an equal modulation of systemic inflammation. A single administration of the carcinogen azoxymethane (AOM) to animals in combination of 4 cycles of treatment with 2% dextran sulfate sodium (DSS) is a reliable chronic inflammation-induced colon cancer model. A single dose of AOM (Fig. 2A and ^{l8, l9} ) or the 4 cycles of DSS treatment are insufficient to induce tumorigenesis ²⁰ . AOM/DSS treatment induced -7-8 tumors in colons of WT mice (FIG. 15(A)). This burden was lower in counterparts but, surprisingly, much lower in mice. The tumors from AOM/DSS-treated mice were not different

from those of similarly treated WT mice (FIG. 15 (B)). High PCNA immunoreactivily (a marker of cell proliferation) was detected in tumors of AOM/DSS-treated WT and PARP-1 mice, which was much lower in tumors of treated PARP-Γ ^" mice (p<0.001 ; FIG. 27). FIG. 15(D) shows thai the colonic mucosa was almost completely absent in affected areas. However, the mucosa of AQM/DSS-treated PARP-Γ or PARP-! mice showed some disorganization and injury, but the colonic crypts were relatively intact or in the process of recovery. The results on colitis are consistent with ^2t . The effects of PARP-1 inhibition on the tumor burden mirrored a decrease in systemic inflammation (FIG. 15(E)). These results show that there are benefits to partially inhibiting PARP-1 and support the notion that aiming at completely inhibiting the enzyme with high doses of PARP inhibitors (in non-BRCA- deficient cancers) may not provide an important clinical value and may become detrimental in the long run.

[00180]

[00181] Partial inhibition of PARP-1 by gene heterozygosity protects against induced tumor burden in mice while complete inhibition by gene KO aggravates it. We next examined the effect of PARP-1 gene dosage on APC ^Min -induced intestinal tumorigenesis. We generated C57BL/6 PARP-1 ' and PARP-1 mice" in the A background (FIG. 21(A)). PARP-1 heterozygosity provided a remarkable protection against the tumor burden (FIG. 21(B)). Surprisingly, PARP-1 KO not only did not protect against the tumor burden, it significantly aggravated it. FIG. 21(C) shows that PARP-1 heterozygosity completely blocked the generation of large tumors (>4 mm) with a significant concomitant decrease in small and middle size tumors. Conversely, PARP-1 KO promoted an increase in the number of small tumors. Weight loss observed in the mice was prevented by PARP-1 heterozygosity but not KO (FIG. 28). PCNA immunoreactivity in tumors of mice with WT or PARP-1 KO was not different (FIG. 21(D)); but, the difference between these 2 groups and that of mice was highly significant (p<0.01). Paradoxically, intra-tumoral inflammation (VCAM-1 and COX -2; FIG. 2 1(D) and FIG. 21(E)) was equally reduced in tumors of mice.

[00182]

[00183] PARP inhibition with a low dose of olaparib (Lynparza™) is protective against -induced tumor burden in mice while a higher dose of the drug is not. We next examined the effect of a low dose (5 mg/kg) and a five times higher dose (25 mg/kg) of olaparib starting at 8 week of age on n tumorigenesis. FIG. 22(A) shows that

although PARP inhibition with the low dose of olaparib provided a good protection against the tumor burden, the higher dose showed high variability but overall it provided no protection against the burden. In fact, some mice showed a tumor burden that was higher than that of the vehicle group. The differential effects of the two doses of olaparib on the tumor burden were mirrored by respective effects on cachexia (FIG. 22(B)). The results are relatively consistent with the differential effects attained using the genetic approach. Note that most, if not all, studies examining the effects of olaparib on carcinogenesis using preclinical models have used doses as high as 300 mg/kg/day. We are confident that higher and more frequent doses of olaparib would aggravate the tumor burden in our experimental model.

[00184] PARP-1 inhibition genetically (by gene heterozygosity or KO) or by olaparib is effective at blocking systemic inflammation in APC ^Min ' mice. Chronic inflammation exists in APC ^Min -driven intestinal carcinogenesis (FIG. 23) and ² '" ²⁵ . All forms of PARP-1 inhibition were able to significantly reduce IL-6 and TNF albeit not to the level of control mice. MCP-1 levels were significantly reduced by PARP-1 inhibition to levels similar to those of WT controls. These results demonstrate that PARP-1 inhibition promote an antiinflammatory environment.

