Compositions and methods of treatment of corneal endothelium disorders

Abstract

Provided herein are compositions and methods of use thereof for treating a corneal endothelium disorder (e.g., Fuchs endothelial corneal dystrophy). Also provided are compositions and methods of use thereof for preserving corneal tissue prior to transplantation.

Description

[0001]Attorney Docket No.00633-0394WO1 / MEEI 2024-199 COMPOSITIONS AND METHODS OF TREATMENT OF CORNEAL ENDOTHELIUM DISORDERS CLAIM OF PRIORITY This application claims the benefit of U.S. Provisional Application Serial No. 63 / 647,665, filed on May 15, 2024. The entire contents of the foregoing are incorporated herein by reference. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. EY020581 awarded by the National Institutes of Health. The Government has certain rights in the invention. TECHNICAL FIELD Provided herein are compositions and methods of use thereof for treating a corneal endothelium disorder (e.g., Fuchs endothelial corneal dystrophy). Also provided herein are compositions and methods of use thereof for preserving corneal tissue prior to transplantation. BACKGROUND Corneal endothelial cells (CEnCs) form a hexagonal monolayer in the posterior cornea and serve a central role in maintaining corneal clarity and hydration by constant pumping of ions between the stroma and aqueous fluid1,2. Derived from the neural crest, CEnCs are arrested in the post-mitotic state3,4and show minimal proliferation in vivo with 4000 cells / mm2at birth that gradually decreases with age. Fuchs endothelial corneal dystrophy (FECD), the most common cause of corneal endothelial degeneration, is an age-related genetically heterogeneous disease resulting in the accelerated loss of CEnCs leading to corneal edema and loss of vision5. FECD affects 4% of the U.S. population over the age of 40 years and is the leading indication for the corneal transplantations performed in the U.S., which is currently the only treatment option available for this disease6-8. It is characterized by mitochondrial dysfunction, profibrotic extracellular matrix deposits forming dome shaped excrescences called guttae protruding from the Descemet’s membrane, and ensuing CEnC apoptosis9-11. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 SUMMARY The DNA damage response (DDR) signal transduction pathway is orchestrated by upstream central kinases such as ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-Related) and DNA-PKcs (DNA–dependent protein kinase) which, upon activation by DNA damage, phosphorylates and activates downstream effectors that are involved in DNA repair, cell-cycle arrest, senescence, or apoptosis16. ATM has been shown to play a central role in controlling the DNA damage response leading to cell cycle activation of postmitotic cells, like neurons17-19. ATM but not ATR is essential for the upregulation of p21 and the G1 cell cycle checkpoint20and activation of senescence21,22. ATM responds to DNA double- stranded breaks (DSBs), which may be caused intrinsically through the collapse of stalled replication fork or extrinsically through exposure to ionizing radiation (IR)23. Despite great advancements in our understanding of ATM signaling and function in recent years, the complex mechanisms involved in its activation and response are not fully resolved. ATM is a 370-kDa serine / threonine kinase belonging to the phosphatidyl inositol 3-kinase (PI3K) family24,25. In its inactive form, ATM forms homodimers which dissociate into catalytically active monomers following rapid intermolecular autophosphorylation of serine1981 (pATM-S1981) upon ATM activation26. Since the initial discovery of its autophosphorylation site and its role in ATM activation, other ATM post-translational modifications have been reported27,28. The MRN (MRE11- RAD50-NBS1) complex recruits ATM to the site of DSBs29, where it acts as a damage sensor that can also form a physical bridge spanning the DSBs30. Activated ATM phosphorylates histone variant H2AX at serine139 (γH2AX-S139) within minutes after DNA damage, forming γH2AX foci which enables the recruitment of numerous DNA repair proteins and chromatin remodeling complexes around DSBs31,32. ATM is generally regarded as the principal mediator of the G1 / S cell cycle checkpoint, which prevents cells with damaged DNA from entering S-phase. In response to induction of DNA-DSBs, ATM directly phosphorylates tumor suppressor protein p53 on serine15 (pp53-S15)33thereby stabilizing p53. Additionally, pATM- S1981 activates cell cycle checkpoint protein CHK2 at threonine68 (pChk2-T68) that in turn phosphorylates p53 at serine20 (pp53-S20)34. This phosphorylation at p53 leads to its stabilization by preventing its Mdm2-mediated ubiquitination and Attorney Docket No.00633-0394WO1 / MEEI 2024-199 degradation34,35. Activated p53 acts as a transcription factor and drives the expression of genes involved in cell cycle checkpoint activation, such as p21, but also several genes which are involved in the induction of apoptosis. resulting in cell cycle arrest36,37. In addition to its role in the G1 / S checkpoint, ATM also contributes to the activation of the intra-S phase and G2 / M cell cycle arrest, as cells deficient in ATM do not reduce DNA synthesis following induction of DNA-DSBs, referred as radioresistant DNA synthesis38,39. In response to oxidative stress, various substrates are phosphorylated in an ATM-dependent manner, thus demonstrating the complexity of the ATM-mediated DDR pathways40,41. Deficiency of ATM in humans results in the neurodegenerative disorder, Ataxia-telangiectasia, characterized by loss of Purkinje neurons, immunodeficiencies, hypersensitivity to ionizing radiations and predisposition to cancer42. Furthermore, reactive oxygen species (ROS) has been shown to directly trigger ATM autophosphorylation and facilitate its localization to DSBs43. While mechanisms of DDR are well studied in proliferating cells, DDR signaling pathways in post-mitotically arrested cells, specifically CEnCs that are particularly prone to oxidative stress-mediated damage, remain unclear. The present disclosure investigated the role of oxidative stress induced by a chemical stressor menadione, as well as a physiological stressor UVA irradiation, in ATM-mediated DDR signaling in normal and diseased CEnCs. Since FECD is more pronounced in females14, the combined effect of UVA and catechol estrogen (4- OHE2), a by-product of estrogen metabolism, in elucidating the role of ATM in the progression of FECD was also examined herein. Studies described herein demonstrated that NQO1 was downregulated in FECD specimens as well as FECD cell lines44compared to controls. Downregulation of NQO1 exacerbated menadione induced oxidative stress leading to endothelial-mesenchymal transition in corneal endothelial cells10. Additionally, NQO1-null mice exhibited greater CE cell loss, greater ROS production, and lack of neutralization of reactive estrogen metabolites due to loss of NQO114, mimicking end-stage FECD. As described herein, NQO1-null (NQO1- / -) stable cell lines10were used as a cellular model of FECD in addition to patient derived cell lines, to study ATM-mediated DDR signaling under acute and chronic oxidative stress. Using in-vitro and in vivo FECD models, data of the present disclosure demonstrated that effectors of DDR signaling determined the extent of cell Attorney Docket No.00633-0394WO1 / MEEI 2024-199 cycle arrest, DNA repair, and / or senescence based on the duration of the stress and DNA damage. By using an ATM inhibitor (e.g., KU-55933 and KU-60019) and a CHK2 inhibitor (e.g., CCT241533), the present disclosure demonstrated that modulation of DDR signaling can have a potential therapeutic application in averting senescence in diseased CEnCs and that ATM-driven modulation of the cell cycle and DNA repair can potentially provide an effective therapeutic strategy against FECD. The present disclosure, using an UVA-mouse model of FECD, demonstrated that ATM inhibition with KU-60019 (i) rescued corneal edema at weeks-2, 3, 4 post- UVA, (ii) rescued corneal endothelial cell loss at weeks-2, 4 and 10 post-UVA and (iii) attenuated multinucleation and senescence at weeks-2, 4 and 10 post-UVA. Once activated, ATM phosphorylates many downstream effectors including checkpoint kinase 2 (CHK2). As a downstream kinase of ATM, CHK2 is particularly important in the response to DNA double-strand breaks. This type of DNA damage plays an important role in corneal damage and dysfunction. Using UVA-mouse model of FECD, the present disclosure demonstrated that CHK2 inhibition with CCT241533 (i) rescued corneal edema at weeks-1, and 2 post-UVA, (ii) rescued corneal endothelial cell loss at weeks-1, and 2 post-UVA and (iii) attenuated multinucleation and senescence at week-2 post-UVA. Optimal ex vivo corneal storage in eye banks is crucial to increase both the number of corneas suitable for graft and their intrinsic quality, mainly the number of viable endothelial cells, which dictates graft survival in recipients. With both passive storage methods used worldwide significant endothelial cell loss is inevitable. (Garcin et al., Am J Transplant.2019 Jun;19(6):1641-1651). As provided herein, ex vivo normal human donor corneal endothelial cells demonstrated markedly reduced corneal endothelial cell death when treated ex vivo with a medium comprising KU- 60019 or CCT241533. The present disclosure provides that use of an ATM inhibitor (e.g., KU-55933 and KU-60019) and / or a CHK2 inhibitor (e.g., CCT241533) can improve upon current eye-banking methods. Provided herein are methods of treating or preventing corneal endothelial cell loss in a subject in need thereof. In some embodiments, a method of treating or preventing corneal endothelial cell loss in a subject in need thereof comprises administering to the subject an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 Also provided herein are methods of treating or preventing corneal edema in a subject in need thereof. In some embodiments, a method of treating or preventing corneal edema in a subject in need thereof comprises administering to the subject an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both. Additionally, provided herein are methods for treating a corneal endothelium disorder in a subject in need thereof. In some embodiments, a method for treating a corneal endothelium disorder in a subject in need thereof comprises administering to the subject an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both. Provided herein are also methods for preventing corneal graft failure in a subject in need thereof. In some embodiments, a method for preventing corneal graft failure in a subject in need thereof comprises administering to the subject an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both. In some embodiments, the effective amount of an ATM inhibitor, a CHK2 inhibitor, or both is administered to the subject before corneal transplant, during corneal transplant, after corneal transplant, or at any combination thereof. Provided herein are methods of transplanting a corneal tissue into a subject in need thereof. In some embodiments, a method of transplanting a corneal tissue into a subject in need thereof comprises: (a) harvesting a cornea, corneal tissue or corneal endothelium from a donor; (b) placing the cornea, corneal tissue or corneal endothelium in a medium comprising an ATM inhibitor, a CHK2 inhibitor, or both; and (c) transplanting the cornea, corneal tissue or corneal endothelium into a subject in need thereof. In some embodiments, the cornea, corneal tissue or corneal endothelium can be stored in the medium comprising an ATM inhibitor, a CHK2 inhibitor, or both for up to 21 days prior to transplanting into a subject in need thereof. In some embodiments, the transplanted cornea, corneal tissue or corneal endothelium can be further treated with an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both following transplantation into the subject. In some embodiments, a subject in need thereof can be suspected of having or has been diagnosed as having a corneal endothelium disorder. In some embodiments, subject in need thereof can be suspected of having or has been diagnosed as having Fuchs endothelial corneal dystrophy (FECD), posterior polymorphous dystrophy, congenital hereditary endothelial dystrophy (CHED), iridocorneal endothelial (ICE) syndrome, or pseudophakic bullous keratopathy (PBK). In some embodiments, a Attorney Docket No.00633-0394WO1 / MEEI 2024-199 subject in need thereof can be suspected of having or has been diagnosed as having FECD. In some embodiments, a subject in need thereof can have or be at risk of having ultraviolet-mediated damage to a cornea. In some embodiments, a subject in need thereof can be in need of a corneal transplantation. In some embodiments, a subject in need thereof can have or be at risk of having corneal endothelial cell loss after a corneal transplantation procedure. In some embodiments, a corneal transplantation procedure can be a full-thickness or partial-thickness corneal transplantation. In some embodiments, a corneal transplantation procedure can be posterior lamellar keratoplasty (PLK), deep lamellar endothelial keratoplasty (DLEK), Descemet stripping endothelial keratoplasty (DSEK), Descemet stripping automated endothelial keratoplasty (DSAEK), DM endothelial keratoplasty (DMEK), DM automated endothelial keratoplasty (DMAEK), and / or deep anterior lamellar keratoplasty (DALK). In some embodiments, a subject in need thereof can have or be at risk of having corneal endothelial cell loss after full-thickness or partial-thickness corneal transplantation, herpes zoster ophthalmicus, uveitis, and / or graft rejection. In some embodiments, the effective amount of an ATM inhibitor, a CHK2 inhibitor, or both can be administered according to any of the methods disclosed herein to the eye of the subject. In some embodiments, the effective amount of an ATM inhibitor, a CHK2 inhibitor, or both can be administered according to any of the methods disclosed herein to the eye of the subject by systemic administration, subconjunctival injection, intraperitoneal injection, intracameral injection, or topical administration to the eye. Also provided herein are methods of preserving or restoring corneal tissue. In some embodiments, a method of preserving or restoring corneal tissue comprises: (a) harvesting a cornea, corneal tissue or corneal endothelium from a donor; and (b) placing the cornea, corneal tissue or corneal endothelium in a medium comprising an ATM inhibitor, a CHK2 inhibitor, or both. In some embodiments, the method of preserving or restoring corneal tissue can further comprise: (c) storing the cornea, corneal tissue or corneal endothelium in a medium comprising an ATM inhibitor, a CHK2 inhibitor, or both. In some embodiments, the method of preserving or restoring corneal tissue can further comprise: (c) storing the cornea, corneal tissue or corneal endothelium for up to 21 days prior to transplant. In some embodiments, the method of preserving or restoring corneal tissue can further comprise: (c) storing the Attorney Docket No.00633-0394WO1 / MEEI 2024-199 cornea, corneal tissue or corneal endothelium for up to 21 days at 2-40°C prior to transplant. In some embodiments, an ATM inhibitor for use in any of the methods disclosed herein can be selected from the group consisting of KU-55933, KU-60019, AZD0156, CP-466722, AZ31, AZ32, AZD1390, and A41. In some embodiments, an ATM inhibitor for use in any of the methods disclosed herein can be KU-60019. In some embodiments, a CHK2 inhibitor for use in any of the methods disclosed herein can be selected from the group consisting of CT241533, LY2606368, LY2880070, hymenialdisine, idoloazepine-6, 2-arylbenzimidazole, NSC1095555, PV-1019, VRX0466617, aminopyridine-7, and PHI-101. In some embodiments, a CHK2 inhibitor for use in any of the methods disclosed herein can be CCT241533. Provided herein are pharmaceutical compositions comprising an ATM inhibitor, a CHK2 inhibitor, or both and at least one pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition disclosed herein can comprise an ATM inhibitor selected from the group consisting of KU-55933, KU- 60019, AZD0156, CP-466722, AZ31, AZ32, AZD1390, and A41. In some embodiments, a pharmaceutical composition disclosed herein can comprise the ATM inhibitor KU-60019. In some embodiments, a pharmaceutical composition disclosed herein can comprise a CHK2 inhibitor selected from the group consisting of CT241533, LY2606368, LY2880070, hymenialdisine, idoloazepine-6, 2- arylbenzimidazole, NSC1095555, PV-1019, VRX0466617, aminopyridine-7, and PHI-101. In some embodiments, a pharmaceutical composition disclosed herein can comprise the CHK2 inhibitor CCT241533. In some embodiments, a pharmaceutical composition disclosed herein can be suitable for administration to the eye of a subject. In some embodiments, a pharmaceutical composition disclosed herein can be an eye drop formulation. Provided herein is also a corneal storage medium. In some embodiments, a corneal storage medium comprises an ATM inhibitor, a CHK2 inhibitor, or both. In some embodiments, a corneal storage medium disclosed herein can comprise an ATM inhibitor selected from the group consisting of KU-55933, KU-60019, AZD0156, CP- 466722, AZ31, AZ32, AZD1390, and A41. In some embodiments, a corneal storage medium disclosed herein can comprise the ATM inhibitor KU-60019. In some embodiments, a corneal storage medium disclosed herein can comprise a CHK2 Attorney Docket No.00633-0394WO1 / MEEI 2024-199 inhibitor selected from the group consisting of CT241533, LY2606368, LY2880070, hymenialdisine, idoloazepine-6, 2-arylbenzimidazole, NSC1095555, PV-1019, VRX0466617, aminopyridine-7, and PHI-101. In some embodiments, a corneal storage medium disclosed herein can comprise the CHK2 inhibitor CCT241533. In some embodiments, a corneal storage medium disclosed herein can further comprise a base preservation medium. Also contemplated in the present disclosure are kits comprising at least one or more materials needed to collect a cornea, corneal tissue or corneal endothelium from a donor and / or a cell culture medium (e.g., a base preservation medium) and an ATM inhibitor, a CHK2 inhibitor, or both. As used herein, unless otherwise specified, the term “about” means plus or minus 10%. