Novel small molecule inhibitors of topbp1-BRCT7 / 8 that exert Anti-MYC activity and synergy with inhibitors of PARP1 / 2 and PARP14

Abstract

Embodiments of the present disclosure pertain to methods of treating or preventing a cancer in a subject by administering to the subject a therapeutically effective amount of a composition that includes a topoisomerase II|3-binding protein 1 (TopBP 1) inhibitor. The composition may also include a poly (ADP-ribose) polymerase (PARP) inhibitor. Additional embodiments of the present disclosure pertain to a composition that includes a TopBP 1 inhibitor. In some embodiments, the composition also includes a PARP inhibitor. In some embodiments, the composition is suitable for use in treating or preventing a cancer in a subject.

Description

TITLENOVEL SMALL MOLECULE INHIBITORS OF TOPBP1-BRCT7 / 8 THAT EXERT ANTI- MYC ACTIVITY AND SYNERGY WITH INHIBITORS OF PARP1 / 2 AND PARP14CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 540,781, filed on September 27, 2023. The entirety of the aforementioned application is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01CA203824, awarded by the National Institutes of Health; W81XWH1810329, awarded by the Department of Defense; and W81XWH19 10369, awarded by the Department of Defense. The government has certain rights in the invention.BACKGROUND

[0003] A need exists for more effective compounds and methods for treating or preventing cancer. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to methods of treating or preventing a cancer in a subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a composition. In some embodiments, the composition includes a topoisomerase II|3-binding protein 1 (TopBPl) inhibitor. In some embodiments, the composition further includes a poly (ADP-ribose) polymerase (PARP) inhibitor.

[0005] Additional embodiments of the present disclosure pertain to a composition that includes a TopBPl inhibitor. In some embodiments, the composition also includes a PARP inhibitor. In some embodiments, the composition is suitable for use in treating or preventing a cancer in a subject.

[0006] In some embodiments, the TopBPl inhibitor includes, without limitation:, derrivatives thereof, or combinations thereof.

[0007] In some embodiments, Ri includes, without limitation, polycyclic aromatic hydrocarbons, naphthalene, biphenyl, fluorene, anthracene, phenanthrene, phenalene, derivatives thereof, or combinations thereof. In some embodiments, R2 includes, without limitation, furan groups, phenyl groups, benzoyl groups, methoxybenzene, methoxybenzene amine, 4-methoxylphenyl, 4- (dimethylamino) benzamide, pyridine, 4-methoxyaniline, thiophene, methylamine, dimethylamine,propylamine, anisole, aniline, toluene, morpholine, phenetole, 4-methoxyacetophenone, tertbutylamine, 1 ,2-dimethoxybenzene, 2-methylformanilide, derivatives thereof, or combinations thereof. In some embodiments, X includes NH or NH2. In some embodiments, Y includes NH, NH2, or O. In some embodiments, Z includes O or S.DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1A-1H show that Cpd 2H3 inhibits TopBP 1-BRCT7 / 8 binding to the pS1159 peptide and induces cell death in breast and ovarian cancer cells. FIG. 1A shows the structure of Cpd 2H3. FIG. IB shows that Cpd 2H3 blocks the interaction between TopBP 1-BRCT7 / 8 and pS1159 peptide in vitro. Purified TopBP 1-BRCT7 / 8 was incubated with various concentrations of Cpd 2H3 and biotinylated phospho-S 1159 peptide or nonphosphorylated peptide (Btn-nP), followed by Streptavidin Sepharose pulldown. The pulldown of TopBPl-BRCT7 / 8 was analyzed by immunoblotting (IB) with an anti-TopBPl antibody recognizing TopBP 1-BRCT7 / 8. FIG. 1C shows the structure of TopBPl- BRCT7 / 8 complexed with pS1159 peptide or Cpd H3. Top: pS1159 peptide was overlayed with TopBP 1-BRCT7 / 8 (PDB code: 3AL3), guided by the structure of TopBPl-BRCT7 / 8 with pBACH peptide, and simulated to a local energy minimum. Bottom: The lowest-energy docking of Cpd 2H3 (ball & stick model) to the pocket of TopBP 1-BRCT7 / 8. FIGS. 1D-1E show that Cpd 2H3 induces cell death in MDAH-2774 (FIG. ID) and TOV-112D (FIG. IE). Cells were treated with vehicle DMSO (Veh) or 2H3 as indicated for 24 h. The viable and dead cells were determined by trypan blue exclusion assay. Data shown are the mean ± SD of triplicates. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle control (two-tailed t test). FIG. IF shows that Cpd 2H3 inhibits the cell viability of MDA-MB-468 cells. Cells were treated with vehicle DMSO or 2H3 as indicated for 48 h. Cell viability was determined by MTT assay. Data shown arc the mean ± SD from three biological replicates. FIG. 1G shows that Cpd 2H3 induces apoptosis in MDAH-2774, TOV-112D, and MDA- MB-468 cancer cells, but not non-transformed AML12 mouse hepatocytes or MCF-10A cells. Cells were treated with vehicle DMSO or 2H3 as indicated for 20 h. Active caspase-3 / 7 was determined by Caspase Gio® 3 / 7 Assay. Data shown are the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle control (two-tailed t test). FIG. 1H shows that Cpd 2H3 inhibits clonogenic survival of TOV-112D cells. Equal amounts of cells were treated with vehicle DMSO or 2H3 as indicated for 24 h. After PBS washing, cells were cultured in fresh growth mediumfor another five days. Viable cells were fixed with 3% formaldehyde followed by staining with 0.5% crystal violet.