[00185]

[00186] PARP-1 inhibition provides a tumor-suppressive environment in MCA-38 cell- based allograft model of colon cancer. Given the above results, it became critical to determine whether PARP-1 plays a role in the host response to tumor development. We took advantage of an allograft model using the colon adenocarcinoma cell line MCA-38 (from a C57BL/6 mouse), which is WT for PARP-1 (FIG. 24(A)). FIG. 24(B) and FIG. 24(C) show that grafting of MCA-38 cells onto WT mice leads to formation of large solid tumors; these tumors were significantly smaller when grafted onto PARP-V or PARPA mice. The anti-tumor effects of PARP-1 gene heterozygosity and KO were accompanied by an efficient reduction in systemic (FIG. 24(C)) and intratumoral (FIG. 24(D)) inflammation as well as reduced CD68' inflammatory cell recruitment (FIG. 24(E)).

[00187]

[00188] PARP-l plays a role in MDSC function and its partial PARP-1 inhibition is sufficient to block the suppressive activity of these cells without affecting the function of dendritic cells. Since PARP-1 seems to play an important role in the host response to tumor formation, it may influence the function of MDSCs. To test this, we isolated MDSCs from MCA-38 cell-based tumors (by enzymatic digestion+puriflcation using EasySep Mouse CDUb Positive Selection). Purity of MDSCs was verified by FACS for CDl lb and Gr- 1 positivity. MDSCs isolated from tumors of WT mice were very effective at suppressing T cell proliferation upon stimulation (FIG. 25(A)). Interestingly, MDSCs derived from tumors of PARP-Γ or PARP-1 mice almost completely failed to suppress the proliferation of these WT T cells. Note that PARP-1 gene heterozygosity was sufficient to impair the function of MDSCs suggesting that they are very sensitive to PARP-1 inhibition.

[00189] Also note that PARP-1 inhibition does not modulate indiscriminately all immune cells as it has little to no effect on CD4 ⁺ T1, CD8+T ²⁷ , NIC ²⁷ , or dendritic (DCs) (FIG. 25(B)) cell populations. We acknowledge that Aldinucci et al. ²⁸ reported a role for PARP in DC maturation; this conclusion was reached using unreasonably high doses of PARP inhibitors (TIQ-A) (20-30μΜ), which we showed to be cytotoxic ²⁹ .

[00190]

[00191] Low concentrations of olaparih are more effective than high concentrations in blocking MCA-38-based tumors in WTmice. Given the above results, we next speculated that low doses of PARP inhibitors (e.g. olaparib) might be more beneficial than high doses in immunocompetent mice. To this end, WT mice were engrafted with MCA-38 cells and as soon as the tumors were palpable (day 6), mice received 0.2, 1, or Smg/kg olaparib or vehicle. Mice that received vehicle increased in si/e in a time-dependent manner and were sacrificed at day 16. The tumors in mice that received Smg/kg olaparib were relatively smaller than those of WT. Remarkably, the tumors in mice that received the lowest dose of olaparib barely increased above the si/e the tumors at the day 5. The middle dose (1 mg/kg) were very effective at blocking tumor growth; at 16 growth was more visible. These results are extremely important because they provide an unexpected and a novel paradigm-shifting concept with high clinical relevance. These results may also explain why high doses of PARP inhibitors Med to be efficacious against several cancers with no BRCA mutation.

[00192]

[00193] (I) To decipher the relationship between PARP-1, its inhibition by decreasing doses of olaparib, and MDSCs differentiation, intm-ttimoral recruitment and function using the MCA-38 cell-based allograft model.