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS FIGS.1A-1F: FECD cells showed greater activation of ATM-mediated DDR than normal CEnCs in response to menadione-induced chemical stress. FIG.1A: Western blot of time course of MN (25 µM)-induced DDR activation showed earlier and greater activation of phospho-ATM (pATM / ATM) and its downstream effector proteins (pH2AX / H2AX, pp53 / p53, and pChk2 / Chk2) in SVF1-73F compared to SVN1-67F cells. FIG.1B: Fold change in densitometry of MN- over un-treated cells showed significantly greater activation of pATM / ATM, pp53 / p53, and pH2AX / H2AX, but not pChk2 / Chk2 proteins in SVF1-73F compared to SVN1-67F at 60 min of treatment with 25 µM MN. *P < 0.05 applying paired t-test. FIG.1C: Attorney Docket No.00633-0394WO1 / MEEI 2024-199 Immunoprecipitation with anti-pATMS1981 or IgG antibody and immunoblotting from whole cell lysates prepared after treatment with 25 µM MN for 60 min showed greater pulldown of pATM / ATM and pp53 / p53 in SVF1-73F compared to SVN1-67F cells. Western blotting of whole cell lysates showed greater pATM / ATM and pp53 / p53 expression levels in SVF1-73F compared to SVN1-67F cells after MN treatment. FIG.1D: Western blotting at 60 min after co-treatment with MN (25 µM) and ATM inhibitor, KU-55933 (20 µM) showed depleted levels of pATM / ATM and pp53 / p53 affirming ATM-driven DDR activation. FIG.1E: Cell cycle phase histograms of normal cells, SVN1-67F, and FECD cells, SVF1-73F and SVF5-54F, stained with propidium iodide and subjected to flow cytometry demonstrated an increase in the S-phase distribution of all three cell lines 24 h after treatment with 50 µM MN compared to no treatment. FIG.1F: Inter-cell line comparison of percentage cells in each phase of the cell cycle showed a significantly elevated S-phase in SVF5- 54F compared to SVN1-67F cells 24 h after MN treatment. ***, * indicates P < 0.001, or < 0.05, respectively, applying one-way ANOVA with Tukey’s Multiple Comparison Test comparing the S-phase of each group. Data represented as mean + SEM of N>3. FIGS.2A-2G: Physiological stressors, UVA and 4-OHE2, caused greater ATM-mediated G2 / M phase arrest in FECD cells. Western blot analysis (FIG.2A) and densitometry (FIG.2B) showed greater activation of phospho-ATM (pATM / ATM) and its downstream effector proteins (pH2AX / H2AX, pChk2 / Chk2, CASP3 / β-actin) in SVF1-73F compared to SVN1-67F after 60 min of treatment with 10 J / cm2UVA (+ 10 µM 4-OHE2). FIG.2C: Cell cycle analysis by propidium iodide staining and flow cytometry after treatment with UVA + 4-OHE2 demonstrated greater G2 / M cell cycle arrest in SVF1-73F compared to normal SVN1-67F cells (center), while this arrest was rescued with 20 µM of ATM inhibitor (KU-55933) (right). FIG.2D: Percentage of cells in each stage of the cell cycle. FIG.2E: Western blot analysis and densitometry of G2 / M marker protein, Cyclin B1, at 24 h post UVA+4-OHE2treatment showed greater G2 / M arrest in SVF1-73F compared to SVN1-67F cells. FIG.2F: Western blot analysis and densitometry of senescence marker proteins, p53 / β-actin and p21 / β-actin, 24 h post UVA+ 4-OHE2treatment showed induction of senescence in SVF1-73F but not SVN1-67F cells. FIG.2G: SA- β-GAL staining of control (top) and 24 h post UVA + 4-OHE2treated (bottom) Attorney Docket No.00633-0394WO1 / MEEI 2024-199 SVN1-67F and SVF1-73F cells. Data presented as mean values ± SE. Scale bars = 100 μm. ****, ***, **, * indicates P < 0.0001, or < 0.001, or < 0.01, or < 0.05, respectively, applying one-way ANOVA test with Tukey’s Multiple Comparison Test. Data represented as mean + SEM of N>3. FIGS.3A-3F: Loss of NQO1, as seen in FECD, led to hyperphosphorylation of ATM and FACS sorting of G2 / M arrested NQO1-null cells showed upregulation of DNA-repair genes upon acute UVA+4-OHE2 treatment. FIG.3A: Timeline of acute treatment of NQO1-wildtype (NQO1+ / +) and NQO1-null (NQO1- / -) cells. FIG.3B: Western blot analysis and densitometry showing greater activation of pATM / ATM, and its downstream effector protein, pChk2 / Chk2, in NQO1- / -compared to NQO1+ / +cells after 1 hour of treatment with 10 J / cm2or 25 J / cm2UVA (+ 10 μM 4-OHE2). FIG.3C: Cell cycle analysis by propidium iodide staining and flow cytometry after treatment with UVA + 4-OHE2 demonstrating greater G2 / M cell cycle arrest in NQO1- / -compared to NQO1+ / +cells (center), while this arrest was rescued with 20 μM of ATM inhibitor (KU-55933) (right). FIG.3D: Percentage of cells in each stage of the cell cycle along with western blot of G2 / M phase marker protein, cyclin B1, showing the G2 / M arrest with UVA + 4-OHE2 and its rescue with addition of 20 μM of ATM inhibitor (KU-55933). FIG.3E: Increased mRNA expression of four DNA- repair genes: LIG3, NEIL2, TOP3A and XPC in G2 / M phase of NQO1- / -compared NQO1+ / +cells, displayed as a delta Ct ratio of UVA+4OHE2 / untreated. FIG.3F: Reduced mRNA expression of LIG3, NEIL2, TOP3A and XPC in G2 / M phase after treatment with ATM inhibitor (KU-55933) in NQO1+ / +and NQO1- / -cells. ****, ***, **, * indicates P < 0.0001, or < 0.001, or < 0.01, or < 0.05, respectively, applying one-way ANOVA test with Tukey’s Multiple Comparison Test. Data represented as mean + SEM of N>3. FIGS.4A-4M: Chronic UVA+4OHE2 treatment in NQO1- / -CEnCs demonstrated severe DNA damage, arrest in G0 / G1 phase of cell-cycle, and degradation of DNA-repair genes, thus aggravating the induction of cytotoxic senescence. FIG.4A: Timeline of chronic treatment of NQO1+ / +and NQO1- / -cells. FIG.4B: Cell cycle analysis by propidium iodide staining and flow cytometry after chronic treatment with UVA + 4OHE2demonstrated greater G0 / G1 cell cycle arrest in NQO1- / -compared to NQO1+ / +cells. FIG.4C: Percentage of cells in each stage of the cell cycle along with western blot of G0 / G1 phase marker protein, cyclin D1, showed Attorney Docket No.00633-0394WO1 / MEEI 2024-199 the G0 / G1 arrest with UVA + 4-OHE2 and its rescue with addition of 5 μM of KU- 55933. Reduced mRNA expression of four DNA-repair genes – LIG3 (FIG.4D), NEIL2 (FIG.4E), TOP3A (FIG.4F), and XPC (FIG.4G) in both G0 / G1 and G2 / M phases of NQO1- / -cells compared NQO1+ / +cells, displayed as a delta Ct ratio of UVA+4-OHE2 / untreated. Upon treatment with ATM inhibitor (KU-55933) during chronic UVA treatment, differential expression was rescued in G2 / M phase for all four genes (LIG3, NEIL2, TOP3A and XPC), while in G0 / G1 phase for only LIG3 and TOP3A in NQO1- / -cells. Increased mRNA expression of TP53 (FIG.4H), and CDKN1A (FIG.4I) genes in G0 / G1 phase of NQO1- / -cells compared NQO1+ / +cells, displayed as a delta Ct ratio of UVA+4-OHE2 / untreated. Upon treatment with ATM inhibitor (KU-55933) during chronic UVA treatment, differential expression was rescued in G2 / M phase for TP53 and CDKN1A genes, in both NQO1+ / +and NQO1- / -cells. FIG.4J: Western blot analysis and densitometry of senescence marker protein p53 / β-actin, 5 days post UVA+ 4-OHE2treatment showed more induction of senescence in NQO1- / -cells compared NQO1+ / +cells. FIG.4K: Increased staining of senescence marker, SA-β-gal, after chronic treatment with UVA + 4OHE2 which was reduced with addition of 20 μM of ATM inhibitor (KU-55933) in NQO1+ / +and NQO1- / -cells. Data presented as mean values ± SE. Scale bars = 50 μm. FIG.4L: Graphic representation of differences between acute (left box) and chronic (right box) UVA treatments in NQO1+ / +(solid black line) and NQO1- / -(dashed line) cells, and rescue with KU-55933 in DNA repair and senescence. FIG.4M: Overview of proposed ATM activation pathway under chronic or acute stress conditions in FECD. ****, ***, **, * indicates P < 0.0001, or < 0.001, or < 0.01, or < 0.05, respectively, applying one-way ANOVA test with Tukey’s Multiple Comparison Test. Data represented as mean + SEM of N>3. FIGS.5A-5F: UVA-induced cell cycle re-entry, senescence and DNA damage was delayed in the absence of Atm in the mouse model of FECD. FIG.5A: Schematic of the timeline of UVA-irradiation and corneal evaluation by HRT and OCT in Atm-WT and Atm-null mice. FIG.5B: Genotyping PCR gel confirming the homozygous knockout of Atm in Atm-null mice. FIG.5C: Western blot analysis showed absence of pAtm and Atm proteins in Atm-null mice corneas pre- and one- day post-UVA. β-Actin was used as a loading control and microsome lysate was used as a positive control. FIG.5D: Whole mount staining of corneal endothelium with Attorney Docket No.00633-0394WO1 / MEEI 2024-199 cell cycle re-entry marker Ki67 (light grey) and DAPI (dark grey) at baseline (no UVA), 2 days, 3 days and 1-week post-UVA in Atm-WT and Atm-null mice. * P < 0.05 applying Unpaired Student’s t-test. Whole mount staining of the corneal endothelium and quantification of percent (%) positive nuclei for (FIG.5E) DNA damage marker pH2AX (light grey), and (FIG.5F) senescence marker H3K9me3 (light grey), in Atm-WT and Atm-null mice at day 3, week 1, week 2, week 4 and week 10 post-UVA irradiation. ***, * indicates P < 0.001, or < 0.05, respectively, applying one-way ANOVA test with Tukey’s Multiple Comparison Test. Scale bars = 50 μm. Data represented as mean + SEM of N>3. FIGS.6A-6B: Menadione-induced oxidative stress caused profibrotic phenotype and did not activate ATR kinase, in normal or FECD CEnCs. FIG.6A: Morphological changes induced in SVN1-67F (normal) and SVF1-73F (FECD) cells at 60 mins post 25 μM Menadione (MN) treatment. FIG.6B: Western blot analysis of pATR and ATR in SVN1-67F (normal) and SVF1-73F (FECD) cells treated with 25 μM MN and lysates prepared at different time points (30 mins, 60 mins, 90 mins and 120 mins) post 25 μM MN treatment, demonstrating that MN treatment did not activate ATR in normal or FECD cells. FIG.7: Immunoprecipitation with anti-ATM antibody showed greater pulldown of p53 in FECD cells. Immunoprecipitation with anti-ATM antibody and immunoblotting from whole cell lysates of cells treated with 25 µM MN for 60 min showed greater pulldown of p53 in SVF1-73F compared to SVN1-67F cells. FIGS.8A-8C: Characterization of ATM inhibitor KU55933 in SVN1-67F normal cells. FIG.8A: Dose response curve of ATM inhibitor KU55933 in SVN1- 67F cells. Percentage of cell viability was assessed for different doses of the inhibitor KU55933, ranging from 1 μM to 100 μM. FIG.8B: Western blot analysis of downstream effected proteins of ATM (pChk2, Chk2, pH2AX, H2AX) in SVN1-67F (normal) and SVF1-73F (FECD) cells treated with 25 μM Menadione (MN). FIG. 8C: Western blotting showed greater pATM and pp53 activation at 60 min of treatment with 25 µM MN in two FECD cell lines, SVF1-73F and SVF5-54F, compared to the normal cell lines, SVN1-67F and SVN2-67F. FIG.9: Cell cycle phases (gating) of normal cells, SVN1-67F, and FECD cells, SVF1-73F and SVF5-54F, stained with propidium iodide and subjected to flow cytometry 24 h after treatment with 50 µM MN compared to no treatment. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 FIG.10: Cell doubling time in hours for the normal SVN1-67F and the two FECD SVF1-73F and SVF5-54F cell lines (n=3 per cell line, One way ANOVA). FIG.11: UVA light dose of 25 J / cm2induced oxidative stress activated pATM in both normal as well as FECD CEnCs. Western blot analysis of pATM (top panel) and ATM (middle panel) in SVN1-67F (normal) and SVF1-73F (FECD) cells treated with 25 J / cm2UVA (+ 10 μM 4-OHE2) and lysates prepared at 1-hour post treatment, demonstrating that pATM was activated in both normal SVN1-67F, as well as FECD SVF1-73F cell line 1 hour of post UVA treatment. Actin (bottom panel) was used as a loading control. FIG.12: Percent expression at baseline of DNA-repair genes in G0 / G1 phase of NQO1+ / +and NQO1- / -cells under acute UVA and 4-OHE2 treatment condition. mRNA expression of four DNA-repair genes – LIG3 (top left panel), NEIL2 (top right panel), TOP3A (bottom left panel), and XPC (bottom right panel) in G0 / G1 and G2 / M phase of NQO1+ / +and NQO1- / -cells during acute UVA treatment. All 4 DNA repair genes (LIG3, NEIL2, TOP3A and XPC) evaluated in this study for acute effect post- UVA+4-OHE2 treatment, did not show a significant difference in expression between G0 / G1 and G2 / M phases of cell cycle at baseline in neither NQO1+ / +nor NQO1- / -cells. This confirmed that the upregulation of these DNA repair genes in G2 / M was caused by exposure to UVA+4-OHE2. FIG.13: Percent expression of DNA-repair genes in G0 / G1 phase of NQO1+ / +and NQO1- / -cells post ATM inhibition under acute UVA and 4-OHE2treatment conditions. mRNA expression of four DNA-repair genes – LIG3, NEIL2, TOP3A and XPC in G0 / G1 and G2 / M phase of NQO1+ / +and NQO1- / -cells, displayed as a delta Ct ratio of UVA+4-OHE2 / untreated, after treatment with 20 μM of ATM inhibitor (KU- 55933) during acute UVA treatment. FIG.14: Cell cycle analysis of NQO1+ / +and NQO1- / -cells with and without chronic (5-days) treatment with UVA, 4-OHE2and ATM inhibitor KU-55933. Cell cycle analysis by propidium iodide staining and flow cytometry after treatment with 25 J / cm2UVA, 10 μM 4-OHE2and 5 μM ATM inhibitor (KU-55933), and controls in NQO1+ / +and NQO1- / -cells. Percentage of cells in each stage of the cell cycle are listed in their respective boxes. FIGS.15A-15B: Characterization of Atm-WT and Atm-null mice at different time points post UVA irradiation. Single 500 J / cm2UVA irradiation dose caused Attorney Docket No.00633-0394WO1 / MEEI 2024-199 morphological changes and damage in mouse cornea and mouse corneal endothelial cells in both Atm-WT and Atm-null mice. FIG.15A: Representative images of Slit lamp (broad beam) imaging, Sodium fluorescein staining and OCT (optical coherence tomography) for ATM-WT and ATM-null mice pre-UVA and at 1-day, 1-week, 2- weeks and 1-month post UVA irradiation. FIG.15B: Line graph showing CCT for ATM-WT and ATM-null mice pre-UVA and at 1-day, 1-week, 2-weeks and 4-weeks post UVA irradiation. n=5, *p<0.05. FIG.16: HRT, pH2AX and H3K9Me3 staining and cell density in Atm-WT and Atm-null mice pre-UVA treatment. Whole mount staining of the corneal endothelium and quantification of percent (%) positive nuclei for DNA damage marker pH2AX (light grey, top panels), and senescence marker H3K9Me3 (light grey, bottom panels), in Atm-WT and Atm-null mice at day-0 or pre-UVA irradiation. Scale bars = 50 µm. FIGS.17A-17B: Schematic of the timeline of UVA-irradiation, KU-60019 (FIG.17A) or CCT241533 (FIG.17B) administration and corneal evaluation by HRT and OCT in female wild-type C57BL / 6 mice. FIGS.18A-18B: (FIG.18A) Representative OCT images of mouse corneas pre-UVA at weeks 1, 2, 3, 4 and 10 post-UVA (500 J / cm2), and (FIG.18B) OCT image-based CCT analysis demonstrating that KU60019 treatment stabilized CCT which was significantly lower at week-2, 3 and 4, compared to controls. Mixed-effect regression analysis was employed to analyze the effect of KU-60019 administration post-UVA. n = 10 for weeks 1 and 2 post UVA, n = 9 for weeks 3 and 4 post UVA and n = 3 for week 10 post UVA. Data are mean ± SEM; P < 0.05. FIGS.19A-19D: (FIG.19A) In vivo confocal HRT images of mouse corneal endothelia showed less enlarged and more hexagonal morphology after KU-60019 injection compared to vehicle injection (IP) at weeks 2, 4, and 10 post-UVA, demonstrating a 19.6%, 23% and 21% CEnC rescue respectively. (FIG.19B) Quantification of corneal endothelial cell number showed a higher cell count in KU- 60019-treated mice compared to vehicle mice at week 2, 4, and 10 post-UVA. Data are mean ± SEM. N≥3 per group per timepoint, **p < 0.01, ****p < 0.0001, two-way ANOVA. (FIG.19C) Representative confocal images of whole mount of mouse CEnCs detecting ZO-1 (light grey) and DAPI (dark grey) immunostaining showed an absence of enlarged cells and greater hexagonality in KU-60019-treated mice Attorney Docket No.00633-0394WO1 / MEEI 2024-199 compared to vehicle-treated mice at week 2, 4, and 10 post-UVA. (FIG.19D) Quantification of number of cells per field based on ZO-1 border showed higher cell density in KU60019-treated mice compared to vehicle-treated mice at week 2, 4, and 10 post-UVA, demonstrating a 13.3%, 12.5% and 14% CEnC per field, respectively. Data are mean ± SEM. Scale bar, 50 μm. N=3 per group per timepoint, *p < 0.05, ***p < 0.001, One-way ANOVA. FIGS.20A-20C: (FIG.20A) Whole mount staining of the corneal endothelium and quantification of percent (%) multinucleated cells (FIG.20B) and percent (%) positive nuclei for senescence marker H3K9me3 (white arrows) (FIG. 20C), in WT C57BL / 6 mice at week 2, week 4 and week 10 post-UVA irradiation. ***, **, * indicates P < 0.001, P < 0.01, or < 0.05, respectively, applying one-way ANOVA test with Tukey’s Multiple Comparison Test. Scale bars = 50 μm. Data represented as mean + SEM of N=3. FIGS.21A-21B: (FIG.21A) Representative OCT images of mouse corneas pre-UVA at weeks 1, and 2 post-UVA (500 J / cm2), and (FIG.21B) OCT image-based CCT analysis demonstrating that CCT241533 treatment stabilized CCT which was significantly lower at week-1, and 2, compared to controls. Mixed-effect regression analysis was employed to analyze the effect of CCT241533 administration post-UVA. n = 5 for weeks 1 and 2 post UVA. Data are mean ± SEM; ***, * indicates P < 0.001, or < 0.05, respectively. FIGS.22A-22D: (FIG.22A) In vivo confocal HRT images of mouse corneal endothelia showed less enlarged and more hexagonal morphology after CCT241533 treatment compared to vehicle injection (IP) at weeks 1, and 2 post-UVA, demonstrating a 21.5% and 28.5% CEnC rescue, respectively. (FIG.22B) Quantification of corneal endothelial