[0009] FIGS. 2A-2F show hit expansion and SAR studies, which identify Cpd 5D4 as a TopBPl- BRCT7 / 8 inhibitor. FIGS. 2A-2B show structures of initial hit 2H3 and its derivatives 5D4 and 5H3. FIG. 2A shows a ball-and-stick structure of each compound in the TopBP 1-BRCT7 / 8 pocket. Yellow dotted circles indicate the modified side chains from parental compounds. FIG. 2B shows the chemical structures of 5D4 and 5H3. FIGS. 2C-2D show that Cpd 5D4 inhibits TopBP 1-BRCT7 / 8 binding to the pS1159 peptide or mutp53(DBD). FIG. 2C shows the effect of 5D4 on the in vitro binding of biotinylated pS1159 peptide to purified TopBPl-BRCT7 / 8, which was determined by Streptavidin Sepharose pulldown as described in FIG. IB. FIG. 2D shows that purified TopBP 1- BRCT7 / 8 was incubated with GST-mutp53-R273H(DBD) in the presence of various concentrations of 5D4, and a GST pulldown assay was performed. The pulldown of TopBP 1-BRCT7 / 8 was detected by immunoblotting using an anti-TopBPl antibody against the C-terminus of TopBPl. FIG. 2E shows a summary of an SAR study. The cytotoxic activity of each compound was evaluated in multiple cell lines, including MDA-MB-468, BT549, MDAH-2774, and TOV-112D. FIG. 2F shows the structure of TopBP 1-BRCT7 / 8 complexed with BACH1 phosphopeptide (PDB: 3AL3), TopBPl phosphopeptide, Cpd 2H3 or 5D4. Yellow solid circles indicate the position of tyrosine (in BACH1), tryptophan (in TopBPl) or a multibenzene ring in 2H3 and 5D4. Yellow dotted circles indicate the position of pT1133 (BACH1), pS1159 (TopBPl) or a side chain of 2H3 and 5D4.