[00194] /. To determine the effects of PARP inhibition on the recruitment of MDSCs using the allograft model. PARP-1 appears to play a role in MDSC function and, without wishing to be bound by theory, this effect may accompany a decrease in MDSCs recruitment to the tumors. For example, PARP-1 inhibition may affect the recruitment of the cells given our reported connection between PARP-1 and CXCR2 ³⁰ , a receptor required for trafficking to tumors' ³¹ . [00195] Specific Experiment J. MCA-38 cells will be engrafted onto mice as shown in FIG. 24. Tumors will be collected from sacrificed mice on day IS (or when the si/e of the tumors is large enough to isolate MDSCs in all groups). Portions of the tumors and spleens will be digested to generate single cell suspension. Cells will be assessed for MDSC numbers by FACS with fluorescently labeled antibodies to CD1 lb, Ly6G (Grl), or Ly6C. The two latter markers will allow us to determine whether the recruited MDSCs are granulocytic (GrMDSCs) (CD1 lb\ Ly6G ^'high , Ly6C ^lowa ) or monocytic (MoMDSCs) (CD1 lb', Ly6C Ly6G ). Recruitment will be assessed as percent MDSCs of the total number of isolated cells. Given that percentages can be misleading and that the tumor size between groups could be very different, we will correct the numbers according to tumor volume. We will also assess the single cell suspension for the prevalence of cytotoxic CDS T-cell populations (IFNy' CDS' CD3' CD45 '). MDSCs from all groups will also be isolated as described above and subjected to RNA extraction followed by quantitative RT-PCR using primer sets for iNOS and Arg 1.

[00196] Specific Experiment 2. To determine whether the tumor microenvironment influenced the differences we may see in the above experiment, a portion of the tumor will be subjected to RNA extraction followed by quantitative RT-PCR using primer sets specific for CXCR2 or CXCR4, GM- CSF, G-CSF, IL-4, IL-6, IL-13, TNF, TGFβ and several others.

[00197]

[00198] 2. To determine whether low doses of olaparib prevent tumor progression by decreasing the recruitment of MDSCs in the allograft model.

[00199] Specific Experiment I: We will repeat the experiment described in FIG. 20 with two major alterations: 1) we will keep measuring tumor sizes for a longer period even after the termination of the groups receiving vehicle. This will allow us to have a better idea on how long the effect of the low doses of olaparib can maintain their effects on tumor progression. 2) We will also determine the lowest dose of the drug that can modulate tumor progression in our allograft model. We will continue using a de-escalation of the doses by five-fold increments as described in FIG.20.

[00200] Specific Experiment 2: While conducting experiment 1, we will assign a group of mice to be sacrificed at day I 5 to conduct the same assays described herein, such as in Goal 1.

[00201]

[00202] 3. To determine the role of PARP-1 and the effect of olaparib on differentiation of MDSCs ex vivo. The results of goal 1 may not differentiate between the effects on MDSCs, other immune cells, and cancer cells. It is, thus, important to determine whether PARP inhibition affects directly differentiation of MDSCs. This becomes even more important when we consider the fact that olaparib as well as other PARP inhibitors affect both PARP-1 and PARP-2. We intend to examine whether PARP-1 expression and/or activity affect differentiation of MDSCs from BM progenitors.

[00203] Specific Experiment 1. BM cells will be harvested from WT,

in ¹⁰ , which will then be cultured with G-CSF, GM-CSF, and IL-6 ³² . MDSCs will be assessed by FACS for numbers and subtypes. MDSCs will also be positively selected to assess their T-cell suppression capacity and their ability to express iNOS and Argl by RT-PCR.

[00204] Specific Experiment 2. A portion of WT or PARP-1" BM cells will be cultured in the presence of 1, 0.2, 0.04μΜ olaparib (at day 0 and day 2). Again, PARP inhibitors may affect PARP-1 and PARP-2 and thus it becomes important to examine whether the effects of olaparib are associated solely with PARP-1 or may also be related to PARP-2. To address this, we will treat PARP-1 ^" BM cells with olaparib as will be done for WT cells. MDSCs from all conditions will be subjected to FACS analysis, purification, T cell suppression capacity, and RT-PCR for iNOS and Argl.

[00205]

[00206] 4. To determine whether partial or complete depletion of PARP- 1 in cancer cells affects recruitment of MDSCs to tumors in the allograft model. We will next address whether PARP-1 plays a role in the ability of tumors to influence MDSC recruitment and activation. We will use shRNA and CRISPR/cas9 approaches on MCA-38 cells for partial and complete depletion, respectively, in a manner similar to that achieved in AS49 cells (FIG. 26). Partial knockdown would mimic PARP-1 heterozygosity and deletion with CRISPR/Cas9 would mimic PARP-1 KO.