cell number showed a higher cell count in CCT241533-treated mice compared to vehicle-treated mice at week 1, and 2 post- UVA. Data are mean ± SEM. N≥3 per group per timepoint ****p < 0.0001, two-way ANOVA. (FIG.22C) Representative confocal images of whole mount of mouse CEnCs detecting ZO-1 (light grey) and DAPI (dark grey) immunostaining showed absence of enlarged cells and greater hexagonality in CCT241533-treated mice compared to vehicle-treated mice at week 1, and 2 post-UVA. (FIG.22D) Quantification of number of cells per field based on ZO-1 border showed higher cell density in CCT241533-treated mice compared to vehicle-treated mice at week 2 post- Attorney Docket No.00633-0394WO1 / MEEI 2024-199 UVA, demonstrating 27% rescue of CEnC per field. Data are mean ± SEM. Scale bar, 50 μm. N>3 per group per timepoint, ****p < 0.0001, one-way ANOVA. FIGS.23A-23C: (FIG.23A) Whole mount staining of the corneal endothelium and quantification of percent (%) multinucleated cells (FIG.23B) and % positive nuclei for senescence marker H3K9me3 (white arrows) (FIG.23C), in WT C57BL / 6 mice at week 2 post-UVA irradiation and CCT241533 administration. *** indicates P < 0.001 applying one-way ANOVA test with Tukey’s Multiple Comparison Test. Scale bars = 50 μm. Data represented as mean + SEM of N>3. FIGS.24A-24B: Cell death analysis of ex vivo normal human donor corneal endothelial cells unstressed and stressed with pro-oxidant menadione, showing the cell rescue with CCT241533 or KU-60019. Tissues were incubated in the cell culture media and stressed with Menadione which caused 33% cell death compared to media alone. ATM inhibitor (KU-60019) and CHK2 inhibitor (CCT241533) rescued cell death by 24% and 29% respectively, when added to the recovery media in addition to menadione. Cells were stained with dead cell marker Ethidium Homodimer (white dots) and DAPI (light grey dots). Microscopy images at 10X magnification (FIG. 24A) and analysis of % positive nuclei for Ethidium Homodimer (FIG.24B). *** indicates P < 0.001 and ** indicates P < 0.01 applying one-way ANOVA test with Tukey’s Multiple Comparison Test. DETAILED DESCRIPTION Due to high metabolic activity, inability to replicate the genome, and lifelong exposure to ultraviolet light, the mitochondria-rich CEnCs are highly susceptible to ROS-mediated DNA damage15. Very little is known about the protective mechanisms by which normal CEnCs detect and repair DNA damage, thereby facilitating their longevity. Cell cycle machinery has been described as a crucial element of DNA damage response (DDR) in post-mitotic cells, like neurons and CEnCs, which either enables repair of damaged DNA or initiates apoptosis upon extensive damage17. In neurons, low levels of reactive oxygen species have been shown to promote cell cycle re-entry as opposed to apoptosis, indicating reparable DNA damage facilitates cell cycle re- entry as a survival mechanism to activate DNA repair pathways such as nonhomologous DNA end joining in post mitotic cells19,54. While FECD is characterized by the loss of CEnCs due to apoptosis, little is understood about the Attorney Docket No.00633-0394WO1 / MEEI 2024-199 status of surviving diseased CEnCs and how the disease progresses. Increased p53- mediated apoptosis was shown in FECD patient specimens and in vitro cell lines55, and UVA irradiation, a physiologically relevant stressor, caused G2 / M arrest in vitro in HCEnCs with both senescent and epithelial-mesenchymal transition (EMT) phenotypes detected in G2 / M arrested cells45. In the present disclosure, we demonstrated that short-term UVA exposure of diseased CEnCs led to an early activation of ATM that resulted in G2 / M phase arrest and upregulation of DNA repair genes. However, during chronic degenerative processes there was an ATM-driven shift to the pro-senescent phenotype that led to G0 / G1 phase arrest and loss of DNA repair capacity. Our findings herein on ATM-driven cell cycle and DNA repair in FECD underscore the mechanism that can be manipulated to incur cryoprotection based on DDR modulation. Following DSBs induced by oxidative stress, DNA repair and cell cycle checkpoints are the main mechanisms of maintenance of genomic integrity56. Cells have several checkpoints that function at various phases of the cell cycle. Specifically, the G0 / G1- and intra-S-phase checkpoints prevent inappropriate DNA replication, whereas the G2 / M checkpoint prevents cells with DNA damage from entering mitosis. In the present disclosure, we first detected that increased phosphorylation of ATM played a central role as an early responder to DSBs in mildly damaged CEnCs due to acute exposure to a chemical stressor, menadione. Early hyperactivation of ATM, and not ATR, in FECD cells phosphorylated p53 as the downstream target molecule and resulted in elevated S-phase. The predominant mechanism of S-phase arrest was the inhibition of firing of late origins of replication57-60. Onset of DNA repair including repair of inter-strand cross link (ICLs) lesions have been detected during S-phase when encountered by the replication machinery61,62. ICLs detected by the replisome initiated a complex set of enzymatic reactions that, with other homologous recombination (HR) events in S-phase, were regulated by Fanconi Anaemia pathway63. ATM and other proteins including the MRN complex and BRCA1 have been shown to be required for activation of this checkpoint by activating signaling cascades of ATM-p53-Cdk2-CyclinE and ATM- BRCA1 / FANCD2 / NBS1 / SMC164-66. FECD is a female predominant disorder characterized by an upregulation of genotoxic estrogen metabolites, mainly in the form of catechol estrogens (4-OHE2) Attorney Docket No.00633-0394WO1 / MEEI 2024-199 and lack of their neutralization due to loss of NQO1 in the end-stage disease44,67. Estrogen metabolites and stress hormone have been shown to activate γH2AX68,69, resulting in induction of DNA damage. ATM has been previously shown to play a role in suppressing the oncogenic effect of estrogens and preventing estrogen receptor positive breast cancer development70. In the present disclosure, we detected that both UVA and 4-OHE2were activators of DDR, especially ATM, as the major sensor of DSBs. Interestingly, loss of NQO1 led to hyperactivation of the ATM-driven response seen in FECD-derived corneal endothelial cells. The hyperactivation of ATM with subsequent Chk2 activation, further upregulated Cyclin B1 leading to G2 / M arrest of cell cycle71, thus preventing cells from undergoing mitosis. As a result, there was marked upregulation of specific base excision repair (BER) genes, that have been shown to be deficient in FECD50, in the G2 / M-sorted NQO1- / -cells as compared to G2 / M-sorted NQO1+ / +cells after acute stress. As shown herein, this effect, including upregulation of the BER gene levels, was mitigated by an ATM inhibitor, KU-55933, after acute stress, indicating that early ATM activation and G2 / M cell cycle arrest were critical for jumpstarting the DNA repair process. However, when the cells were exposed to chronic stress, the ensuing persistent DNA damage led to an ATM-driven restriction of cell cycle transition from G1 to S phase, culminating in a G0 / G1 arrest, which was significantly more pronounced in NQO1- / -cells. Likewise, the cell cycle- dependent transcriptional analysis of DNA repair genes detected reduction in all four repair genes (LIG3, NEIL2, TOP3A, and XPC) in the G0 / G1 phase of NQO1- / -compared to NQO1+ / +cells, notably different from the findings after acute stress. Furthermore, ATM inhibition decreased G0 / G1 phase in chronically stressed cells and restored DNA repair back into G2 / M phase in both NQO1+ / +and NQO1- / -cells, mitigating the effects of sustained DNA damage. As shown herein, ATM inhibition also decreased the senescence markers in the G0 / G1 phase in both cell lines; however, the rescue of senescence was greater in the NQO1- / -cells, indicating the greater effect of ATM in the diseased state. Prolonged cell cycle arrest has been previously described to activate senescence72and persistence of DDR was known to initiate this process73. Progression to senescence involved cell cycle arrest and the process was mediated through the p53-p21 pathway74, whose sustained activation has been shown to be sufficient to induce senescence49,75. p53-mediated differential transactivation of genes has been shown to Attorney Docket No.00633-0394WO1 / MEEI 2024-199 help the cell decide between apoptosis or survival76. The canonical understanding suggested that senescence predominantly transpired in the G1 or sometimes in the G2 phases of the cell cycle, with quiescence prevailing in the G0 phase77. Under acute stress conditions, we detected a transient cell cycle arrest in G2 / M that triggered the initial activation of p53-p21 initiating premature senescence as a cytoprotective mechanism. Our chronic cellular FECD model induced a substantial DNA damage response, upregulating the p53 (TP53 gene) and p21 (CDKNA1 gene) pathway and transitioning the cells to the state of mature cytotoxic senescence. Remarkably, inhibition of ATM by use of KU-55933 inhibitor attenuated senescence in our chronic cellular FECD model via reduction in SA-β-Gal positivity78. We have previously shown that UVA-irradiation of wildtype mouse corneal endothelial cells in vivo induced cell cycle reentry to G2 / M phase and progressed to senescence45. Similar cell-cycle re-entry has also been described in other post-mitotic cells, like neurons, which contributed to mild cognitive impairment and early Alzheimer's disease pathology, as well as other neurodegenerative diseases79-81. In corroboration with our in vitro findings described herein, we noted that in vivo, UVA irradiation activated the cell cycle and increased Ki67 expression in response to DNA damage, in the post-mitotic cells of the ocular tissue. However, the cell cycle activation gradually diminished with time, with a faster decrease in Ki67 positivity in Atm-null mice (at day 3) compared to Atm-WT (at week 1). Furthermore, Atm-WT, but not Atm-null mice, showed a greater increase in H3K9me3 positivity at weeks-1 and -2 and then again at week 10 post-UVA, suggesting the onset of both premature senescence and cytotoxic senescence in the presence of functional ATM. Our data described herein showed that G2 / M checkpoint played a pivotal role in regulating the effects of DNA damage in normally quiescent CEnCs after UVA exposure, as seen in FECD degeneration. Interestingly, UVA exposure also triggered substantially greater pH2AX foci formation in Atm-WT compared to Atm-null mouse CE. Likewise, a previously published study has demonstrated that H2AX phosphorylation was significantly reduced in Atm- / - compared to WT mouse embryonic fibroblasts, indicating that ATM was the major DDR kinase involved in phosphorylation of H2AX and was one of the earliest kinases to be activated in the cellular response to double strand breaks31. The presence of fewer and enlarged nuclei at week 4 after UVA exposure in Attorney Docket No.00633-0394WO1 / MEEI 2024-199 both Atm-WT and Atm-null suggested that the cells may have undergone a mitotic defect, possibly failed cytokinesis which may result in the development of polyploidy82. Targeting ATM, the master regulator of the DDR, has demonstrated neuroprotective effects. Caffeine, a nonspecific inhibitor of ATM and other phosphatidylinositol 3-kinase family members, protects against etoposide-induced DNA damage and cell death in neurons in vitro, and genetic reduction of ATM gene dosage was neuroprotective in mouse models of Huntington’s disease83,84. Recent studies have demonstrated that targeting the central ATM-Chk2 pathway, which regulates the double-strand breaks, slows down neural decline in Drosophila models of chronic neurodegeneration85. Administration of clinically relevant Atm-Chk2 inhibitors to rats, following spinal cord and optic nerve injury, is shown to result in significant axon regeneration / sprouting and a marked recovery of lost sensory, motor, and visual function85. Additionally, since ATM hyper-activation during oxidative stress promotes photoreceptor cell death in progressive retinitis pigmentosa and age- related macular degeneration86, loss of ATM and 53BP1 in mice photoreceptors has been shown to lower the activation of cell death signaling in post-mitotic neurons87. Recently, it was also reported that terminally differentiated and post-mitotic osteoclast cells show better survival in the absence of ATM88. In the present disclosure, we demonstrated that the effects of acute and chronic oxidative stress-induced cell cycle arrest and DNA repair were ATM-driven by using an ATM inhibitor, KU-55933, in our in-vitro FECD cellular models. KU-55933 was the first potent and selective ATM inhibitor that has been demonstrated to confer sensitization to IR and DNA DSB-inducing chemotherapeutics46. Importantly, in cells derived from ataxia-telangiectasia patients, which express no functional ATM, no radiosensitization was observed, validating the selectivity of KU-55399 compound towards ATM46. In the presence of DSB, KU-55933 has been shown to significantly block HR repair signals by γ-H2AX and RAD51 focal reduction in human melanoma cells89. Although this compound was commonly used as an ATM kinase inhibitor, it has been described to have limited utility in vivo owing to its high lipophilicity90. In our study described herein, co-treatment of KU-55933 with UVA and 4-OHE2in an acute cellular FECD model showed an abrogation of the observed upregulation of DNA repair genes in G2 / M phase cells, thus signifying the role of ATM as a key Attorney Docket No.00633-0394WO1 / MEEI 2024-199 regulator of this process. However, in our chronic cellular FECD model, co-treatment of KU-55933 with UVA and 4-OHE2,displayed a protective mechanism which averted the G0 / G1 phase arrest and restored the expression of DNA repair genes in the G2 / M phase. This indicated that chronic ATM activation rendered the cells prone to accumulation of DNA damage due to deficient DNA repair during the G0 / G1 phase arrest. Our data provided herein revealed that ATM was not only a key activator of DDR in CEnCs due to oxidative stress, but also a master controller of cell cycle progression, transcriptional regulation of DNA repair genes, and senescence. The in vitro UVA FECD models used in the studies of the present disclosure highlighted the potential of ATM inhibition in delaying DDR, promoting cell cycle progression, restoring DNA repair processes, and forestalling senescence in diseased CEnCs. Senescent cells are known to secrete many factors such as cytokines, growth factors, matrix remodeling proteins, proteases, and chemokines, which are collectively referred to as the senescence-associated secretory phenotype (SASP)91. The most prominent component of SASP is interleukin-6 (IL-6), a pleiotropic pro-inflammatory cytokine whose secretion is shown to increase due to DDR-dependent senescence in a variety of cell types92-94. Interestingly, inhibition of ATM is shown to prevent IL-6 from contributing to the proliferation of glioblastoma cells after IR95. Based on the present disclosure, ATM inhibition could also be efficacious in the setting of diseases like FECD involving oxidative stress and pro-inflammatory cytokine signaling. Inhibition of ATM after its early activation along with specific cell cycle checkpoints can be an effective combinatorial therapy to improve corneal function by enhancing DNA repair and delaying senescence. In summary, the present disclosure provides an understanding of the role of ATM activation in DDR signaling cascade and regulation of cell cycle in FECD. Based on our findings disclosed herein, ATM-driven modulation of the cell cycle and DNA repair can potentially provide an effective therapeutic strategy against FECD. Provided herein are methods for treating a corneal endothelium disorder as well as methods for treating or preventing corneal endothelial cell loss and methods of treating or preventing corneal edema in a subject by administration of an ATM inhibitor, a CHK2 inhibitor, or both. Further provided are methods of treating corneal Attorney Docket No.00633-0394WO1 / MEEI 2024-199 tissue ex vivo with an ATM inhibitor, a CHK2 inhibitor, or both for purposes of preserving or restoring corneal tissue for transplantation. ATM Inhibitors ATM is a member of the PIKK family and participates in the DNA damage response. ATM is activated by DNA damage and phosphorylates several key proteins that initiate the DNA damage checkpoint, cell cycle arrest, DNA repair or apoptosis (see, e.g., Lovejoy et al., DNA Repair (Amst).2009 Sep 2;8(9):1004-8). Thus, excessive activation of the DNA damage response can lead to degeneration of corneal endothelial cells as demonstrated herein. As used herein, the term “ATM inhibitor(s)” refers to any compound, drug, agent and the like that is capable of decreasing and / or inhibiting the function of ataxia telangiectasia mutated (ATM), a core component of the DNA repair system. Non- limiting examples of ATM inhibitors which can be used according to the present disclosure include KU-55933, Dactolisib (BEZ235), KU-60019, KU-59403, AZ31, AZ32, AZD0156, AZD1390, VE-821, CP-466722, Berzosertib (VE-822), AZD7648, CGK733, SJ573017, SKLB-197, Lartesertib (M4076), M3541, caffeine, XRD-0394, ATM-IN-1, WSD0628, (S)-WSD0628, and A41. KU-55933 is a potent ATM inhibitor with an IC50and Kiof 12.9 and 2.2 nM, respectively, and is highly selective for ATM as compared to DNA-PK, PI3K / PI4K, ATR and mTOR. This inhibitor was utilized in the in vitro studies of the present disclosure where it was demonstrated that inhibiting ATM activation with KU-55933 restored DNA repair in G2 / M phase and attenuated senescence in a chronic cellular model of FECD lacking NQO1. KU-60019 is a selective inhibitor of the ATM protein with an IC50 value of 6.3 nM that is an analog of KU-55933. KU- 60019 was also utilized in the