[0010] FIGS. 3A-3J show that Cpd 5D4 induces apoptosis and inhibits cell viability in 2D and 3D cultured breast and ovarian cancer cells. FIGS. 3A-3B show that Cpd 5D4 induces apoptosis in breast and ovarian cancer cells, but only has minimal effect on non-transformed AML12 mouse hepatocytes and MCF10A. Different cell lines as indicated were treated with vehicle DMSO or 5 pM 2H3, 5H3 or 5D4 for 18-22 h. Apoptosis was determined by Caspase-Gio® 3 / 7 Assay. Data shown are the mean ± SD of three or four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle control (two-tailed t test). FIG. 3C shows that Cpd 5D4 inhibits clonogenic survival of MDA- MB-468 cells. Equal amounts of cells were treated with vehicle DMSO or different concentrations of 5D4 for 24 h. After PBS washing, cells were cultured in fresh growth medium for another four days, and then fixed with 3% formaldehyde. Viable cells were stained with 0.5% crystal violet. The right panel shows the relative colony formation compared to the vehicle control. The relative colonyformation was analyzed using ImageJ to determine the IC50 of 5D4 (= 0.2 pM). FIGS. 3D-3E show that Cpd 5D4 inhibits the growth of breast cancer tumor spheres. Equal amounts of MDAMB-468 cells were seeded on 6-wcll plates with a ccll-rcpcllcnt surface. When the diameter of tumor spheres reached 30-50 pm, spheres were treated with vehicle DMSO or different concentrations of 5D4 on day 0, 3 and 6 (FIG. 3D). The images were captured 24 h after each treatment, and the diameter of tumor spheres was measured. Data shown are the mean ± SEM of 40-60 spheres (*P < 0.05 versus 5D4- treated cells). FIG. 3E shows that BCM2665 PDX tissues were trypsinized and cells were cultured in Complete DMEM / F12 Media for Patient-Derived in vitro and Organoid Cultures as described in NCI Patient-Derived Models Repository (SOP30101). After ten days of culturing, equal amounts of tumor spheres were treated with DMSO vehicle or 5 pM 5D4 every other day for 6 days, followed by CCK-8 assay to determine the viability of tumor spheres. Data shown are the mean ± SD of two experiments done in triplicates. FIG. 3F shows a cellular thermal shift assay. MDA-MB-468 cells were treated with vehicle DMSO or 5D4 (10 pM) at 37 °C for 2 h. Cell lysates were heated for 3 min at temperatures ranging from 42 to 48.4 °C, followed by western blot analysis. Quantification is shown in the bottom panel. FIGS. 3G-3H show that TopBP 1 depletion decreases cell viability and blunts the response to 5D4 in MDA-MB-468 cells. FIG. 3G shows that the early-passage MDA-MB-468 cells stably harboring a scrambled shRNA (shScr) or one of the TopBPl shRNAs (shTopBPl #1 or #2) were treated with indicated concentrations of 5D4 for 19 h. The percentage of live cells was measured by trypan blue exclusion assay. Data shown are the mean ± SD of four biological replicates. *P < 0.05, **P < 0.01 compared with shTopBPl (two-tailed t test). Upper panel: immunoblots confirming the depletion of TopBPl . The relative intensities of TopBPl were quantified by ImageJ and normalized to loading control HSP90. FIG. 3H shows that the carly-passagc MDA-MB-468 cells harboring shScr or shTopBPl (#1) were treated with different concentrations of 5D4 for 24 h. Cell viability was determined by CCK-8 assay. Data shown are the mean ± SD of four biological replicates. **P < 0.01, ***P < 0.001 versus vehicle control (two-tailed t test). The intensities of TopBPl on the immunoblot were quantified by ImageJ and normalized to GAPDH. FIG. 31 shows that the depletion of TopBPl increases the basal caspase-3 / 7 activity, but attenuates 5D4-induced caspase-3 / 7 activation. MDA-MB-468 cells stably expressing shScr or shTopBPl were treated with 3 pM 5D4 for 20 h, followed by caspase-3 / 7 activity assay. Data shown are the mean ± SD of nine biological replicates from four independent experiments. **P < 0.01, ***P < 0.001 versus treated and vehicle control cells,respectively (two-tailed t test). FIG. 3J shows that transient depletion of TopBPl elevates the basal caspase-3 / 7 activity and blocks the further response to 5D4. Hl 299 cells stably expressing mutp53(R273H) were transiently transfected with a pSUPER vector harborinig shScr or shTopBPl. After 48 h, cells were treated with 5 pM 5D4 for 21 h, followed by caspase-3 / 7 activity assay. Data shown are the mean ± SD of three biological replicates, and are representative out of three independent experiments. ***P <0.001 versus vehicle control (two-tailed t test).

[0011] FIGS. 4A-4F shows that Cpd 5D4 treatment blocks the interaction of TopBPl with E2F1, mutp53, MIZ1, CIP2A or PLK1, but has no impact on its binding to RPA2, Rad9 or Treslin. FIGS. 4A-4B show co-immunoprecipitation experiments, which show that 5D4 treatment inhibits TopBPl interaction with E2F1 (FIG. 4A) or mutp53 (FIG. 4B) in MDA-MB-468. MDA-MB-468 cells were treated with vehicle DMSO or 5D4 (5 pM) for 22-24 h. Cells were harvested and subjected to immunoprecipitation using an anti-TopBPl mouse monoclonal antibody. Co-immunoprecipitated E2F1 or mutp53 was detected by immunoblotting using a rabbit antibody specific to E2F1 or mutp53. The relative intensities of immunoprecipitated E2F1 or mutp53 were quantified using Image J and normalized to the immunoprecipitated TopBPl in the same samples. FIGS. 4C-4F show that MDA- MB-468 cells (FIGS. 4C-4D) and MDAH-2774 cells (FIG. 4F) were treated with vehicle DMSO or 5D4 (2 pM in FIGS. 4C-4D and 3 pM in FIG. 4F) for 20 h, and then harvested for coimmunoprecipitation. Endogenous TopBPl in the whole cell lysates was immunoprecipitated with an anti-TopBPl mouse monoclonal antibody or a control mouse IgG, followed by immunoblotting using a rabbit antibody specific to the indicated protein. Data shown in FIG. 4E are the relative fold changes of TopBPl binding to its interacting partners in 5D4-treated MDA-MB-468 cells compared to that in vehicle treated cells (n = 4 biological replicates for MIZ1, CIP2A, PLK1 or mutp53; n = 2 for Rad9; n = 1 for Treslin). ***P < 0.001 versus vehicle (two-tailed t test).