[00207] Specific Experiment. MCA-38 cells that were subjected to partial knockdown (MCA38- ) and those to CRISPR/Cas9 (MCAOS- will be engrafted with their respective controls onto opposing flanks of WT mice. Tumor volumes will be assessed as in FIG. 24. At day 15 (or when the size of the tumors is large enough to isolate MDSCs in all groups), tumors will be harvested after sacrificing the mice. The tumors will be divided into equal portions by weight. A portion will be digested to generated single cell suspensions, which will be assessed for MDSCs populations by FACS or for MDSC isolation by positive selection to assess their T-cell suppression capacity or their ability to express iNOS and Argl. The second portion of the tumors will be assessed for CXCR2 or CXCR4 and a number of inflammatory factors (described in Goal 1 of Aim 1) to determine whatever difference we see between the groups can be attributed to the ability of the cancer cells to produce the factors that are critical for MDSC recruitment and activation.

[00208] [00209] (2) To examine whether myeloid-specific or colon epithelial-specific deletion of PARI'- 1 influences tumorigenesis and MDSC trafficking in a mouse model of AP -driven colon cancer.

[00210] Using spontaneous models will provide us with critical information that is of great relevance to the human condition. For the following studies, we have several options, which include different models of colon cancer representing the many aspects and complexity of the human disease. Our lab is very well versed in mouse model. APC gene mutations are

attributed to cases of familial adenomatous polyposis (FAP) as well as approximately -70% of sporadic colorectal cancer" ^'35 . We are also versed with a colon cancer model that is exclusively induced by repeated administrations of the carcinogen DMH ¹⁶ or its metabolite AOM. We also have the AOM/DSS model that is induced by chronic colon inflammation (FIG. 15). We will focus on the Α model because of its simplicity; however, we can use any of these models as needed, which altogether encompass many aspects of human colon cancer. We will use our new PARP-1- floxed mice under different Cre-strains. The mutant strain was generated (germ line transmission and Neo cassette removal) by Cyagen. We will be crossing the mutant mice with several Cre- strains. We succeeded in generating the floxed-PARP-1 under the control of Tek-Cre (Fig.10) and termed PARP- for heterozygous and KO, respectively. We should complete the generation of termed PARP- strain rather shortly. These strains will then be crossed with as in FIG.21.

[00211]

[00212] l.To examine whether myeloid-specific PARP- 1 gene heterozygosity or KO reduces APi ^Mm -induced tumor burden and determine whether it alters intra- tumoral MDSC trafficking and function.

[00213] Specific Experiment. mice will be sacrificed at 16 wks of age; we will use the liitermates as WT PARP-1 animals. The tumor burden in the intestinal track will be assessed (numbers and sizes) as described in FIG. 21. Tumors will be collected and subdivided according to their position in the intestinal track. Two third of the tumors and spleens will be digested for single cell suspension. The remaining portion will be processed for RNA and DNA extraction. A small portion of the single suspensions will be assessed by FACS analysis for the number and subtypes of MDSCs. The remaining will be used for MDSC isolation as described above. Isolated MDSCs will be assessed for their T cell suppression capacity or for RNA extraction to assess the expression levels of iNOS and Argl. Extracted RNA (directly from the tumors or spleens) will be assessed by qPCR with primer sets specific for CXCR2 or CXCR4 and a number of inflammatory factors (described in Goal I of Aim 1). [00214]

[00215] 2. To examine whether colon epithelial cell-specific PARP-1 gene heterozygosity or KO alter ¹ -induced tumor burden by influencing MDSC trafficking and function within the tumor microenvironment.

[00216] Specific Iixuerimenl.

processed the same way as described in Goal

1, above.

[00217] 3. To examine the potential differential effects of colon epithelial cell-specific PARP-1 gene heterozygosity and KO on p -induced tumor burden is associated with changes in genomic instability in tumor cells. We believe that the primaiy reason for PARP-1 gene KO for aggravating the tumor burden in A mice is the accumulation of genomic instability in addition to that induced by the mutation.

[00218] Specific Kxperiment. DNA extracted from the tumors of the different experimental groups will be assessed by aCGH essentially as done in our published report ¹⁶ . The samples will be analyzed using the Agilent DNA microarray platform. Data including Copy Number Variations will be assessed by Agilent Feature Extraction software 12 and analyzed with Agilent Genomic Workbench software 7.0 using the statistical algorithms / score.