in vitro studies of the present disclosure. KU-60019 was shown to be useful as a monotherapy, have good pharmacokinetics, and was well-tolerated in mouse studies. KU-60019 radiosensitizes h-TERT-immortalized normal fibroblasts but not A-T fibroblasts, suggesting it is a specific ATM kinase radiosensitizer (Golding et al., Mol Cancer Ther.2009 Oct;8(10):2894-902). KU- 60019 is also currently in clinical trials for kidney cancers (NCT03571438). Other ATM inhibitors that are well suited for the methods disclosed herein include: (1) CP-466722, a reversible ATM inhibitor with transient ATM inhibition (IC50value of 410 nM); (2) AZ31 and AZ32 which have low blood brain barrier Attorney Docket No.00633-0394WO1 / MEEI 2024-199 (BBB) penetration (IC50 value of 46 and 6.2 nM respectively); (3) KU-59403, which has no anti-tumor effect alone, but does in combination with chemotherapy (IC50value of 3.0 nM); (4) AZD1390 is a potent and highly selective compound with excellent BBB penetration (IC50value of 0.78 nM); (5) 2-[({2-[(2,4- dichlorobenzyl)oxy]-1-naphthyl}methyl)amino]ethanol (A41) is a relatively new ATM inhibitor and is described in Dou et al., Eur J Med Chem.2022 Apr 5;233:114196 (IC50 value of 33.4 nM); and (6) AZD0156, a first in class orally available ATM inhibitor (Kurt et al., Cancer Res 15 July 2016; 76 (14_Supplement): 4859) with an IC50 value of 0.58 nM. In some embodiments, an ATM inhibitor suitable for use herein is selected from the group consisting of KU-55933, KU-60019, AZD0156, CP-466722, AZ31, AZ32, AZD1390, A22 and A41. In some embodiments, an ATM inhibitor suitable for use herein is KU-55933 or KU-60019. In some embodiments, an ATM inhibitor suitable for use herein is KU-60019. CHK2 Inhibitors Checkpoint kinase 2 (CHK2) is a multifunctional enzyme whose functions are central to the induction of cell cycle arrest and apoptosis by DNA damage induced by, for example, ionizing radiation. Upon activation, CHK2 relays the checkpoint activation signal to a number of effectors, which mediate many of the phenotypic characteristics provoked by DNA damage including cell cycle arrest and apoptosis. (See, e.g., Ahn et al., DNA Repair (Amst).2004 Aug-Sep;3(8-9):1039-47; Matthews et al., Expert Opin Drug Discov.2013 Jun;8(6):621-40). As used herein, the term “CHK2 inhibitor(s)” refers to any compound, drug, agent and the like that is capable of decreasing and / or inhibiting CHK2 kinase activity. Non-limiting examples of CHK2 inhibitors which can be used according to the present disclosure include PV-1019 (NSC 744039), CCT241533, VRX0466617, AZD7762, BML-277, prexasertib (LY2606368), LY2880070, hymenialdisine, idoloazepine-6, 2-arylbenzimidazole, NSC1095555, VRX0466617, aminopyridine-7, PV1162, and PHI-101 (Lasmotinib). CCT241533 is a very potent CHK2 inhibitor with an IC50 of 3 nM and a Ki of 1.16 nM. It is useful as monotherapy with good pharmacokinetics, oral bioavailability of 38-54%, and is well-tolerated in mouse studies (Taylor et al., Sci Adv.2022 Sep 16;8(37):eabq2611). Prexasertib (LY2606368) is a selective, ATP-competitive Attorney Docket No.00633-0394WO1 / MEEI 2024-199 second-generation CHK1 inhibitor with a Ki of 0.9 nM and an IC50 of <1 nM. It also inhibits CHK2 (IC50=8 nM) and RSK1 (IC50=9 nM). It was developed by Eli Lilly as a CHK1 / 2 inhibitor and is currently in clinical trial Phase II for tumors in patients with certain breast, ovarian or prostate cancers. (Taylor et al., Sci Adv.2022 Sep 16;8(37):eabq2611). PV1162 is a selective CHK2 inhibitor with an IC50 of 0.29 nM. PV1162 inhibits ATP binding to CHK2 by targeting the gatekeeper-dependent hydrophobic pocket, which is specific to CHK2 and located behind the ATP-binding site (adenine-binding region), thereby inhibiting the phosphorylation activity of CHK2 (Lountos et al., FEBS Lett.2011 Oct 20;585(20):3245-9). Other available CHK2 inhibitors suitable for use herein include: (1) hymenialdisine which is a natural compound initially identified as MEK1 inhibitor, (IC50value of 42 nM); (2) a derivative of hymenialdisine – idoloazepine-6 (IC50value of 8 nM); (3) ABI (2-arylbenzimidazole) which is a highly selective inhibitor for CHK1 with ~42% CHK2 inhibition reported (IC50value of 15 nM); (4) NSC1095555, which acts as an ATP-competitive inhibitor (IC50 value of 240 nM); (5) an analog of NSC1095555 – PV-1019 (IC50 value of 24 nM; also refered to as NSC 744039); (6) VRX0466617 (IC50 value of 140 nM) (Carlessi et al., Mol Cancer Ther.2007 Mar;6(3):935-44); (7) aminopyridine-7 (IC50 value of 28 nM); (8) PHI-101 (Lasmotinib) as described in Park et al., BMC Cancer.2022 Jan 3;22(1):28; and (9) LY2880070, an orally active CHK1 / 2 inhibitor, IC50＜1 nM that can be used as an anticancer agent for combination with DNA damaging agents. In some embodiments, a CHK2 inhibitor suitable for use herein is selected from the group consisting of CT241533, LY2606368, LY2880070, hymenialdisine, idoloazepine-6, 2-arylbenzimidazole, NSC1095555, PV-1019, VRX0466617, aminopyridine-7, and PHI-101. In some embodiments, a CHK2 inhibitor suitable for use herein is CT241533. In some embodiments, a CHK2 inhibitor suitable for use herein is LY2606368. Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of pharmaceutical compositions comprising or consisting of at least one ATM inhibitor, at least one CHK2 inhibitor, or both as active ingredients. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes Attorney Docket No.00633-0394WO1 / MEEI 2024-199 saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. A listing of active compounds and specific drugs suitable for use herein as supplementary active compounds can be found in The Merck Index Online; Royal Society of Chemistry, 2025; rsc.org / merck-index (accessed March 12, 2025), and the United States Pharmacopeia-47 / National Formulary-47, published by the United States Pharmacopeial Convention, Inc., Rockville Md., 2024. In some embodiments, a supplementary active compound can be one known in the art to treat and / or alleviate a symptom associated with a corneal endothelium disorder (e.g., FECD) and / or corneal endothelium damage / cell loss resulting from surgery or ultraviolet radiation-mediated damage. The present pharmaceutical compositions provided herein can be formulated for administration by a variety of routes and in a variety of dosage forms including those for oral, rectal, parenteral (such as subcutaneous, intramuscular, and intravenous), epidural, intracameral, intrathecal, intra-articular, topical (e.g., ocular) and buccal administration. In some embodiments, the pharmaceutical compositions provided herein are formulated for oral delivery. In some embodiments, the pharmaceutical compositions provided herein are formulated for intraperitoneal delivery. In some embodiments, the pharmaceutical compositions provided herein are formulated for ophthalmic delivery (i.e., ocular delivery). Pharmaceutical compositions provided herein can be formulated for administration to the eye (i.e., ocular delivery). Pharmaceutically acceptable carriers and excipients for use in a formulation for ocular delivery of active agents (e.g., ATM inhibitors, CHK2 inhibitors) are generally known in the art (see, e.g., Banou et al., Yale J Biol Med.2024 Dec 19;97(4):491-503) and are suitable for use herein. In some embodiments, pharmaceutical compositions provided herein can be formulated for topical application to the eye. In some embodiments, pharmaceutical compositions provided herein can be formulated for intravitreal, subretinal, and / or suprachoroidal injection. In some embodiments, pharmaceutical compositions disclosed herein can be formulated for administration to the eye of the subject by systemic administration, subconjunctival injection, intraperitoneal injection, intracameral injection, or topical administration to the eye. In some embodiments, the pharmaceutical compositions are Attorney Docket No.00633-0394WO1 / MEEI 2024-199 formulated for topical administration to the eye or region of the eye. In some embodiments, the pharmaceutical compositions are eye drops. Eye drop formulations contemplated herein can be formulated with or without one or more tear substitutes. A variety of tear substitutes are known in the art and include, but are not limited to: monomeric polyols, such as, glycerol, propylene glycol, and ethylene glycol; polymeric polyols such as polyethylene glycol; cellulose esters such hydroxypropylmethyl cellulose, carboxy methylcellulose sodium and hydroxy propylcellulose; dextrans such as dextran 70; water soluble proteins such as gelatin; vinyl polymers, such as polyvinyl alcohol, polyvinylpyrrolidone, and povidone; and carbomers, such as carbomer 934P, carbomer 941, carbomer 940 and carbomer 974P. In some embodiments, an eye drop formulation disclosed herein can comprise a cyclodextrin (for example alpha-, beta- or gamma-cyclodextrin, e.g., alkylated, hydroxyalkylated, carboxyalkylated or alkyloxycarbonyl- alkylated derivatives, or mono- or diglycosyl-alpha-, beta- or gamma-cyclodextrin, mono- or dimaltosyl-alpha-, beta- or gamma-cyclodextrin or panosyl-cyclodextrin). In some embodiments, an eye drop formulation disclosed herein can comprise hydroxypropyl- b-cyclodextrin (HPβCB). In some embodiments, an eye drop formulation disclosed herein can comprise at least one nanoparticle. In some embodiments, the nanoparticles contemplated herein can be based on different polymers. In some embodiments, the nanoparticles for use in the eye drops contemplated herein can be comprised of poly(lactic-co-glycolic acid) (PLGA). In some embodiments, an eye drop formulation disclosed herein can comprise at least one dendrimer. Dendrimers are defined as nano-sized, radially symmetric molecules having a well-defined, homogenous and monodisperse structure (Abbasi et al., Nanoscale Research Letters, 2014; 9(1):247-257). Several types of dendrimers can be synthesized including, but not limited to, Poly(propyleneimine) dendrimers (PPI), Poly(amidoamine) dendrimers (PAMAM), Poly 2,2-bis(methylol)propionic acid (PBisMPA), Poly(benzyl ether) dendrimers (PBzE), poly(lysine) dendrimers (PLL), and polymelamine, (triazine) dendrimers. (Busy et al., CHAPTER 16—THERAPEUTIC APPLICATIONS, ADVERSE EFFECTS OF ENGINEERED NANOMATERIALS, 2012, pages 296-301). In some embodiments, the at least one dendrimer can comprise third polyamidoamine dendrimer (PAMAM G3.0). In some embodiments, an eye drop formulation disclosed herein can comprise at least one nanomicelle. A “nanomicelle” can refer to Attorney Docket No.00633-0394WO1 / MEEI 2024-199 an aggregate (or cluster) of surfactant molecules, where surfactants are chemicals that are amphipathic, which means that they contain both hydrophobic and hydrophilic groups. Nanomicelles are colloidal particles with nanometer size ranges, forming spherical structures of amphiphilic molecules in water. In some embodiments, an eye drop formulation disclosed herein can comprise a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PVCL-PVA-PEG) nanomicelle formulation. In some embodiments, a composition disclosed herein can be an ocular irrigating solution. An ocular irrigating solution is a sterile cleansing solution used to rinse or flush the eye, typically for removing foreign objects, debris, or after chemical exposure. It can also be used to maintain the eye's natural condition during eye surgery. Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) 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 should be stable under the conditions of Attorney Docket No.00633-0394WO1 / MEEI 2024-199 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, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), 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 particle size 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, thimerosal, and the like. In many cases, it will be preferable 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 that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, 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 above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The pharmaceutical compositions of the present disclosure can be included in a container, pack, or dispenser together with instructions for administration. Methods of Treatment The compositions described herein can be used to treat subjects with a corneal endothelium disorder. In some embodiments, a corneal endothelium disorder can be Fuchs endothelial corneal dystrophy (FECD), posterior polymorphous dystrophy, congenital hereditary endothelial dystrophy (CHED), iridocorneal endothelial (ICE) syndrome, or pseudophakic bullous keratopathy (PBK). In some embodiments, compositions described herein can be used to treat subjects suspected of having or has been diagnosed as having Fuchs endothelial corneal Dystrophy (FECD). As used herein, the terms "patient" and "subject" and similar phrases can be used Attorney Docket No.00633-0394WO1 / MEEI 2024-199 interchangeably and are intended to refer to subjects who are at risk for and / or have been diagnosed with one or more a corneal endothelium disorders (e.g, FECD). Preferable, the subject is human, but the methods can be used in other mammals, e.g., non-human veterinary subjects such as non-human primates, cats, dogs, horses, cows, goats, and rabbits. Methods of diagnosing a corneal endothelium disorder (such as FECD), are known in the art and suitable for used herein (see, e.g, Moshirfar et al., Corneal Dystrophy.2023 Aug 7. In: StatPearls [Internet - accessed on May 13, 2025]. Treasure Island (FL) StatPearls Publishing; 2025 Jan. PMID: 32491788; and Gurnani et al., Fuchs Endothelial Dystrophy.2025 Apr 6. In: StatPearls [Internet - accessed on May 13, 2025]. Treasure Island (FL): StatPearls Publishing; 2025 Jan. PMID: 31424832). In some embodiments, compositions described herein can be used to treat subjects who have or are at risk of having corneal endothelial cell loss after full- thickness or partial-thickness corneal transplantation, herpes zoster ophthalmicus, uveitis, and / or graft rejection. Corneal transplant procedures involve the transplant of only the corneal endothelium, as in Descemet stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK), rather than replacing the full thickness cornea as in penetrating keratoplasty (PK). DSAEK and DMEK are indicated whenever the corneal dysfunction is limited to the endothelium, while other corneal tissues are not primarily affected. Unfortunately, endothelial cell density (ECD) post-transplant drops by 25-37% within 6 months after DSAEK and / or DMEK, wherein this cell loss can occur during surgery. (see, e.g., Maghsoudlou et al., Cornea Transplantation.2024 Feb 24. In: StatPearls [Internet – accessed on May 13, 2025]. Treasure Island (FL): StatPearls Publishing; 2025 Jan. PMID: 30969512). In some embodiments, a corneal transplantation procedure can be posterior lamellar keratoplasty (PLK), deep lamellar endothelial keratoplasty (DLEK), Descemet stripping endothelial keratoplasty (DSEK), Descemet stripping automated endothelial keratoplasty (DSAEK), DM endothelial keratoplasty (DMEK), DM automated endothelial keratoplasty (DMAEK), and / or deep anterior lamellar keratoplasty (DALK) (see, e.g., Maghsoudlou et al.). In some embodiments, compositions described herein can be used to treat subjects who have or are at risk of having ultraviolet-mediated damage to their cornea. Ultraviolet radiation (UVR) carries higher energy than visible light and high Attorney Docket No.00633-0394WO1 / MEEI 2024-199 dose exposure to UVR causes direct cellular damage to the eye. Ultraviolet-mediated damage can result in various corneal pathologies, including those characterized by loss of corneal endothelium cell density (See, e.g., Ivanov et al., J Biophotonics.2018 Jul;11(7):e201700377). The methods generally include administering a therapeutically effective amount of an ATM inhibitor, a CHK2 inhibitor, or both. As used herein the terms “administer,” “administering,” and “administration” are intended to mean introducing at least one agent (e.g., an ATM inhibitor, a CHK2 inhibitor) into a subject. When administration is for the purpose of treatment, the agent (e.g., an ATM inhibitor, a CHK2 inhibitor) can be provided before, during, and / or after the onset of or progression of a symptom or sign of the corneal endothelium disorder and / or corneal endothelium cell damage / loss. The methods can include administration of one or more doses of an ATM inhibitor, a CHK2 inhibitor, or both to treat a corneal endothelium disorder in the subject. The methods disclosed herein can also include administration of one or more doses of an ATM inhibitor, a CHK2 inhibitor, or both to treat corneal endothelial cell loss and / or corneal edema in a subject following one or more ocular surgeries. Multiple factors such as corneal distortion, irrigation solution turbulence, mechanical trauma by instruments, nuclear fragments, intraocular lens contact, and free oxygen radicals have been implicated in causing corneal damage during ocular surgery (see, e.g., Singh et al., Indian J Ophthalmol.2022 Nov;70(11):3791-3796). In some embodiments, methods disclosed herein can include administration of one or more doses of an ATM inhibitor, a CHK2 inhibitor, or both to prevent and / or treat corneal endothelial cell loss and / or corneal edema in a subject following one or more ocular surgeries (e.g., full-thickness or partial-thickness corneal transplantation). In some embodiments, one or more doses of an ATM inhibitor, a CHK2 inhibitor, or both are administered before an ocular surgery to prevent and / or treat corneal endothelial cell loss and / or corneal edema in a subject following the ocular surgery. In some embodiments, one or more doses of an ATM inhibitor, a CHK2 inhibitor, or both are administered after an ocular surgery to prevent and / or treat corneal endothelial cell loss and / or corneal edema in a subject following the ocular surgery. In some embodiments, an ATM inhibitor, a CHK2 