[0012] FIGS. 5A-5F show that Calcein AM and 5D4 enhance the expression of E2F1 target genes in apoptosis and autophagy as well as p53 / p63 / p73 target genes, but reduce the expression of MYC target genes. FIG. 5A shows that MDA-MB-468 cells were treated with CalAM or 5D4 (5 pM) for 6 h. RNA was isolated and subjected to RNA-seq. FPKM: fragments per kilobase of exon per million mapped fragments. Shown are mean ± SD (n = 4 biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle control (two-tailed t test). FIGS. 5B1-5B2 show gene-set enrichment analysis, which shows that 5D4 treatment inhibits the mRNA expression of MYC target genes in MDA-MB-468 cells. NES: normalized enrichment score; FDR: false discovery rate. FIGS. 5C-5E show that MDA-MB-468 (FIG. 5C), MDAH-2774 (FIG. 5D) or A2780cis (FIG. 5E) cells were treated with different concentrations of CalAM or 5D4 for 18 h. The whole cell lysates were subjected to immunoblotting using antibodies specific to the indicated proteins. FIG. 5F shows depletion of TopBPl increases p21Cipl expression but decreased the levels of PLK1, Rad51, and Cyclin DI, and attenuates 5D4-induced p62 expression. MDA-MB-468 cells stably expressing shScr or shTopBPl were treated with 1.5 M or 3 pM 5D4 for 20 h, followed by immunoblotting to detect the expression of the indicated proteins in the whole cell lysates.

[0013] FIGS. 6A-6H show that Cpd 5D4 treatment enhances the MIZ1 binding to the p21Cipl promoter. FIGS. 6A-6D show the results of a ChIP assay that was performed to determine the MIZ1 binding to the p21Cipl promoter in vehicle (Veh)-treated control cells, or MDA-MB-468 cells treated with 2 pM 5D4 for 6 h (FIG. 6A), MDAH- 2774 cells treated with 4 pM 5D4 for 6 h (FIG. 6B), HCC95 cells treated with 4 pM 5D4 for 4 h (FIG. 6C), or H2170 cells treated with 2 pM 5D4 for 4 h (FIG. 6D). MIZ1 binding to |3-Actin promoter serves as a negative control (n = 4). ***P < 0.001 versus vehicle control (two-tailed t test). FIGS. 6E-6F shows the depletion of MIZ1 attenuates the effect of 5D4 on the inhibition of cell viability. MDAMB-468 (FIG. 6E) or MDAH-2774 (FIG. 6F) cells stably expressing shScr or a MIZ1 shRNA (shMIZl#l or #2) were treated with vehicle DMSO or different concentrations of 5D4 for 45 h. The relative cell viability was determined by CCK-8 assay. Data shown are the mean ± SD done in quadruplicates. *P < 0.05, **P < 0.01, ***P < 0.001 versus shScr (two-tailed t test). FIGS. 6G-6H show that the depletion of MIZ1 dampens the effect of 5D4 on the induction of p21Cipl. MDA-MB-468 (FIG. 6G) or H2170 cells (FIG. 6H) stably expressing shScr or shMIZl (#1 or #2) were treated with vehicle DMSO or 5D4 for 20 h. Immunoblotting was performed using an antibody specific to p21Cipl, MIZ1 or GAPDH.

[0014] FIGS. 7A-7C show that Cpd 5D4 significantly decreases tumor growth of ovarian and breast cancer xenografts. FIGS. 7A-7B show NSG mice bearing MDAH-2774 (n = 5 in vehicle group; n = 6 in 5D4 group) or MDA-MB-468 (n = 5 per group) xenografts were administered with 5D4 (40 mg / kg, i.p., q3d x 3 doses) or vehicle. Data shown are the mean tumor volumes ± SD (left panel) and the mean tumor weights ±SD (right panel). FIG. 7C shows NSG mice bearing BCM2665 PDX were administered with 5D4 (40 mg / kg, i.p., twice weekly x 3 weeks) or vehicle; n = 7 per group. ***P <0.001 versus vehicle (two-tailed t test). The x-axis represents the day after injection of cells or implantation of PDX.

[0015] FIGS. 8A-8D show that Cpd 5D4 and PARP1 / 2 inhibitors synergistically inhibit cell viability in breast and ovarian cancer cells. MDA-MB-468 or MDAH-2774 cells were treated with 5D4, rucaparib, or a combination of 5D4 with rucaparib as indicated for 45 h (FIGS. 8A-8B) or 20 h (FIGS. 8C-8D). Cell viability was determined by CCK-8 assay. Data shown in FIGS. 8A and 8B are the mean ± SD done in quadruplicates. Active caspase-3 / 7 was determined by Caspase-Gio® 3 / 7 Assay. Data shown in FIGS. 8C and 8D are the mean ± SEM from three or four independent experiments. Combination index (CI) values and Fa (Fraction affected)-CI plots were generated using CompuSyn software. CI < 1 indicates synergism.