[00219] Expected outcomes: Without wishing to be bound by theory, we expect that partial PARP-1 inhibition would be effective in reducing the recruitment of MDSCs and their function. It is rather possible that low doses of olaparib block the function of MDSCs but not their recruitment. These potential differential effects are likely given our previous findings showing that low doses of olaparib interferes with T cell-response to CD3/CD28-stimulated production of Th2 inflammatory factors without affecting the signal leading to their proliferation". We may also expect that inhibition of PARP in MDSCs may change their phenotype to acquire an anti-tumor trait in a manner similar to that observed when Chop protein was depleted in MDSCs ³⁸ . This is based on the finding (FIG. 30) that PARP-1 inhibition decreases Chop expression in CD68' cells in response to oxidized cholesterols.

[00220] The effects of PARP-1 inhibition may be even more enhanced if its efficiency in increasing the efficacy of adoptive T cell and checkpoint blockade (e.g. PD-1 or PD-Ll>based therapies is examined. Clinical ³⁹ and preclinical trials ⁴⁰ have looked into the combination of PARP inhibitors and anti-PDl therapy but again using high daily doses of the drugs or immunodeflcient mice. In some studies, the dose of the PARP inhibitor was as high as 300mg/kg/day, which is unreasonable and certainly nontherapeutic, as these high doses can be extremely toxic to all cell types including the cancer cells themselves. We believe that here resides the novelty of our hypothesis.

[00221] Without wishing to be bound by theory, we expect that the tumor burden to be significantly lower in both PARP-l mice compared to the PARP- (on Apc background) controls. This is based on the potential that myeloid cells that are partially or completely deficient in PARP-1 would be unable to induce inflammation even if the tumor cells would produce inflammatory cues. The associated MDSCs would also be incapable of suppressing T cells. It is possible that PARP-1 KO may have some effect on T cell function and if this possibility presents itself, we believe that APC ^M mice would have a higher tumor burden than that of APC mice but would be significantly less than that of the APC ^Min /PARP- l controls. As for the APC mice, it would be difficult to predict the outcome. If we consider that the complete absence of PARP-1 in CECs would prevent the production of inflammatory factors necessary for MDSC recruitment; these same cues may be necessary for recruiting cytotoxic T cells. The net outcome would depend on the effect of PARP-1 gene deletion on the cancer cell. If high genomic instability is reached, then one would expect to observed high incidence of tumors (size and/or numbers) in the colon while the tumor burden in the small intestine would remain the same as in the control mice.

[00222] References Cited in this Example:

1. Berger NA, Besson VC, Boulares AH, Burkle A, Chiarugi A, Clark RS, Curtin NJ, Cuzzocrea S, Dawson TM, Dawson VL, Hasko G, Liaudet L, Moroni F, Pacher P, Radermacher P, Sal/man AL, Snyder SH, Soriano FG, Stros/najder RP, Sumegi B, Swanson RA, S/abo C. Opportunities for the repurposing of parp inhibitors for the therapy of non-oncological diseases. Br J Pharmacol. 2017

2. George A, Kaye S, Banerjee S. Delivering widespread brca testing and parp inhibition to patients with ovarian cancer. Nature reviews. Clinical oncology. 2016

3. Chen EX, Jonker DJ, Siu LL, McKeever K, Keller D, Wells J, Hagerman L, Seymour L. A phase i study of olaparib and irinotecan in patients with colorectal cancer: Canadian cancer trials group ind 187. Invest New Drugs. 2016;34:450-457

4. Leichman L, Groshen S, CNeil BH, Messersmilh W, Berlin J, Chan E, Leichman CG, Cohen SJ, Cohen D, Lenz HJ, Gold P, Boman B, Fielding A, Locker G, Cason RC, Hamilton SR, Hochster HS. Phase ii study of olaparib (azd-2281 ) after standard systemic therapies for disseminated colorectal cancer. Oncologist. 2016;21 : 172- 177 5. Pommier Y, O'Connor MJ, de Bono J. Laying a trap to kill cancer cells: Parp inhibitors and their mechanisms of action. Sci Transl Med. 2016;8:362ps317