inhibitor, or both are administered during an ocular surgery to prevent and / or treat corneal endothelial cell loss and / or corneal Attorney Docket No.00633-0394WO1 / MEEI 2024-199 edema in a subject following the ocular surgery. In some embodiments, an ocular irrigating solution comprising an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both is administered during an ocular surgery to prevent and / or treat corneal endothelial cell loss and / or corneal edema in a subject following the ocular surgery. Also contemplated herein are methods of preventing corneal graft failure and / or delaying onset of corneal graft failure following administration of one or more doses of an ATM inhibitor, a CHK2 inhibitor, or both. At present, corneal transplantation is the only method that can cure corneal endothelial dysfunction, including Fuchs’ endothelial corneal dystrophy (FECD), posterior polymorphous corneal dystrophy (PPCD), aphakic or pseudophakic bullous keratopathy (ABK / PBK), endothelial dysfunction caused by penetrating or blunt trauma, congenital hereditary endothelial dystrophy (CHED), iridocorneal endothelial (ICE) syndrome, refractory glaucoma, previous failed corneal grafts, herpes simplex virus endotheliitis, and failed penetrating keratoplasty (PK) (see, e.g., Feizi, Ther Adv Ophthalmol.2018 Dec 7;10:2515841418815802). Graft failure through corneal endothelial cell loss is a constant threat throughout the lifetime of a corneal graft (see, e.g., Claerhout et al., Int Ophthalmol.2008 Jun;28(3):165-73). In some embodiments, methods of preventing corneal graft failure and / or delaying onset of corneal graft failure disclosed herein can include administration of one or more doses of an ATM inhibitor, a CHK2 inhibitor, or both before a corneal transplant. In some embodiments, methods of preventing corneal graft failure and / or delaying onset of corneal graft failure disclosed herein can include administration of one or more doses of an ATM inhibitor, a CHK2 inhibitor, or both after a corneal transplant. In some embodiments, methods of preventing corneal graft failure and / or delaying onset of corneal graft failure disclosed herein can include administration of an ATM inhibitor, a CHK2 inhibitor, or both during a corneal transplant. In some embodiments, methods of preventing corneal graft failure and / or delaying onset of corneal graft failure disclosed herein can include administration of an ocular irrigating solution comprising an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both during a corneal transplant. Administration of a therapeutically effective amount of a treatment described herein can result in a reduction in corneal endothelial cell loss compared to the Attorney Docket No.00633-0394WO1 / MEEI 2024-199 amount of corneal endothelial cell loss in a subject that was not treated with a composition of the present disclosure. In some embodiments, administration of a therapeutically effective amount of a treatment described herein can attenuate corneal endothelial cell loss from about 25% to about 50% in a subject after treatment. Administration of a therapeutically effective amount of a treatment described herein can result in a reduction in the amount of senescent corneal endothelial cells compared to the amount of senescent corneal endothelial cells in a subject that was not treated with a composition of the present disclosure. In some embodiments, administration of a therapeutically effective amount of a treatment described herein can attenuate the number of senescent corneal endothelial cells from about 20% to about 40% in a subject after treatment. Administration of a therapeutically effective amount of a treatment described herein can result in less corneal edema compared to the amount of corneal edema in a subject that was not treated with a composition of the present disclosure. In some embodiments, administration of a therapeutically effective amount of a treatment described herein can prevent the onset of corneal edema in a subject after treatment. Compositions and Methods of Use in Preserving Corneal Tissue Ex Vivo Optimal ex vivo corneal storage in eye banks is crucial to increase both the number of corneas suitable for graft and their intrinsic quality, mainly the number of viable endothelial cells, which dictates graft survival in recipients. As demonstrated herein, adding an ATM inhibitor or a CHK2 inhibitor ex vivo to the recovery media (also referred to herein as corneal storage medium) markedly reduced corneal endothelial cell death in human donor corneal tissues. Provided herein are corneal storage medium compositions. Corneal storage medium compositions can comprise an ATM inhibitor, a CHK2 inhibitor, or both. In some embodiments, corneal storage medium compositions disclosed herein can further comprise a base preservation medium. Corneal preservation mediums (e.g., base preservation mediums) are known in the art (see., e.g., Sharma et al., Indian J Ophthalmol.2021 Sep;69(9):2452-2456) and are suitable for supplementation with an ATM inhibitor, a CHK2 inhibitor, or both as disclosed herein. Non limiting examples of base preservation mediums suitable for use herein can include Optisol, Optisol-GS, Cornisol, Cornea Cold, Eusol-C, and Life 4°C. Corneal storage medium compositions disclosed herein can comprise a base medium and one or more Attorney Docket No.00633-0394WO1 / MEEI 2024-199 additional optional agents as needed. Optional agents that may be included in the corneal storage medium compositions contemplated herein can include, but are not limited to, serum (either natural or synthetic), chondroitin sulfate, dextran, insulin, a buffer such as HEPES buffer, non-essential amino acids, or sodium bicarbonate. Methods of preserving or restoring corneal tissue ex vivo are also provided herein. Methods can comprise at least (a) harvesting a cornea, corneal tissue or corneal endothelium from a donor; and (b) placing the cornea, corneal tissue or corneal endothelium in a medium (e.g., a corneal storage medium composition) comprising an ATM inhibitor, a CHK2 inhibitor, or both. Methods of harvesting cornea, corneal tissues are generally known in the art and are suitable for use herein (see, e.g., Maghsoudlou et al., Cornea Transplantation.2024 Feb 24. In: StatPearls [Internet – accessed on May 13, 2025]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 30969512). The cornea, corneal tissue or corneal endothelium harvested from a donor can be stored prior to transplantation into a subject. In some embodiments, the donor tissue can be stored for up to 21 days, up to 14 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day prior to transplant. In some embodiments, the donor tissue can be stored for up to 21 days, up to 14 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day at 2-40°C prior to transplant. In some embodiments, the donor tissue can be stored for up to 21 days, up to 14 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day at 20-40°C prior to transplant. The compositions and methods described herein can increase the short or intermediate term (corneal storage) and / or long term (e.g., post-transplant) health, function and / or viability of corneas, and corneal tissue including the corneal endothelium. For example, the compositions described herein increase the health, function and / or viability of corneas, and corneal tissue including the corneal endothelium which are stored, after procuring and / or culturing prior to transplant. In some embodiments, a cornea, corneal tissue or corneal endothelium harvested from a donor can have less corneal endothelial cell loss after storage in a medium (e.g., a corneal storage medium composition) comprising an ATM inhibitor, a CHK2 inhibitor, or both as compared to a cornea, corneal tissue or corneal endothelium harvested from a donor and stored in a medium that does not have an ATM inhibitor, Attorney Docket No.00633-0394WO1 / MEEI 2024-199 a CHK2 inhibitor, or both according to the methods disclosed herein. In some embodiments, a cornea, corneal tissue or corneal endothelium harvested from a donor can have about 20% to about 30% attenuated corneal endothelial cell loss after storage in a medium (e.g., a corneal storage medium composition) comprising an ATM inhibitor, a CHK2 inhibitor, or both according to the methods disclosed herein. In some embodiments, a kit disclosed herein can comprise at least one or more materials needed to collect a cornea, corneal tissue or corneal endothelium from a donor and / or a cell culture medium (e.g., a base preservation medium) and an ATM inhibitor, a CHK2 inhibitor, or both. EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Materials and Methods The following materials and methods were used in the Examples below. Cell lines and cell culture Corneal endothelial cell lines were derived from Descemet’s membrane and endothelium collected from either a normal cadaveric donor (67 year-old female with 15 / 16 CTG repeats - SVN1-67F) or FECD patients that underwent endothelial keratoplasty (73 year-old female with 16 / 74 CTG repeats - SVF1-73F and 54 year-old female with 11 / 73 CTG repeats - SVF5-54F) by immortalization using SV40 T Antigen Cell Immortalization Kit (#CILV01, Alstem Cell Advancements, Richmond, CA) as described in Benischke et al., Sci Rep.7, 6656 (2017) and Ong Tone et al., Ophthalmol Sci.1, 100006 (2021). NQO1+ / +and NQO1− / −cells were generated from a normal HCEnC-21T cell line (derived from a 21-year-old normal male cadaveric donor) using the CRISPR-Cas9 system as described in Katikireddy et al., Free Radic Biol Med.116, 19-30 (2018). Cells were cultured in Chen’s medium containing OptiMEM-I (#51985091, Life Technologies), 8% fetal bovine serum (#10082147, Life Technologies), 5 ng / mL epidermal growth factor (#01-101, Millipore), 66 µg / mL bovine pituitary extract (#500-102, Gemini Bioproducts), 200 mg / L mg / L calcium chloride (#C7902, Sigma-Aldrich), 0.08% chondroitin sulfate (#C9819, Sigma- Aldrich), 50 mg / mL gentamicin (#15750078, Life Technologies), and 1:100 diluted antibiotic / antimycotic solution (#15240062, Life Technologies). Sub-culturing of Attorney Docket No.00633-0394WO1 / MEEI 2024-199 CEnCs was performed using 0.05% Trypsin (#25300120, Life Technologies) for 5 min at 37°C. In vitro acute treatment with menadione For treatments with menadione (MN), MN powder (#M5750, Sigma) was freshly dissolved in sterile distilled water to make a 10 mM working stock. Cells were seeded in complete Chen’s medium at a density of 0.35×106cells on FNC (#0407, Athena ES)-coated 6-well plates overnight at 37°C with 5% CO2. To assay the levels of DDR proteins, cells were incubated with 25 µM MN diluted in serum-free DMEM (#10567014, Life Technologies) followed by harvesting after 30, 60, 90, or 120 min for western blotting. For ATM inhibitor treatment, 20 µM KU-55933 (#118500, Calbiochem) was used to pre-treat cells for 30 min in Chen’s medium, then co-treat with MN in DMEM, and then was added to OptiMEM-I during the 24 h recovery period. To determine the cell cycle fate after MN stress, cells were treated with 50 µM MN for 60 min in DMEM followed by a 24 h recovery period in OptiMEM-I and then harvested for flow cytometry analysis. In vitro acute treatment with UVA light and 4-OHE2For acute treatments with ultraviolet-A light plus 4-hydroxyestradiol (UVA+4-OHE2), 0.4×105cells were seeded in estrogen-free Chen’s medium (modifications-phenol red-free OptiMEM (#11058021, Life Technologies) and 8% charcoal-stripped fetal bovine serum (FBS) (#100-119, Gemini Bio-products) on FNC-coated 6-well plates overnight. For UVA irradiation, growth medium was replaced with sterile phosphate buffered saline (PBS) and cells were exposed to two 19.5-inch UVA tubes (XX-15L; Analytik Jena US LLC) emitting 365 nm light (irradiance: 14.77 mW / cm2) to deliver a fluence of either 10 J / cm2(11 minutes) or 25 J / cm2(27 minutes), followed by a 24 h recovery period in phenol red-free OptiMEM. For 4-OHE2 treatments, 4-OHE2 powder (#E2500-000, Steraloids) was dissolved in 100% ethanol to make a 50 mM master stock which was freshly diluted in sterile PBS and added to cells immediately after UVA irradiation at 10 µM concentration in phenol red-free OptiMEM. For ATM inhibitor treatment, cells were pre-treated with 20 µM KU-55933 for 30 min in estrogen-free Chen’s medium and then re-added to cells in phenol red-free OptiMEM immediately after UVA irradiation. To assay the levels of DDR proteins, cells were harvested at 1 h post UVA or UVA+4-OHE2 treatments for western blotting. To determine the cell cycle fate, Attorney Docket No.00633-0394WO1 / MEEI 2024-199 cells were harvested 24 h post UVA+4-OHE2 with or without KU-55933 treatment for cell cycle analysis and cell sorting by flow cytometry (FIG.3A). In vitro chronic treatment with UVA light and 4-OHE2 For chronic UVA+4-OHE2 treatments, 0.1×105cells were seeded in estrogen- free Chen’s medium on FNC-coated 6-well plates overnight. Cells were pre-treated with 5 µM KU-55933 for 30 min and then exposed to 25 J / cm2UVA followed by addition of 10 µM 4-OHE2with or without re-addition of 5 µM KU-55933 as described above. Media was replaced with fresh phenol red-free OptiMEM every 48 h and cells were harvested for cell cycle analysis and cell sorting by flow cytometry on day 5 (FIG.4A). UVA irradiation of mouse cornea and drug administration 8 week (wk) old female C57BL / 6 mice were irradiated on the right eye with UVA (500 J / cm2) while the left eye served as a control. For KU-60019 administration: 1-wk post-UVA, mice were administered (IP) thrice up to 10 weeks with the specific ATM inhibitor, KU60019 (5 mg / kg) or vehicle, DMSO (FIG.17A). For CCT241533 administration: mice were administered (IP) 1-hr pre-UVA and thrice up to 2 weeks with the specific CHK2 inhibitor, CCT241533 (5 mg / kg) or vehicle, captisol (FIG.17B). UVA irradiation of the mouse corneas was performed as described in Liu et al., Proc Natl Acad Sci U S A 2020. Briefly, a UVA LED source (M365LP1; Thorlabs, USA) with an emission peak of 365 nm light, 8 nm bandwidth (FWHM), and irradiance of 398 mW / cm2was focused down to a 4 mm diameter illumination spot onto the mouse cornea. The time of UVA exposure was adjusted to deliver the appropriate fluence (20 minutes 57 s for 500 J / cm2) as measured with a thermal power sensor head (S425C, Thorlabs, USA) and energy meter console (PM100D, Thorlabs). The right eye (OD) was irradiated, while the contralateral eye (OS) was covered with retention drapes (SpaceDrapes, Inc., USA) and served as untreated control eye. Immunoprecipitation and western blotting Whole cell lysates were prepared by lysis in RIPA buffer containing HALT protease and phosphatase inhibitors (#78440, Life Technologies) for 30 min on ice. Protein concentration was determined using the BCA assay kit (#23225, Life Technologies). For immunoprecipitation, cells were lysed in immunoprecipitation Attorney Docket No.00633-0394WO1 / MEEI 2024-199 lysis buffer for 15 min and centrifuged at 14000 rpm for 10 min at 4°C. Total protein concentration was determined as described above. Protein samples were first precleared with protein A or G beads for 30 min and then incubated with 2 µg anti- pATM antibody (#05-740, Millipore) for 2 h at 4°C followed by the addition of 35 μl of protein A- or G-sepharose slurry and rotating for 1 h. Protein A / G beads were collected and washed with lysis buffer four times. Immunoprecipitates or whole cell lysates were resolved by loading onto a 4-12% Bis-Tris gel for SDS-PAGE and blotted onto a polyvinylidene difluoride membrane (#IPVH00010, Millipore) which was blocked in 5% nonfat milk (#1706404, Bio-Rad) or 5% bovine serum albumin (#BP1600, Fisher Scientific) in tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h and incubated overnight with primary antibody at 4°C. Refer to Table 1 for a list of primary antibodies used in these experiments. Table 1: Primary Antibodies used for Western Blotting Antibody Dilution Company Catalog# S1981-ATM 12000 Milli r 05-740 3 Blots gG (#sc- 2357) from Santa Cruz Biotechnology Inc., USA for 1 h. The membrane was developed with SuperSignal West Pico or Femto (#34577 or # 34096, Life Technologies, USA) plus chemiluminescent substrate. Densitometry was analyzed with ImageJ software (Schneider, Rasband & Eliceiri (2012) Nature Methods, 9(7), 671–675), and protein content was normalized relative to β-actin protein content. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 Experiments were repeated a minimum of three times. Results were averaged and SEM values were calculated. Cellular viability and morphology Phase-contrast microscopy (Leica DM IL LED) was employed to visualize cell morphology. Cell Titer Glo reagent (#G9241, Promega, Madison, WI) was used to determine the cell viability, according to the manufacturer's protocol. The luminescence was determined by a luminometer (Turner Biosystems, Sunnyvale, CA). Cell cycle analysis and cell sorting Cell cycle analysis and cell sorting were performed as described in White et al., Free Radic Biol Med.164, 34-43 (2021). Briefly, CEnCs were fixed with 70% ethanol for 20 min, treated with 100 μg / ml RNase, and stained with 50 μg / ml propidium iodide. Cell cycle data was acquired using a BD LSR II flow cytometer. Single cells were identified by measuring forward and side scatter, and cell doublets were excluded. The combined gates were applied to a forward scatter versus propidium iodide signal (PE channel) to produce a histogram plot. Quantification of cells in each phase of the cell cycle was carried out using FlowJo cell cycle analysis (v10.6.2, FlowJo, LLC). For FACS (Fluorescence-Activated Cell Sorting), G0 / G1 and G2 / M phase cells were sorted and collected in PBS, using Cytomation MoFlo cell sorter and resuspended in Trizol (Invitrogen) for RT-PCR analysis. RT-PCR RNA was extracted from cultured CEnCs using Trizol (Invitrogen) and RNeasy Micro Kit (Qiagen, Valencia, CA) according to manufacturer’s protocol. RNA quality and quantity were measured using NanoDrop spectrophotometer (Life Technologies). iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) was used to reverse-transcribe RNA. RT-PCR was performed by TaqMan gene expression assays (Applied Biosystems, Foster City, CA) with 2X Kapa Probe Fast master mix (#KK4703, Roche) for detection of mRNA expression of all genes. Results were normalized to GAPDH internal control and relative expression expressed as 2^ΔΔ(− CT). The TaqMan assays used for RT-PCR are listed in Table 2. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 Table 2: TaqMan assays used for DNA repair gene expression analysis in sorted CEnCs after treatment with UVA+4-OHE2 and ATM inhibitor T H MAN SA-β-GAL staining Cell lines were stained for SA-β-GAL using a senescence histochemical staining kit (#CS0030, Sigma-Aldrich) according to the manufacturer’s protocol, with an incubation period of 16 h at 37oC. Images of stained cells (10 images acquired at ×20 magnification for each well) were captured using bright field microscopy (EVOS XL Core). For quantification, positively stained cells were manually counted and reported as a percentage of total cells. Atm-wild type and -null mice Wildtype Atmtm1Mmpl+ / +(Atm-WT), heterozygous Atmtm1Mmpl / +(Atm-het), and knockout Atmtm1Mmpl / tm1Mmpl(Atm-null) were as described in Campbell et al., Hum Mol Genet.24, 6331-49 (2015). The strain was maintained by setting up monogamous pairs consisting of one male and one female mouse crossed as per a heterozygous X heterozygous breeding scheme and the litters were genotyped by PCR to identify knockouts and wildtypes for the experiments. Mice were housed at Schepens Eye Research Institute, Boston, USA, in a controlled environment with constant temperature, 12-hour light / dark cycle, and food and water available ad libitum. Mice were anesthetized with a combined dose of ketamine (100 mg / kg) and xylazine (20 mg / kg) administered intraperitoneally (IP). Animal studies were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research as well as the NIH Guide for the Care and Use of Animals and were performed at Schepens Eye Research Institute (SERI) with approval from SERI Institutional Animal Care and Use Committees IACUC. UVA Irradiation of Mouse Cornea and in vivo imaging UVA irradiation of the mouse corneas was performed as described in Liu et al., Proc Natl Acad Sci U S A.117, 573-583 (2020). Briefly, a UVA LED source Attorney Docket No.00633-0394WO1 / MEEI 2024-199 (M365LP1; Thorlabs, USA) with an emission peak of 365 nm light, 8 nm bandwidth (FWHM), and irradiance of 398 mW / cm2was focused down to a 4 mm diameter illumination spot onto the mouse cornea. The time of UVA exposure was adjusted to deliver the appropriate fluence (20 minutes 57 s for 500 J / cm2) as measured with a thermal power sensor head (S425C, Thorlabs, USA) and energy meter console (PM100D, Thorlabs). The right eye (OD) was irradiated, while the contralateral eye (OS) was covered with retention drapes (SpaceDrapes, Inc., USA) and served as untreated control eye). Mouse eyes were enucleated in sterile PBS, and corneal were isolated, snap-frozen, and sent for estrogen metabolite analysis or western blot following treatment with vehicle or drugs. For in vivo imaging, mice were anesthetized as described earlier, and visual assessment of their corneas was performed as described in our prior publication14pre- UVA and 1 day, 3 days, 1 week, 2 weeks, and 4 weeks post-UVA. Briefly, a slit lamp biomicroscope attached to a camera (SL-D4, Topcon Healthcare, The Netherlands) was used to assess the epithelial cell integrity. Anterior segment images were taken using anterior segment optical coherence tomography (OCT) (Bioptigen Spectral Domain Ophthalmic Imaging System Envisu R2200) with a 12 mm telecentric lens to scan the cornea and measure central corneal thickness. At week 2, 4 and 10 post-UVA for KU-60019 treated mice and at week 1 and 2 for CCT241533 treated mice, we assessed: (i) central corneal thickness (CCT) with AS-OCT, and (ii) endothelial cell density using HRT. Mice were anesthetized, and the anterior segment images were taken using anterior segment optical coherence tomography (OCT) (Bioptigen Spectral Domain Ophthalmic Imaging System Envisu R2200) with a 12 mm telecentric lens to scan the cornea. Central corneal thickness was measured using the inbuilt software in the OCT. For imaging the CE, mice were wrapped with heat retention drapes on the platform that held them securely and then imaged by laser scanning in vivo confocal microscopy using the Heidelberg Retina Tomograph III (HRT III) with Rostock Corneal Module (RCM) (Heidelberg Engineering). The laser confocal microscope acquired 2D images representing a coronal section of 400 × 400 μm (160,000 μm2) at selectable corneal depth. Acquired images comprised 384 X 384 pixels with a lateral resolution of 1 μm per pixel. Digital images were stored on a computer workstation at 3 frames per sec. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 Immunohistochemistry Mouse eyes were enucleated either at day 1, day 3, week 1, week 2, week 4, or week 10 post irradiation in sterile, ice-cold PBS. Under a stereo-zoom microscope (MZ6, Leica), the eyeball was penetrated with a 26-gauge syringe needle to create an incision at the corneo-scleral junction. Curved Vannas capsulotomy scissors, 33 / 8’’ (5677E, Ambler Surgical) were inserted into the incision to snip out the corneal cup from which the lens was removed, and remaining iris and loose tissue was scraped. The corneal cups were fixed in 4% paraformaldehyde (PF) for 20 min at room temperature (RT). For H3K9me3 and Ki67 staining, the corneal cup was permeabilized and blocked in 3% BSA + 0.1% TX-100 + 1% donkey serum in PBS for 1 h at RT with gentle shaking followed by an additional 10 min of blocking in 10% donkey serum. For Ki67, an additional permeabilization step with 1% TX-100 for 10 min was applied prior to blocking. The corneal cups were incubated with primary antibodies at 4°C overnight and with rhodamine conjugated donkey anti- rabbit secondary antibody next day for 1 h at RT. For pH2AX staining, the corneal cups were permeabilized in 0.1% Triton X100 + 3% BSA in PBS for 30 min at RT with gentle shaking, followed by incubation with primary antibody at 4°C overnight and then with Fluorescein Isothiocyanate (FITC) conjugated goat anti-mouse antibody next day for 1 h at RT. Refer to Table 3 for a list of primary antibodies used in these experiments. Table 3: Primary Antibodies used for Immunohistochemistry Antibody Dilution Company Catalog# H3K9me3 1:100 Thermo Fisher Scientific PA5-31910 The ti ntifade mounting medium (#H-1000-10, Vector Labs) and imaged on Leica SP8. At week 2, 4 and 10 post-UVA for KU-60019 treated mice and at week 1 and 2 for CCT241533 treated mice, we evaluated the development of multiple enlarged nuclei in cells, possibly due to failed cytokinesis, resulting in polyploidy (multinucleation) using ZO-1 immunostaining, and the development of senescence by H3K9me3 immunostaining. Mouse eyes were enucleated either at week 2, week 4, or week 10 post irradiation in sterile, ice-cold PBS. Under a stereo-zoom microscope (MZ6, Leica), the eyeball was penetrated with a 26-gauge syringe needle to create an Attorney Docket No.00633-0394WO1 / MEEI 2024-199 incision at the corneo-scleral junction. Curved Vannas capsulotomy scissors, 33 / 8’’ (5677E, Ambler Surgical) were inserted into the incision to snip out the corneal cup from which the lens was removed, and the remaining iris and loose tissue was scraped. The corneal cups were fixed in 4% paraformaldehyde (PF) for 20 min at room temperature (RT). For H3K9me3 and ZO1 staining, the corneal cup was permeabilized and blocked in 3% BSA + 0.1% TX-100 + 1% donkey serum in PBS for 1 h at RT with gentle shaking followed by an additional 10 min of blocking in 10% donkey serum. The corneal cups were incubated with primary antibodies at 4°C overnight and with rhodamine conjugated donkey anti-rabbit secondary antibody next day for 1 h at RT. The tissues were incubated with DAPI dye, mounted onto glass slides with antifade mounting medium (#H-1000-10, Vector Labs), and imaged on Leica SP8. Statistical Analysis Statistical analysis was carried out using GraphPad Prism V10 (GraphPad Software Inc, CA) using Student’s t-test for comparing two groups and one- or two- way analysis of variance (ANOVA) with Tukey’s post hoc test for comparing 3 or more groups. All data is reported as the mean ± SEM. Significance for all measures was set at P < 0.05. Example 1: Menadione-induced oxidative stress caused earlier and greater activation of ATM-mediated DDR, leading to elevated S-phase in FECD cells. Previously, we established that chemical stressor, menadione (MN) induced intracellular ROS in CEnCs and caused the pathophysiological changes seen in FECD9. To determine the time-kinetics of ROS-induced DDR activation in CEnCs, we treated SVN1-67F (normal) and SVF1-73F (FECD) cells, with 25 μM MN and assessed the levels of DDR proteins by western blotting at 30, 60, 90 and 120 minutes (min) of treatment. MN induced a time-dependent activation of ATM via autophosphorylation at S1981 (pATM), DNA damage marker H2AX via phosphorylation at S139 (pH2AX), and cell-cycle checkpoint regulators p53 and Chk2 via phosphorylation at S15 (pp53) and T68 (pChk2) respectively (FIG.1A). In SVF1-73F cells, levels of pATM / ATM, pH2AX / H2AX, pp53 / p53, and pChk2 / Chk2 exhibited peak activation as early as at 60 min and declined at 90 and 120 min, while in SVN1-67F cells, pATM / ATM levels peaked only later at 120 min without significant activation of pH2AX / H2AX and pp53 / p53 yet, thereby establishing 60 Attorney Docket No.00633-0394WO1 / MEEI 2024-199 min as the timepoint of greatest differential ATM-mediated DDR activation between the two cell lines (FIG.1A). At 60 min, MN caused cell elongation and formation of rosette-like structures and long processes, characteristic of FECD morphology9,45, distinctly in SVF1-73F cells (FIG.6A) and induced a marked increase in the levels of pATM / ATM (4-fold), pH2AX / H2AX (4-fold), and pp53 / p53 (3.6-fold), predominantly in SVF1-73F cells, whereas MN upregulated pChk2 / Chk2 levels in both SVF1-73F (1.6-fold) and SVN1-67F (2.8-fold) cells at this timepoint (FIG.1B). We found lack of activation of another key DDR kinase, ATR, after MN treatment as indicated by the unchanged pATR / ATR levels across all timepoints in both SVN1- 67F and SVF1-73F cells (FIG.6B). Furthermore, an immunoprecipitation assay using pATM antibody, but not IgG antibody (negative control) at 60 min of MN treatment resulted in an increased pull down of pATM along with an enhanced co- immunoprecipitation of pp53-S15, a direct substrate of pATM, in SVF1-73F compared to SVN1-67F cells (FIG.1C). Additionally, immunoprecipitation with total ATM antibody demonstrated an increased pull down of total p53 in SVF1-73F compared to SVN1-67F cells (FIG.7). We further employed a specific and potent ATM kinase inhibitor, KU-5593346, to examine whether MN-induced activation of p53 was dependent on the ATM protein kinase. KU-55933, at 20 μM concentration, determined from a dose response curve (FIG.8A), completely ablated the MN- induced phosphorylation of ATM (S1981) and p53 (S15) (FIG.1D) as well as H2AX (S139) and Chk2 (T68) (FIG.8B) in both SVN1-67F and SVF1-73F cells, indicating that ATM played a major role in transducing the MN-induced DNA damage signaling in CEnCs. We confirmed our finding of increased pATM / ATM activation in FECD compared to normal cells using an additional normal cell line, SVN2-67F, as well as an additional FECD cell line, SVF5-54F treated with MN (FIG.8C). ATM was known to be involved in mediating the halting of cell cycle progression in response to oxidative stress-mediated DNA damage47, however, little was known about its role in cell cycle regulation in the context of FECD pathogenesis. We therefore assayed the cell cycle profiles post-MN treatment of normal SVN1-67F cells, and FECD SVF1- 73F and SVF5-54F cells. Treatment with 50 μM MN for 1 hour followed by a 24 hour (h) recovery period caused a marked increase in the S-phase population of FECD cells SVF1-73F (3-fold) and SVF5-54F (6-fold), compared to normal cells SVN1-67F (2- fold) (FIGS.1E-1F, FIG.9). There was no difference in the cell doubling times of Attorney Docket No.00633-0394WO1 / MEEI 2024-199 the normal cell line, SVN1-67F, and the two FECD cell lines, SVF1-73F and SVF5- 54F, confirming that the differential S-phase accumulation was not an effect of different proliferations rates between the cell lines (FIG.10). Taken together, these results demonstrated the involvement of the ATM-p53-H2AX axis which was activated earlier and to a greater extent in FECD leading to an elevated S-phase in response to MN-induced oxidative stress. Example 2: UVA light-induced oxidative stress caused ATM-mediated G2 / M- phase arrest in FECD cells. We sought to determine whether the environmental stressor UVA light also caused an early ATM activation in FECD cells compared to normal CEnCs. The effect of female sex was assessed by treatment of cells with catechol estrogen found to be increased in FECD14. Both SVN1-67F (normal) and SVF1-73F (FECD) cells were exposed to UVA (10 J / cm2or 25 J / cm2) with and without 4-OHE2 (10 μM). Western blot analysis with 10 J / cm2, but not 25 J / cm2, showed that UVA+4-OHE2caused greater activation of pATM / ATM (2-fold, p<0.01) and pChk2 / Chk2 (2.5-fold, p<0.001) in SVF1-73F cells, but not in SVN1-67F cells compared to no treatment (FIGS.2A-2B, FIG.11). To investigate the cell cycle status of treated cells compared to untreated, cells in each phase of the cell cycle were quantified using flow cytometry based on fluorescence intensity of propidium iodide labelled DNA content (FIG.2C). Exposure to UVA+4-OHE2 compared to no treatment induced a marked increase in G2 / M phase arrest in SVN1-67F (2-fold; 44% vs 22%) and in SVF1-73F (2.7-fold; 51% vs 19%) at 24 h (FIGS.2C-2D). To investigate the role of ATM activation in G2 / M arrest, we co-treated SVN1-67F and SVF1-73F cells with 20 μM of KU-55933 and UVA+4-OHE2 and found significant reduction in G2 / M arrest compared to treatment with UVA+4-OHE2in both SVN1-67F (0.7-fold; 30% vs 44%) and SVF1-73F (0.6-fold; 31% vs 51%) cells (FIG.2D). Cell cycle is tightly controlled by cyclins and cyclin dependent kinase (CDK) enzymes and the progression from G2- to M-phase is driven by activation of the Cdk1 / Cyclin B1 complex48. We observed an increase in Cyclin B1 levels in SVF1- 73F (5-fold, p<0.0001) which was 1.5-fold greater than the increase in SVN1-67F (2- fold, p<0.01) compared to no treatment, confirming a significantly higher G2 / M phase arrest in FECD compared to normal cells at 24 h post UVA+4-OHE2 treatment (FIG.2E). Cell cycle arrest has been linked to progression of cellular senescence, a Attorney Docket No.00633-0394WO1 / MEEI 2024-199 process mediated through the p53-p21 pathway49. We next tested for evidence of senescence due to the G2 / M phase arrest seen in SVN1-67F and SVF1-73F cells post UVA+4-OHE2 treatment. While p53 / β-actin was elevated in both SVN1-67F normal and SVF1-73F FECD cells, the protein levels of p21 / β-actin were greater in SVF1- 73F cells (2.5-fold, p<0.01) compared to SVN1-67F cells (FIG.2F). SA-β-Gal staining at 24 h post UVA+4-OHE2treatment showed an increase in the percentage of senescent cells in both SVN1-67F and SVF1-7F cells indicating an onset of premature senescence (FIG.2G). Example 3: Loss of NQO1 resulted in ATM-mediated G2 / M phase arrest, leading to upregulation of DNA repair following acute stress with UVA light. NQO1 catalyzed the reduction of reactive quinones to form non-toxic catechol estrogens (4-OHE2). Our previous data demonstrated that loss of NQO1 rendered CEnCs highly susceptible to UVA light44, leading to increased reactive oxygen species (ROS) levels and estrogen genotoxicity, thus causing DNA damage and apoptosis14. As we detected the UVA-induced G2 / M phase cell cycle arrest in FECD CEnCs, we next sought to investigate whether lack of NOQ1, seen in FECD, also exhibited greater ATM activation and G2 / M phase arrest. To establish an acute stress cellular model, we utilized our previously generated NQO1-wildtype (NQO1+ / +) and NQO1-null (NQO1- / -) stable cell lines10and exposed them to UVA light (10 J / cm2or 25 J / cm2) and 4-OHE2 (10 µM), with and without an ATM inhibitor (KU-55933), and recovered for 60 min or 24 hours in low-serum media (FIG.3A). Western blot analysis showed greater and significant activation of pATM / ATM and its downstream target pChk2 / Chk2 at 60 min after both UVA doses (10 J / cm2and 25 J / cm2) with 4- OHE2, (10 µM) compared to control (FIG.3B). Next, we examined the effect of greater UVA-mediated ATM activation due to lack of NQO1 on cell cycle progression. Cell cycle analysis after treatment with UVA (25 J / cm2) and 4-OHE2 followed by recovery for 24 h showed an increase in G2 / M phase in NQO1+ / +cells (2.2-fold; 34% vs 15%) and NQO1- / -cells (3.1-fold; 43% vs 14%) compared to no treatment (FIGS.3C-3D). The cell cycle arrest was further established by increased levels of Cyclin B1 in NQO1+ / +and NQO1- / -cells after UVA+4-OHE2 exposure as seen by western blotting (FIG.3D). We determined that the G2 / M arrest was ATM- driven by co-treating cells with KU-55933 and UVA+4-OHE2 which demonstrated significant reduction in the G2 / M phase arrest in both NQO1+ / +cells (0.6-fold; 19% Attorney Docket No.00633-0394WO1 / MEEI 2024-199 vs 34%) and NQO1- / -cells (0.5-fold; 20% vs 43%) along with a downregulation of Cyclin B1 protein levels compared to no treatment in both cell lines (FIGS.3C-3D). Furthermore, to examine if G2 / M arrest under acute stress jumpstarted DNA repair, we sorted both untreated as well as UVA+4-OHE2treated and NQO1- / -cells from G0 / G1 and G2 / M phases using