[0016] FIGS. 9A-9D show that Cpd 5D4 and PARP-14 inhibitor GeA-69 synergistically inhibit cell viability in breast and ovarian cancer cells. MDA-MB-468 or MDAH-2774 cells were treated with different concentrations of 5D4, GeA-69 or both for 45 h (FIGS. 9A-9B) or 20 h (FIGS. 9C-D). Cell viability and active caspase-3 / 7 were determined as described in FIGS. 8A-8D. Data shown in FIGS. 9A and 9B are the mean ± SD done in quadruplicates. Data shown in FIGS. 9C and 9D are the mean ± SEM from three independent experiments. Right panels are corresponding Fa-CI plots.

[0017] FIGS. 10A-10F show that Cpd 5D4 and talazoparib synergistically inhibit tumor growth in two breast cancer PDX models. FIGS. 10A-10B show that NSG mice bearing BCM2665 or BCM3107 PDX were administered with DMSO vehicle, 5D4 (40 mg / kg, i.p., twice weekly) and / or talazoparib (0.5 mg / kg, daily x 5 per week) for three or four weeks. Shown are mean tumor volumes ± SD (P values calculated for linear mixed models) and mean tumor weights ± SD, (two-tailed t test). In BCM2665 PDX experiment, n = 7 mice in each group; in BCM3107 PDX experiment, n = 9 mice in Veh group and 6 mice in the other groups; n.s, not significant. The x-axis represents the day after implantation of PDX. FIGS. 10C-10E show results from administration with the last dose of vehicle DMSO, 5D4 and / or talazoparib in mice that were euthanized on the following day. Mouse intestine and PDX tumor samples were harvested and subjected to IHC staining of cleaved PARP1 (FIG. 10C). Shown are representative images using 10X objective. Lower panel: The percentage of cleaved PARP1 -positive cells was determined by ImageJ. Twelve images from three xenografts per group were assessed. FIG. 10D shows a Ki67 IHC staining of mouse intestinal epithelium using a 20X objective. FIG. 10E shows that CETSA was performed using the lysates of BCM2665 PDX frommice treated with 5D4 or vehicle DMSO. FIG. 10F is a schematic diagram showing the anticancer activity of 5D4 and its synergy with PARPi.

[0018] FIG. 11 shows a typical calibration curve for 5D4 in mouse blood (linear range 0.5-250 ng / mL, R=0.9999).

[0019] FIGS. 12A1-12C2 provide representative MRM chromatograms for 5D4, including: double blank mouse blood (FIGS. 12A1-12A2); standard sample of 0.5 ng / mL 5D4 in blank mouse blood (the retention times of 5D4 and IS: 2.21 min and 2.84 min, respectively) (FIGS. 12B1-12B2); and a blood sample at 60 min after 40 mg / kg oral dose of 5D4 in a mouse (FIGS. 12C1-12C2).

[0020] FIG. 13 shows a mean blood concentration vs. time profile of 5D4 in mouse blood after a 40 mg / kg of 5D4 oral administration (normal scale) (n=4).DETAILED DESCRIPTION

[0021] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0022] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, arc hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0023] Cancer development involves many steps of genetic alterations and signaling pathway deregulation. Regardless of the events that initiate the development of cancer, during progression, most cancers exhibit deregulation of at least one of the common signaling pathways, such as Rb, p53 and PI3(K) / Akt. Previously, Applicant identified topoisomerase I I[3-bi nd i ng protein 1 (TopBP 1) as a therapeutic target that functions at a convergent point of these common oncogenic pathways.Applicant then identified calcein as a lead inhibitor that binds the 7th-8th BRCA1 carboxyl-terminal (BRCT) domains of TopBP 1 (TopBP 1-BRCT7 / 8). Because calcein lacks membrane permeability, Applicant treated cancer cells with Calcein AM (acctoxymcthyl ester) (CalAM), a cell permeable derivative that can be rapidly hydrolyzed to form calcein inside the cells, and demonstrated its in vivo anticancer activity.

[0024] TopBPl contains nine BRCT domains responsible for many protein interactions. Through the diverse protein-protein interactions, TopBPl is involved in DNA replication, ATR checkpoint activation, DNA repair, mitosis and transcriptional regulation. During Gl / S phase entry, E2F1 transcriptionally induces the expression of TopBPl, which in turn interacts with Treslin to promote the initiation of DNA replication. In late S and G2 phases, activated Akt phosphorylates TopBPl and switches the TopBPl -interacting partner from Treslin to E2F1, resulting in the termination of replication initiation and inhibition of E2F1 -mediated apoptosis.

[0025] During mitosis, TopBPl interacts with SLX4, PLK1, MDC1, topoisomerase Ila and CIP2A (Cancerous Inhibitor of Protein Phosphatase 2A) to maintain genome integrity. Upon DNA replication stress, TopBPl is recruited to stalled replication forks. It then activates ATR (Ataxia telangiectasia mutated and Rad3 related) through a conserved ATR-activating domain. In addition to ATR checkpoint activation, TopBPl plays a crucial role in regulating homologous recombination (HR) DNA repair. With its function in cell cycle progression and DNA damage response, TopBPl is frequently overexpressed in cancers and contributes to cancer progression.