6. Wang ZQ, Auer B, Stingl L, Berghamnier H, Haidacher D, Schweiger M, Wagner EF.

Mice lacking adprt and poly(adp-ribosyl)ation develop normally but are susceptible to skin disease. Genes & development. 1995;9:509-520

7. Oumouna-Benachour K, Hans CP, Suzuki Y, Naura A, Datta R, Belmadani S, Fallon K, Woods C, Boulares AH. Poly(adp-ribose) polymerase inhibition reduces atherosclerotic plaque si/e and promotes factors of plaque stability in apolipoprotein e-deficient mice: Effects on macrophage recruitment, nuclear factor-kappab nuclear translocation, and foam cell death. Circulation. 2007; 1 15:2442-2450

8. Boulares AH, Zoltoski AJ, Sherif ZA, Jolly P, Massaro D, Smulson ME. Gene knockout or pharmacological inhibition of poly(adp-ribose) polymerase- 1 prevents lung inflammation in a murine model of asthma. Am J Respir Cell Mol Biol. 2003;28:322-329

9. Oumouna M, Datta R, Oumouna-Benachour K, Suzuki Y, Hans C, Matthews K, Fallon K, Boulares H. Poly(adp-ribose) polymerase- 1 inhibition prevents eosinophil recruitment by modulating th2 cytokines in a murine model of allergic airway inflammation: A potential specific effect on il-5. J Immunol. 2006;177:6489-6496

10. Ghonim MA, Pyakurel K, Ibba SV, Al-Khami AA, Wang J, Rodriguez P, Rady HF, El-Bahrawy AH, Lammi MR, Mansy MS, Al-Ghareeb K, Ramsay A, Ochoa A, Naura AS, Boulares AH. Parp inhibition by olaparib or gene knockout blocks asthma-like manifestation in mice by modulating cd4(+) t cell function. Journal of translational medicine. 2015; 13:225

1 1. Ghonim MA, Pyakurel K, Ibba SV, Wang J, Rodriguez P, Al-Khami AA, Lammi MR, Kim H, Zea AH, Davis C, Okpechi S, Wyczechowska D, Al-Ghareeb K, Mansy MS, Ochoa A, Naura AS, Boulares AH. Parp is activated in human asthma and its inhibition by olaparib blocks house dust mite-induced disease in mice. Clin Sci (Lond). 2015;129:951-962

12. Gabrilovich DI. Myeloid-derived suppressor cells. Cancer immunology research.

2017;5:3-8

13. Al-Khami AA, Rodriguez PC, Ochoa AC. Metabolic reprogramming of myeloid- derived suppressor cells (mdsc) in cancer. Oncoimmunohgy. 20l6;5:el20077l 14. Mei Z, Liu Y, Liu C, Cui A, Liang Z, Wang G, Peng H, Cui L, Li C. Tumour- infiltrating inflammation and prognosis in colorectal cancer: Systematic review and meta-analysis. Br J Cancer. 2014; 1 10: 1595- 1605

15. Oumouna-Benachour K, Oumouna M, Zerfaoui M, Hans C, Fallon K, Boulares AH.

Intrinsic resistance to apoptosis of colon epithelial cells is a potential determining factor in the susceptibility of the a/j mouse strain to dimethylhydra/ine-induced colon tumorigenesis. Mol Carcinog. 2007;46:993-1002

16. Errami Y, Brim H, Oumouna-Benachour K, Oumouna M, Naura AS, Kim H, Ju J, Davis CJ, Kim JG, Ashktorab H, Fallon K, Xu M, Zhang J, Del Valle L, Boulares AH. lead deficiency in human colon cancer and predisposition to colon tumorigenesis: Linkage to apoptosis resistance and genomic instability. PLoS One. 2013;8:e57871

17. Kanai M, Tong WM, Wang ZQ, Miwa M. Haploinsufficiency of poly(adp-ribose) polymerase- 1 -mediated poly(adp-ribosyl)ation for centrosome duplication. Biochemical and biophysical research communications. 2007;359:426-430

18. Nambiar PR, Girnun G, Lillo NA, Guda K, Whiteley HE, Rosenberg DW.

Preliminary analysis of azoxymethane induced colon tumors in inbred mice commonly used as transgenic/knockout progenitors. Int. I Oncol. 2003;22: 145-150