a cell sorter and extracted the total RNA. Transcription of specific DNA repair genes LIG3, NEIL2, TOP3A and XPC, that were downregulated in FECD ex vivo specimens50, was quantified for each phase of the cell cycle. None of the four DNA repair genes showed a significant difference in expression between G0 / G1 and G2 / M phases of cell cycle at baseline in NQO1+ / +or NQO1- / -cells (FIG.12). Calculating the ratio of mRNA expression in UVA+4- OHE2 / untreated cells showed an upregulation in the expression of all four DNA repair genes (LIG3, NEIL2, TOP3A and XPC) in G2 / M phase of both NQO1+ / +and NQO1- / -cells compared to untreated cells under acute stress upregulation was significantly greater in G2 / M arrested NQO1- / -cells—LIG3 (2.5- fold, p<0.01), NEIL2 (4-fold, p<0.0001), TOP3A (19-fold, p<0.0001), and XPC (5- fold, p<0.001)—as compared to NQO1+ / +cells (FIG.3E). To examine the effect of ATM inhibition on DNA repair in G2 / M phase cells, NQO1+ / +and NQO1- / -cells co- treated with KU-55933 and UVA+4-OHE2 were sorted into G0 / G1 and G2 / M phases. Gene expression analysis after KU-55933 co-treatment in NQO1+ / +and NQO1- / -cells showed lack of upregulation of DNA repair genes in G2 / M phase indicating that ATM-driven G2 / M arrest was essential in activating the DNA repair under cytotoxic stress (FIG.3F). There was no significant change noted in the gene expression of DNA repair genes in the G0 / G1 phase of NQO1+ / +and NQO1- / -cells after KU-55933 treatment (FIG.13). Example 4: Chronic stress with UVA light led to a G0 / G1 cell cycle arrest, downregulation of DNA repair genes, and cytotoxic senescence, which was reversed by ATM inhibition. Since FECD is a chronic, age-related disorder, that spans many decades for clinical presentation, next we aimed to determine the effect of prolonged (chronic) stress with UVA and 4-OHE2 on ATM-mediated cell cycle arrest, DNA repair, and senescence in NQO1+ / +and NQO1- / -cells. For the chronic stress cellular model, NQO1+ / +and NQO1- / -cells were exposed to UVA light (25 J / cm2) and 4-OHE2 (10 µM), with or without KU-55933 (5 µM) and recovered for 5-days in low-serum Attorney Docket No.00633-0394WO1 / MEEI 2024-199 medium (FIG.4A). Chronic stress caused an increase in the G0 / G1 phase in both NQO1+ / +(1.3-fold; 58% vs 43%) and NQO1- / -(1.4-fold; 64% vs 45%) cells, and a decrease in G2 / M compared to no treatment (FIGS.4B-4C, FIG.14). Addition of KU-55933 reduced G0 / G1 phase by 0.8-fold in NQO1+ / +and 0.6-fold in NQO1- / -cells (FIGS.4B-4C, FIG.11). G0 / G1 arrest was additionally shown by an increase in Cyclin D1 levels which was also reduced after addition of KU-55933 in both NQO1+ / +and NQO1- / -cells, as shown by western blotting (FIG.4C). To further determine the effect of G0 / G1 cell cycle arrest on DNA repair efficiency following chronic stress, we sorted the G0 / G1 and G2 / M phases of untreated, UVA+4-OHE2treated, and UVA+4-OHE2+KU-55933 treated NQO1+ / +- / and NQO1 - cells and evaluated the expression profiles of four DNA repair (LIG3, NEIL2, TOP3A, and XPC) using RT-PCR. Post chronic stress with UVA+4-OHE2 in NQO1+ / +, the mRNA expression of LIG3, NEIL2, and XPC was predominantly in the G0 / G1 phase, albeit significantly lower than that seen in G2 / M phase after acute stress (FIGS.4D-4G, FIG.3E). Notably, in NQO1- / -cells, the expression of LIG3, NEIL2, and XPC was significantly reduced in G0 / G1 and G2 / M phase cells collectively compared to NQO1+ / +cells, mimicking end-stage FECD in ex-vivo specimens50(FIGS.4D-4G). After addition of KU-55933 during the chronic stress, the expression of the DNA repair genes was redistributed between G0 / G1 and G2 / M in both NQO1+ / +and NQO1- / -cells, with greater expression noted in the G2 / M phase, compared to without KU-55933 treatment (FIGS.4D-4G). Remarkably, in NQO1- / -cells, after treatment with KU-55933, the expression of LIG3 and TOP3A was found to be significantly greater in both G0 / G1 (3- and 7.5-fold) and G2 / M (4- and 6-fold) phases, while that of NEIL2 and XPC was significantly greater in G2 / M (3.5- and 9.5-fold) phase only, compared to their expression in the respective phases without KU-55933 treatment. This indicated that the observed G0 / G1 arrest and diminished DNA repair in NQO1- / -cells was indeed ATM-driven and inhibition of ATM forestalled the depletion of DNA repair triggered due to chronic stress. Chronic stress with UVA+4-OHE2also resulted in a greater upregulation of senescence markers TP53 (p53 protein; 2-fold) and CDKN1A (p21 protein; 1.5-fold) in NQO1- / -cells compared to NQO1+ / +cells, indicating a greater induction of senescence in a diseased state (FIGS.4H-4I). After treatment with KU-55933, the expression of TP53 and CDKN1A was found to be 0.16- and 0.14-fold lower, Attorney Docket No.00633-0394WO1 / MEEI 2024-199 respectively, in G0 / G1 phase compared to without KU-55933 treatment in NQO1- / -cells (FIGS.4H-4I). This was confirmed by western blot showing a lower protein level of p53 post UVA+4-OHE2 and KU-59933 treatment in NQO1- / -cells (FIG.4J). We also noted a greater positive SA-β-Gal staining following chronic UVA+4-OHE2treatment in both NQO1+ / +and NQO1- / -cells, indicating the presence of senescent cells, which was rescued in both cell lines by the addition of KU-55933 (FIG.4K). Correlating our findings of how NQO1+ / +and NQO1- / -cells behave under acute vs chronic stress with UVA+4-OHE2and UVA+4-OHE2+KU-55933, we demonstrated that NQO1- / -cells (dashed line) exhibited (a) significantly greater (14%) G2 / M arrest under acute stress (left box), and (b) significantly greater (15%) G0 / G1 arrest under chronic stress (right box), compared to NQO1+ / +cells (solid line). ATM inhibition with KU-55933 under chronic stress restored DNA repair gene expression and reduced senescence, thus favoring cell survival (FIG.4L). Our study demonstrated that following acute stress, ATM activation resulted in mild and repairable DNA damage, where cells triggered a series of signaling cascades leading to cell cycle arrest in G2 / M, increased turnover of DNA repair enzymes and premature / cytoprotective senescence, promoting cell survival (FIG.4M). However, under chronic stress, sustained ATM activation initiated a DNA damage cascade which led to cell cycle arrest in G0 / G1, downregulation of DNA repair enzymes, and cytotoxic senescence, thus progressing to a diseased state as seen in FECD (FIG.4M). Example 5: UVA-induced cell cycle re-entry, DNA damage, and senescence were reduced in Atm-null mice. To understand the involvement of ATM in the oxidative stress response in vivo, we irradiated the corneas of Atm-wildtype (Atm-WT) and Atm-knockout (Atm- null) mice with 500 J / cm2UVA light, a dose that induced the FECD phenotype in mice14. We evaluated their corneal endothelia visually, as well as biochemically, at four recovery timepoints: 3 days, 1 week, 2 weeks, and 4 weeks post-UVA (FIG. 5A). The Atm-null mice genotype was determined by PCR analysis (FIG.5B). Additionally, western blot analyses demonstrated an activation of Atm in Atm-WT mice 1-day post-UVA treatment, which was absent in Atm-null mice (FIG.5C). The opacity and corneal edema were evaluated in both Atm-WT and Atm-null mice post- UVA using slit lamp bio-microscopy, followed by measurement of central corneal Attorney Docket No.00633-0394WO1 / MEEI 2024-199 thickness using Anterior Segment Optical Coherence Tomography (AS-OCT) (FIG. 15A). An epithelial defect and corneal swelling were observed at day-1 post UVA in both genotypes (FIGS.15A-15B). We have previously demonstrated that UVA- induced DNA damage in vivo activated the cell cycle, resulting in G2 / M arrest and induction of senescence45. To examine the role of DNA damage in cell cycle activation in vivo, we assayed the nuclear expression of Ki67, an indicator of cell cycle re-entry, in the corneas of Atm-WT and Atm-null mice at baseline (i.e., no UVA) and at day 2, day 3, and week 1 post-UVA by immunohistochemistry staining (FIG.5D). At baseline, corneal endothelial cells showed no Ki67 staining in Atm-WT and Atm-null mice. As an immediate response to UVA, both Atm-WT and Atm-null mouse corneal endothelial cells showed 25% Ki67 positivity at day 2 compared to no UVA. However, the Ki67 positivity decreased at day 3 to 11% in Atm-WT and 2.7% in Atm-null and, at week 1, to 4.5% in Atm-WT and 2.6% in Atm-null mice compared to day 2 post-UVA. Therefore, the reduction in Ki67 positivity from day 2 to day 3 was 34% greater in Atm-null compared to Atm-WT mice, indicating lower cell cycle activation due to the loss of ATM (FIG.5D). Furthermore, we sought to determine the extent of UVA-induced DNA damage by staining for phosphorylated H2AX, a marker of double-strand DNA breaks51. Time-dependent increase in DNA damage was detected in Atm-WT mice marked by an increase in percent pH2AX positive nuclei at day 3 (25%), week 1 (35%), week 2 (56%), and week 4 (59%) post-UVA. Atm-null mice presented with a lower pH2AX percent positivity at week 1 (20%), week 2 (20%), and week 4 (44%) compared to Atm-WT mice, which was suggestive of lower DNA damage response in the absence of ATM (FIG.5E, FIG.16). Next, we checked if UVA-induced cell cycle re-entry and ATM activation progressed to development of senescence detected by increased heterochromatinization, visualized by the presence of tri-methylation of Lys9 on histone H3 (H3K9me3). In Atm-WT mice, senescent cells, quantified by calculating percent H3K9me3 positive nuclei, persisted from day 3 (8%) through week 1 (13%) and week 2 (12%), and significantly decreased at week 4 (3%) post- UVA, which was suggestive of stress-induced premature senescence.52,53In Atm-null mice, senescent cells were significantly lower than those of Atm-WT mice at all the tested time-points (day 3 (4%), week 1 (1%), week 2 (3%), and week 4 (2%) post- UVA), indicating that absence of Atm mitigated the onset of senescence (FIG.5F, Attorney Docket No.00633-0394WO1 / MEEI 2024-199 FIG.16). Interestingly, at a later time-point of week 10 post UVA, the H3K9me3 positivity was significantly increased in Atm-WT (15%) but not in Atm-null mice (4%), implying that Atm played a role in the reappearance of prolonged senescence after chronic accumulation of oxidative stress. Example 6: Use of ATM kinase inhibitor (KU-60019) for rescuing corneal endothelial cell loss, corneal edema and attenuating multinucleation and senescence in the UVA-mouse model of FECD. 8 week (wk) old female C57BL / 6 mice were irradiated on the right eye with UVA (500J / cm2) while the left eye served as control. For KU-60019 administration, 1-wk post-UVA, mice were administered (IP) thrice up to 10 weeks with the specific ATM inhibitor, KU60019 (5 mg / kg) or vehicle, DMSO (FIG.17A). UVA irradiation of the mouse corneas was performed as described herein (see Materials and Methods). At week 2, 4 and 10 post-UVA for the KU-60019 treatted mice, we assessed for: (i) central corneal thickness (CCT) with AS-OCT, and (ii) endothelial cell density using HRT according to the methods described herein (see Materials and Methods). At week 2, 4 and 10 post-UVA for the KU-60019 treated mice, we evaluated the development of multiple enlarged nuclei in cells, possibly due to failed cytokinesis, resulting in polyploidy (multinucleation) using ZO-1 immunostaining, and the development of senescence by H3K9me3 immunostaining according to the methods described herein (see Materials and Methods). ATM inhibition with KU-60019 rescued corneal edema at week-2, 3, 4 post- UVA. KU60019 treatment stabilized CCT which was significantly lower at week-2 (106.5 ± 5.31 μM vs 142±7.11 μM, p<0.001); week-3 (101 ± 2.22 μM vs 148 ± 5.48 μM, p<0.001) and week-4 (103.2 ± 3.3 μM vs 146.5 ± 10.3 μM, p<0.01), compared to controls (FIGS.18A-18B). ATM inhibition with KU-60019 rescued CE cell loss at week-2, 4 and 10 post- UVA. KU60019-treated mice showed decreased CEnC loss by HRT, compared to DMSO-treated mice at week-2 (38.4% vs 58%), week-4 (38% vs 61%), and week-10 (44% vs 65%), demonstrating a 19.6%, 23% and 21% CEnC rescue at weeks 2-, 4- and 10 post-UVA, respectively (FIGS.19A-19B). The rescue in CEnC loss by using ATM inhibitor was further confirmed by ZO1 staining demonstrating a 13.3%, 12.5% and 14% CEnC per field at weeks 2-, 4- and 10 post-UVA, respectively (FIGS.19C- 19D). Attorney Docket No.00633-0394WO1 / MEEI 2024-199 ATM inhibition with KU-60019 attenuated multinucleation and senescence at week-2, 4 and 10 post-UVA. Junctional staining with ZO-1 revealed a progressive increase in multinucleation post-UVA, which was attenuated with KU-60019 at week- 2, -4 and -10 (16.8% vs 6.8%; 20.5% vs 9.9%; and 19.8% vs 5.9%; respectively; p<0.01) (FIGS.20A-20B). Additionally, use of ATM inhibitor substantially reduced the number of UVA-induced senescent cells from 29.5% to 12.6% at week-2 (p<0.001); from 25% to 8.6% at wk-4 (p<0.05), and from 23.2% to 5.5% at wk-10 (p<0.05), thus confirming our findings of reduced senescence in Atm-null mice, compared to WT (FIGS.20A, 20C). These data herein demonstrated that, using an UVA-mouse model of FECD, ATM inhibition with KU-60019 (i) rescued corneal edema at weeks-2, 3, 4 post- UVA, (ii) rescued corneal endothelial cell loss at weeks-2, 4 and 10 post-UVA, and (iii) attenuated multinucleation and senescence at weeks-2, 4 and 10 post-UVA. Example 7: Use of CHK2 inhibitor (CCT241533) for rescuing corneal endothelial cell loss, corneal edema and attenuating multinucleation and senescence in the UVA-mouse model of FECD. 8 week (wk) old female C57BL / 6 mice were irradiated on the right eye with UVA (500J / cm2) while the left eye served as control. For CCT241533 administration, mice were administered (IP) 1-hr pre-UVA and thrice up to 2 weeks with the specific CHK2 inhibitor, CCT241533 (5 mg / kg) or vehicle, captisol (FIG.17B). UVA irradiation of the mouse corneas was performed as described herein (see Materials and Methods). At week 1 and 2 for CCT241533 treated mice we assessed for: (i) central corneal thickness (CCT) with AS-OCT, and (ii) density using HRT according to the methods described herein (see Materials and Methods). At week 1 and 2 for CCT241533 treated mice, we evaluated the development of multiple enlarged nuclei in cells, possibly due to failed cytokinesis, resulting in polyploidy (multinucleation) using ZO-1 immunostaining, and the development of senescence by H3K9me3 immunostaining according to the methods described herein (see Materials and Methods). CHK2 inhibition with CCT241533 rescued corneal edema at week-1, and -2 post-UVA. CCT241533 treatment stabilized CCT which was significantly lower at week-1 (88.6 ± 0.44 μM vs 130.6 ± 15.1 μM, p<0.05); and week-2 (87.2 ± 2.01 μM vs 120.16 ± 11.74 μM, p<0.001), compared to controls (FIGS.21A-21B). Attorney Docket No.00633-0394WO1 / MEEI 2024-199 CHK2 inhibition with CCT241533 rescued CE cell loss at week-1, and 2 post- UVA. CCT241533-treated mice showed decreased CEnC loss by HRT, compared to Captisol-treated mice at week-1 (25.5% vs 47%), and week-2 (28% vs 57%), demonstrating a 21.5% and 28.5% CEnC rescue at weeks 1-, and 2-post-UVA, respectively (FIGS.22A-22B). The rescue in CEnC loss by using CHK2 inhibitor was further confirmed by ZO1 staining demonstrating a notable 27% rescue of CEnC per field at week 2 post-UVA (FIGS.22C-22D). CHK2 inhibition with CCT241533 attenuated multinucleation and senescence at week-2 post-UVA. Junctional staining with ZO-1 revealed a progressive increase in multinucleation post-UVA, which was attenuated with CCT241533 at week-2 (14.8% vs 1.5%; p<0.01) post-UVA (FIGS.23A-23B). Additionally, the use of CHK2 inhibitor substantially reduced the number of UVA-induced senescent cells from 32% to 1.1% at week-2 (p<0.001) (FIG.23C). These data herein demonstrated that, using an UVA-mouse model of FECD, CHK2 inhibition with CCT241533 (i) rescued corneal edema at weeks-1, and 2 post- UVA, (ii) rescued corneal endothelial cell loss at weeks-1, and 2 post-UVA and (iii) attenuated multinucleation and senescence at week-2 post-UVA. Example 8. ATM inhibition and CHK2 inhibition rescued corneal endothelial cell death in ex vivo normal donor human corneal tissues post-oxidative stress. Optimal ex vivo corneal storage in eye banks is crucial to increase both the number of corneas suitable for graft and their intrinsic quality, mainly the number of viable endothelial cells, which dictates graft survival in recipients. With both passive storage methods used worldwide significant endothelial cell loss is inevitable. (Garcin et al., Am J Transplant.2019 Jun;19(6):1641-1651). Normal human donor corneal tissues were incubated in cell culture media and stressed with pro-oxidant Menadione which caused 33% cell death compared to media alone (FIGS.24A-24B). ATM inhibitor (KU-60019) and CHK2 inhibitor (CCT241533) rescued cell death by 24% and 29% respectively, when added to the recovery media in addition to menadione (FIGS.24A-24B). 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M., Losick, V.P., Jurkunas, U.V. Polyploidy leads to senescence in an UVA-based mouse model of Fuchs Dystrophy. ARVO Annual Meeting Abstract (2024). Attorney Docket No.00633-0394WO1 / MEEI 2024-199 83. Kruman, II & Schwartz, E.I. DNA damage response and neuroprotection. Front Biosci 13, 2504-15 (2008). 84. Lu, X.H. et al. Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington's disease. Sci Transl Med 6, 268ra178 (2014). 85. Taylor, M.J., Thompson, A.M., Alhajlah, S., Tuxworth, R.I. & Ahmed, Z. Inhibition of Chk2 promotes neuroprotection, axon regeneration, and functional recovery after CNS injury. Sci Adv 8, eabq2611 (2022). 86. Ruzickova, S. & Stanek, D. Mutations in spliceosomal proteins and retina degeneration. RNA Biol 14, 544-552 (2017). 87. Bhatia, V., Valdes-Sanchez, L., Rodriguez-Martinez, D. & Bhattacharya, S.S. 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OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 WHAT IS CLAIMED IS:

1. A method of treating or preventing corneal endothelial cell loss in a subject in need thereof, the method comprising administering to the subject an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both.

2. A method of treating or preventing corneal edema in a subject in need thereof, the method comprising administering to the subject an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both.

3. A method for treating a corneal endothelium disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both.

4. The method of any one of claims 1-3, wherein the subject in need thereof is suspected of having or has been diagnosed as having Fuchs endothelial corneal dystrophy (FECD), posterior polymorphous dystrophy, congenital hereditary endothelial dystrophy (CHED), iridocorneal endothelial (ICE) syndrome, or pseudophakic bullous keratopathy (PBK), preferably wherein the subject in need thereof is suspected of having or has been diagnosed as having FECD.

5. The method of claim 1 or claim 2, wherein the subject in need thereof has or is at risk of having ultraviolet-mediated damage to a cornea.

6. The method of claim 1 or claim 2, wherein the subject in need thereof has or is at risk of having corneal endothelial cell loss after full-thickness or partial- thickness corneal transplantation, herpes zoster ophthalmicus, uveitis, and / or graft rejection.

7. A method for preventing corneal graft failure in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 8. The method of claim 7, wherein the effective amount of an ATM inhibitor, a CHK2 inhibitor, or both is administered before corneal transplant, during corneal transplant, after corneal transplant, or at any combination thereof.

9. The method of any one of claims 1-8, wherein the ATM inhibitor is selected from the group consisting of KU-55933, KU-60019, AZD0156, CP-466722, AZ31, AZ32, AZD1390, and A41, preferably wherein the ATM inhibitor is KU- 60019.

10. The method of any one of claims 1-8, wherein the CHK2 inhibitor is selected from the group consisting of CT241533, LY2606368, LY2880070, hymenialdisine, idoloazepine-6, 2-arylbenzimidazole, NSC1095555, PV-1019, VRX0466617, aminopyridine-7, and PHI-101, preferably wherein the CHK2 inhibitor is CCT241533.

11. The method of any one of claims 1-10, wherein the effective amount of an ATM inhibitor, a CHK2 inhibitor, or both is administered to the eye of the subject.

12. The method of claim 11, wherein the effective amount of an ATM inhibitor, a CHK2 inhibitor, or both is administered to the eye of the subject by systemic administration, subconjunctival injection, intraperitoneal injection, intracameral injection, or topical administration to the eye.

13. A pharmaceutical composition comprising an ATM inhibitor, a CHK2 inhibitor, or both and at least one pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, wherein: (i) the ATM inhibitor is selected from the group consisting of KU-55933, KU-60019, AZD0156, CP-466722, AZ31, AZ32, AZD1390, and A41, preferably wherein the ATM inhibitor is KU-60019; and / or (ii) the CHK2 inhibitor is selected from the group consisting of CT241533, LY2606368, LY2880070, hymenialdisine, idoloazepine-6, 2- arylbenzimidazole, NSC1095555, PV-1019, VRX0466617, Attorney Docket No.00633-0394WO1 / MEEI 2024-199 aminopyridine-7, and PHI-101, preferably wherein the CHK2 inhibitor is CCT241533.

15. The pharmaceutical composition of claim 13 or claim 14, wherein the pharmaceutical composition is suitable for administration to the eye of a subject.

16. The pharmaceutical composition of any one of claims 13-15, wherein the pharmaceutical composition is an eye drop formulation.

17. A method of preserving or restoring corneal tissue, the method comprising: (a) harvesting a cornea, corneal tissue or corneal endothelium from a donor; and (b) placing the cornea, corneal tissue or corneal endothelium in a medium comprising an ATM inhibitor, a CHK2 inhibitor, or both.

18. The method of claim 17, further comprising: (c) storing the cornea, corneal tissue or corneal endothelium for up to 21 days prior to transplant, optionally wherein the cornea, corneal tissue or corneal endothelium is stored for up to 21 days at 2-40°C prior to transplant.

19. A method of transplanting a corneal tissue into a subject in need thereof, the method comprising: (a) harvesting a cornea, corneal tissue or corneal endothelium from a donor; (b) placing the cornea, corneal tissue or corneal endothelium in a medium comprising an ATM inhibitor, a CHK2 inhibitor, or both; and (c) transplanting the cornea, corneal tissue or corneal endothelium into a subject in need thereof.

20. The method of claim 19, wherein the cornea, corneal tissue or corneal endothelium is stored in the medium comprising an ATM inhibitor, a CHK2 inhibitor, or both for up to 21 days prior to transplanting into a subject in need thereof. Attorney Docket No.00633-0394WO1 / MEEI 2024-199 21. The method of claim 19 or claim 20, wherein the transplanted cornea, corneal tissue or corneal endothelium is further treated with an effective amount of an ATM inhibitor, a CHK2 inhibitor, or both following transplantation into the subject.

22. The method of any one of claims 17-21, wherein the ATM inhibitor is selected from the group consisting of KU-55933, KU-60019, AZD0156, CP-466722, AZ31, AZ32, AZD1390, and A41, preferably wherein the ATM inhibitor is KU- 60019.

23. The method of any one of claims 17-21, wherein the CHK2 inhibitor is selected from the group consisting of CT241533, LY2606368, LY2880070, hymenialdisine, idoloazepine-6, 2-arylbenzimidazole, NSC1095555, PV-1019, VRX0466617, aminopyridine-7, and PHI-101, preferably wherein the CHK2 inhibitor is CCT241533.

24. A corneal storage medium comprising an ATM inhibitor, a CHK2 inhibitor, or both.

25. The corneal storage medium of claim 24, wherein the ATM inhibitor is selected from the group consisting of KU-55933, KU-60019, AZD0156, CP-466722, AZ31, AZ32, AZD1390, and A41, preferably wherein the ATM inhibitor is KU- 60019.

26. The corneal storage medium of claim 24, wherein the CHK2 inhibitor is selected from the group consisting of CT241533, LY2606368, LY2880070, hymenialdisine, idoloazepine-6, 2-arylbenzimidazole, NSC1095555, PV-1019, VRX0466617, aminopyridine-7, and PHI-101, preferably wherein the CHK2 inhibitor is CCT241533.

27. The corneal storage medium of any one of claims 24-26, further comprising a base preservation medium.

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