[0026] TopBPl is an E2F target and its expression is constantly induced when Rb / E2F or p53 pathway is de-regulated. Indeed, TopBPl overexpression is found in most types of cancer (TCGA datasets), and is an independent poor-prognostic factor in breast cancer and ovarian cancer.

[0027] Applicant’s prior studies have elucidated several mechanisms by which TopBPl overexpression promotes cancer development: (i) inhibiting E2Fl-mediated apoptosis, (ii) promoting mutant p53 (mutp53) gain of function (GOF) through transcriptional regulation of NF-Y and p63 / p73, (iii) bypassing the requirement of active Cdk2 to promote DNA replication, and (iv) paradoxically hindering ATR / Chkl activation.

[0028] The activity of TopBPl in repressing E2F1 -mediated apoptosis is regulated by Akt. Akt phosphorylates TopBPl at Seri 159, which in turn induces its oligomerization through TopBPl- BRCT7 / 8 domains. Oligomerization of TopBPl promotes its binding to E2F1 and MIZ1 (MYC-interacting zinc finger protein 1), thereby inhibiting E2F1 -dependent apoptosis and MIZ1 -dependent p21Cipl expression. Thus, compounds that bind and block TopBP 1-BRCT7 / 8 domains, such as CalAM, can reactivate E2F1 -dependent apoptosis and induce p21Cipl expression. In addition to mediating TopBPl binding to wild-type p53 to repress p53 function, TopBP 1-BRCT7 / 8 can also bind mutp53 to promote mutp53 GOF and tumor growth by facilitating mutp53 interaction with NF-Y and p63 / p73. On the other hand, some p53 contact mutants bind both TopBPl and Treslin and facilitate their interaction, thereby overriding the requirement of Cdk2 and promoting DNA replication in late G1 phase.

[0029] Through its BRCT7 / 8 domains, TopBPl recruits PLK1 to phosphorylate Rad51 and facilitates the chromatin loading of Rad51 to promote DNA repair. Thus, TopBP 1-BRCT7 / 8 appears to be an attractive target to develop small molecule inhibitors for cancer therapy.

[0030] In sum, a need exists for more effective compounds and methods for treating or preventing cancer, including cancers associated with over-expressed TopBPl. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0031] In some embodiments, the present disclosure pertains to methods of treating or preventing a cancer in a subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a composition. In some embodiments, the composition includes a topoisomerase II[3-binding protein 1 (TopBPl ) inhibitor. In some embodiments, the composition further includes a poly (ADP-ribose) polymerase (PARP) inhibitor.

[0032] Additional embodiments of the present disclosure pertain to a composition that includes a TopBPl inhibitor. In some embodiments, the composition also includes a PARP inhibitor. In some embodiments, the composition is suitable for use in treating or preventing a cancer in a subject.

[0033] As set forth in more detail herein, the methods and compositions of the present disclosure can have numerous embodiments.

[0034] TopBPl inhibitors

[0035] The compositions of the present disclosure may include various TopBPl inhibitors. Additionally, the methods of the present disclosure may utilize various TopBPl inhibitors to treat or prevent cancer in a subject.

[0036] In some embodiments, the TopBPl inhibitor includes, without limitation:derivatives thereof, or combinations thereof. In some embodiments, the amine group of the quinuclidine group is in protonated form (as shown). In some embodiments, the amine group of the quinuclidinc group is in dcprotonatcd form.

[0037] In some embodiments, Ri includes, without limitation, polycyclic aromatic hydrocarbons, naphthalene, biphenyl, fluorene, anthracene, phenanthrene, phenalene, derivatives thereof, or combinations thereof. In some embodiments, Ri includes naphthalene. In some embodiments, the

[0038] In some embodiments, R2 includes, without limitation, furan groups, phenyl groups, benzoyl groups, methoxybenzene, methoxybenzene amine, 4-methoxylphenyl, 4-(dimethylamino) benzamide, pyridine, 4-methoxyaniline, thiophene, methylamine, dimethylamine, propylamine, anisole, aniline, toluene, morpholine, phenetole, 4-methoxyacetophenone, tert-butylamine, 1,2-dimethoxybenzene, 2- methylformanilide, derivatives thereof, or combinations thereof. In some embodiments, X includes NH or NH2. In some embodiments, Y includes NH, NH2, or O. In some embodiments, Z includes O or S.

[0039] In some embodiments, the TopBP 1 inhibitor includes, without limitation,derivatives thereof, or combinations thereof.

[0040] In some embodiments, the TopBP 1 inhibitor includes the following structure:

[0041] Without being bound by theory, the TopBP 1 inhibitors of the present disclosure may inhibit TopBP 1 in various manners. For instance, in some embodiments, the TopBP 1 inhibitor targets the BRCT7 domain of TopBP 1. In some embodiments, the TopBP 1 inhibitor targets the BRCT8 domain of TopBPl. In some embodiments, the TopBPl inhibitor targets the BRCT7 and BRCT8 domains of TopBP 1.