19. Bissahoyo A, Pearsall RS, Hanlon K, Amann V, Hicks D, Godfrey VL, Threadgill DW. Azoxymethane is a genetic background-dependent colorectal tumor initiator and promoter in mice: Effects of dose, route, and diet. 'lexicological sciences : an official journal of the Society of Toxicology. 2005;88:340-345

20. Tanaka T, Kohno H, Suzuki R, Yamada Y, Sugie S, Mori H. A novel inflammation- related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate. Cancer science. 2003;94:965-973

21. Larmonier CB, Shehab KW, Laubilz D, Jamwal DR, Ghishan FK, Kiela PR.

Transcriptional reprogramming and resistance to colonic mucosal injury in poly(adp- ribose) polymerase 1 (parpl)-deficient mice. The Journal of biological chemistry. 2016;291 :8918-8930

22. Datta R, Naura AS, Zerfaoui M, Errami Y, Oumouna M, Kim H, Ju J, Ronchi VP, Haas AL, Boulares AH. Parp-1 deficiency blocks il-5 expression through calpain- dependent degradation of stat-6 in a murine asthma model. Allergy. 2011;66: 853-861

23. Serebrennikova OB, Tsatsanis C, Mao C, Gounaris E, Ren W, Siracusa LD, Eliopoulos AG, Kha/aie K, Tsichlis PN. Tpl2 ablation promotes intestinal inflammation and tumorigenesis in apcmin mice by inhibiting il-10 secretion and regulatory t-cell generation. Proc Natl Acad Sci USA. 2012;109:E1082-1091

24. McClellan JL, Davis JM, Steiner JL, Day SD, Steck SE, Carmichael MD, Angela Murphy E. Intestinal inflammatory cytokine response in relation to tumorigenesis in the apcmin/+ mouse, cytokine. 2012;57:1 13-1 19

25. Gounaris E, Erdman SE, Restaino C, Gurish MF, Friend DS, Gounari F, Lee DM, Zhang G, Glickman JN, Shin K, Rao VP, Poutahidis T, Weissleder R, McNagny KM, Kha/aie K. Mast cells are an essential hematopoietic component for polyp development. Proc Natl Acad Sci USA. 2007; 104: 19977- 19982

26. Hossain F, Al-Khami AA, Wyc/echowska D, Hernandez C, Zheng L, Reiss K, Valle LD, Trillo-Tinoco J, Maj T, Zou W, Rodriguez PC, Ochoa AC. Inhibition of fatty acid oxidation modulates immunosuppressive functions of myeloid-derived suppressor cells and enhances cancer therapies. Cancer Immunol Res. 2015;3: 1236-1247

27. Rosado MM, Bennici E, Novelli F, Pioli C. Beyond DNA repair, the immunological role of parp-l and its siblings. Immunology. 2013;139:428-437

28. Aldinucci A, Gerlini G, Fossati S, Cipriani G, Ballerini C, Biagioli T, Pimpinelli N, Borgognoni L, Massacesi L, Moroni F, Chiarugi A. A key role for poly(adp-ribose) polymerase- 1 activity during human dendritic cell maturation. J Immunol. 2007; 179:305-312

29. Kim H, Naura AS, Errami Y, Ju J, Bou lares AH. Cordycepin blocks lung injury- associated inflammation and promotes brcal -deficient breast cancer cell killing by effectively inhibiting parp. Mol Med. 201 1

30. Zerfaoui M, Naura AS, Errami Y, Hans CP, Rezk BM, Park J, Elsegeiny W, Kim H, Lord K, Kim JG, Boulares AH. Effects of parp-l deficiency on airway inflammatory cell recruitment in response to Ips or tnf: Differential effects on cxcr2 ligands and dufiy antigen receptor for chemokines. J Leukoc Biol. 2009;86: 1385-1392

31. Highfili SL, Cui Y, Giles AJ, Smith JP, Zhang H, Morse E, Kaplan RN, Mackall CL.

Disruption of cxcr2-mediated mdsc tumor trafficking enhances anti-pdl efficacy. Science translational medicine. 2()14;6:237ra267