[0042] PARP inhibitors

[0043] In some embodiments, the compositions of the present disclosure may include one or more PARP inhibitors. Additionally, the methods of the present disclosure may utilize one or more PARP inhibitors to treat or prevent cancer in a subject.

[0044] The compositions and methods of the present disclosure may utilize various PARP inhibitors. For instance, in some embodiments, the PARP inhibitor inhibits PARP1, PARP2, PARP14, or combinations thereof. In some embodiments, the PARP inhibitor includes, without limitation, talazoparib, rucaparib, olaparib, vcliparib, niraparib, pamiparib, GcA-69, RBN-3143, RBN012759, or combinations thereof. In some embodiments, the PARP inhibitor includes talazoparib.

[0045] Modes of administration

[0046] The compositions of the present disclosure may be administered to subjects in various manners. For instance, in some embodiments, the administration occurs by a method that includes, without limitation, oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, intrathecal injection,intra-articular administration, topical administration, central administration, peripheral administration, aerosol-based administration, nasal administration, transmucosal administration, transdermal administration, parenteral administration, or combinations thereof. In some embodiments, the administration occurs by intravenous administration. In some embodiments, the administration occurs by intraperitoneal administration. In some embodiments, the administration occurs by oral administration.

[0047] Subjects

[0048] The compositions of the present disclosure may be administered to various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject is a non-human mammal, such as mice, rats, dogs, cats, horses, cows, and / or pigs. In some embodiments, the subject is suffering from a cancer. In some embodiments, the subject is vulnerable to a cancer.

[0049] Cancers

[0050] The methods and compositions of the present disclosure may be utilized to treat or prevent various cancers. For instance, in some embodiments, the cancer includes breast cancer, ovarian cancer, lung cancer, a cancer associated with TopBPl over-expression, or combinations thereof. In some embodiments, the methods and compositions of the present disclosure may treat a cancer. In some embodiments, the methods and compositions of the present disclosure may prevent a cancer. In some embodiments, the methods and compositions of the present disclosure may treat and prevent a cancer.

[0051] Compositions

[0052] The compositions of the present disclosure may have various formulations. For instance, in some embodiments, the compositions of the present disclosure may be formulated for administration in one or more doses. In some embodiments, the compositions of the present disclosure also include one or more stabilizers. In some embodiments, the stabilizers include, without limitation, antioxidants, sequestrants, ultraviolet stabilizers, or combinations thereof.

[0053] In some embodiments, the compositions of the present disclosure also include one or more surfactants. In some embodiments, the surfactants include, without limitation, anionic surfactants, sugars, cationic surfactants, zwitterionic surfactants, non-ionic surfactants, or combinations thereof.

[0054] In some embodiments, the compositions of the present disclosure also include one or more excipients. In some embodiments, the excipients include, without limitation, lactose, sucrose, starchpowder, cellulose esters of alkanoic acids, trehalose, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, trehalose, sodium alginate, polyvinylpyrrolidone, polyvinyl alcohol, or combinations thereof.

[0055] In some embodiments, the compositions of the present disclosure include a delivery vehicle, such as a particle. In some embodiments, the particle includes, without limitation, lipid-based particles, carbon-based particles, metal-based particles, or combinations thereof. In some embodiments, the active agents of the present disclosure (e.g., TopBPl and / or PARP inhibitors) are encapsulated in the particles.

[0056] In some embodiments, the compositions of the present disclosure can include additional components. For instance, in some embodiments, the composition can further include dithiothreitol (threo-1,4 Dimercapto-2,3-butanediol), ethylenediaminetetraacetic acid sodium, glycerol (1,2,3- Propanetriol), an ammonium carbonate buffered solution, or combinations thereof.

[0057] In some embodiments, the compositions of the present disclosure can have various pH levels. In some embodiments, the compositions have a pH of about 6.5. In some embodiments, the compositions have a pH of about 7. In some embodiments, the compositions have a pH of about 7.5. In some embodiments, the compositions have a pH in the range of about 6.5 to about 7.5.

[0058] Additional embodiments

[0059] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0060] Example 1. A novel small molecule inhibitor of TopBPl exerts anti-MYC activity and synergy with PARP inhibitors

[0061] Applicant previously identified TopBPl as a promising target for cancer therapy, given its role in the convergence of Rb, PI(3)K7Akt and p53 pathways. Based on this, Applicant conducted a large- scale molecular docking screening to identify a small molecule inhibitor that specifically targets the BRCT7 / 8 domains of TopBPl, which Applicant named 5D4. Applicant’s studies show that 5D4 inhibits TopBPl interactions with E2F1, mutant p53 and CIP2A. This leads to the activation of E2Flmediated apoptosis and the inhibition of mutant p53 gain of function.