32. Al-Khami AA, Rodriguez PC, Ochoa AC. Energy metabolic pathways control the fate and function of myeloid immune cells. J Leukoc Biol. 2017;102:369-380

33. Fearnhead NS BM, Bodmer WF. The abc of ape. Human Molecular Genetics.

2001;10:721-733 34. Powell SM. Ape mutations occur early during colorectal tumorigenesis. Nature. 1992;359

35. Kinzler KW. Lessons from hereditary colorectal cancer. Ceil. 1996;87: 159- 170

36. Fahs SA, Hille MT, Shi Q, Weiler H, Montgomery RR. A conditional knockout mouse model reveals endothelial cells as the principal and possibly exclusive source of plasma factor viii. Blood. 2014;123:3706-3713

37. Schlaeger TM, Bartunkova S, Lawitts JA, Teichmann G, Risau W, Deutsch U, Sato TN. Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice. Proc Natl Acad Sci U SA. 1997;94:3058-3063

38. Thevenot PT, Sierra RA, Raber PL, Al-Khami AA, Trillo-Tinoco J, Zarreii P, Ochoa AC, Cui Y, Del Valle L, Rodriguez PC. The stress-response sensor chop regulates the function and accumulation of myeloid-derived suppressor cells in tumors. Immunity. 2014;41 :389-401

39. Lee JM, Cimino-Mathews A, Peer CJ, Zimmer A, Lipkowitz S, Annunziata CM, Cao L, Harrell MI, Swisher EM, Houston N, Botesteanu DA, Taube JM, Thompson E, Ogurtsova A, Xu H, Nguyen J, Ho TW, Figg WD, Kohn EC. Safely and clinical activity of the programmed death-ligand 1 inhibitor durvalumab in combination with poly (adp-ribose) polymerase inhibitor olaparib or vascular endothelial growth factor receptor 1-3 inhibitor cediranib in women's cancers: A dose-escalation, phase i study. J Clin Oncol. 2017;35:2193-2202

40. Jiao S, Xia W, Yamaguchi H, Wei Y, Chen MK, Hsu JM, Hsu JL, Yu WH, Du Y, Lee HH, Li CW, Chou CK, Lim SO, Chang SS, Litton J, Arun B, Hortobagyi GN, Hung MC. Parp inhibitor upregulates pd-l l expression and enhances cancer-associated immunosuppression. Clin Cancer Res. 2017;23:371 1-3720

EXAMPLE 4

[00223] Use of low doses of PARP inhibitors as adjuvant therapy for immunotherapeutic approaches and treatment strategies that do not target DNA repair/damage mechanisms

[00224] PARP inhibitors such as olaparib (LYNPARZA™) and others (under clinical trials) are used to target cancer cells with deficiencies in DNA repair enzymes (e.g. BRCA mutations) with a goal to achieve synthetic lethality (specific death of cancer cells) . Our results show that partial PARP inhibition is very effective at reducing cancer-related inflammation and promoting a tumor-suppressive environment by interfering with the tumor promoting activity of MDSCs. Without wishing to be bound by theory, one can use PARP inhibitors at a dose that can be gaged according cancer type and affected patient to achieve a better clinical outcome with immunotherapy approaches or with therapies whose targets do not include DNA repair/damage enzymes.

[00225] The PARP inhibitor olaparib (LYNPARZA™) is currently being used as an oral treatment for women with BRCA-mutated advanced ovarian cancer; other PARP inhibitors are under clinical trials for other cancers with DNA repair deficiency. As described herein, PARP inhibitors, at low doses, can be used to increase the efficacy of immunotherapy approaches and other approaches that do not target DNA repair/damage en/ymes. See FIG. 31 , for example. Thus PARP inhibitors can be used for the treatment of a variety of cancers that can benefit from immunotherapy.

[00226] The current concept in the use of PARP inhibitors is to achieve maximal inhibition of the enzyme (PARP) with synthetic lethality (i.e . death of mutated cancer cells) as the ultimate goa 1. Embodiments as described herein enhance the immune system in such a way to improve the efficacy of immunotherapy or approaches that do not target DNA repair/damage en/ymes in fighting cancer. An additional but important aspect with this approach is that low doses of the drugs may lead to fewer side effects.

EQUIVALENTS

[00227] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.