[0062] In addition, 5D4 disrupts the interaction of TopBPl with MIZ1, which in turn allows MIZ1 to bind to its target gene promoters and repress MYC activity. Moreover, 5D4 inhibits the association of the TopBPIPLKl complex and prevents the formation of Rad51 foci. When combined with inhibitors of PARP1 / 2 or PARP14, 5D4 synergizes to effectively block cancer cell proliferation.

[0063] Applicant’s animal studies have demonstrated the antitumor activity of 5D4 in breast and ovarian cancer xenograft models. Moreover, the effectiveness of 5D4 is further enhanced when combined with a PARP1 / 2 inhibitor talazoparib. Taken together, Applicant’s findings strongly support the potential use of TopBPlBRCT7 / 8 inhibitors as a targeted cancer therapy.

[0064] In particular, Applicant has performed a molecular docking screen. Through hit expansion, Applicant identified several novel compounds that target TopBPl-BRCT7 / 8. Applicant has shown the in vivo anticancer activity of one of the compounds, specifically 5D4, and demonstrates its anti- MYC activity and synergy with PARP inhibitors (PARPi).

[0065] Example 1,1. A molecular docking screen identifies a lead compound 2H3 that targets TopBP 1-BRCT7 / 8

[0066] Based on the crystal structure of TopBPl-BRCT7 / 8, Applicant previously performed a small scale of molecular docking to screen more than 2,000 known compounds and identified CalAM, which could block the interactions of TopBPl-BRCT7 / 8 with pS1159-TopBPl phosphopeptide and DNA- binding domain (DBD) of p53. CalAM inhibited tumor growth in breast cancer xenograft models and induced apoptosis in cancer cells highly expressing TopBPl. Chowdhury P, el al. (2014) Targeting TopBPl at a convergent point of multiple oncogenic pathways for cancer therapy. Nat Commun 5:5476. These data provide proof of concept for targeting TopBPl in cancer.

[0067] Applicant then expanded screening to test 200,000 compounds from 3 selected libraries (druglike, natural product derivatives, and compounds tested in men) and identified compounds with best docking scores (close to or < -8.0). Applicant shortlisted the compounds based on availability, predicted cell permeability (polar surface area < 140), and no known targets (in SEA, ZINC, PubChem and ChemBank databases). Thereafter, Applicant prioritized 99 compounds for testing.

[0068] Applicant performed an MTT assay to evaluate the effects of the prioritized compounds on cell viability in BT549 and MDA-MB-468 triple-negative breast cancer (TNBC) cell lines, and carried out biochemical binding assays to assess their activity in blocking the interaction of TopBP 1-BRCT7 / 8with pS 1159-TopBPl peptide. Among these compounds, Cpd 2H3 showed the most active cytotoxic activity.

[0069] Cpd 2H3, a mcthylpyrazolc-quinuclidin derivative (FIG. 1A), blocked TopBP 1-BRCT7 / 8 binding to the pSl 159-TopBPl phosphopeptide in vitro (IC50 = 20 nM) (FIG. IB) and attenuated its association with full-length TopBPl in HEK293T cells. The BRCT7 and BRCT8 domains of TopBPl form a pocket that binds pSl 159-TopBPl phosphopeptide (FIG. 1C, top panel).

[0070] Molecular docking shows that Cpd 2H3 can be docked well into the pocket (FIG. 1C, bottom panel). Cpd 2H3 exhibited cytotoxic activity in MDAH-2774 and TOV-112D ovarian cancer cells (FIGS. 1D-E and G) and MDA-MB-468 breast cancer cells (FIGS. 1F-1G), but only had a minimal effect in non-transformed MCF10A cells and AML12 mouse hepatocytes (FIG. 1G). Cpd 2H3 also inhibited clonogenic viability of TOV-112D cells (FIG. 1H). It is predicted that Cpd 2H3 has more favorable drug-like properties than CalAM according to Lipinski’s rules.

[0071] Example 1,2. Hit expansion identifies Cpd 5D4 as a more potent TopBPl inhibitor

[0072] To determine whether there are common features among the lead compounds capable of targeting TopBPl-BRCT7 / 8, Applicant compared the docking of these lead compounds. Indeed, 2H3 and calcein possess multi-benzene rings to dock into a pocket within TopBPl-BRCT7 / 8, which normally binds to the +4 Tryptophan residue of pS1159-TopBPl phosphopeptide or the +4 Tyrosine of pT1133-BACHl phosphopeptide.

[0073] Comparing the sequences from different species, this TopBP 1-BRCT7 / 8 pocket binds a conserved S / TxxxQ motif with an aromatic a.a. (Q, such as W / Y / F) in the +4 position. The structurebased compound docking suggests that the polycyclic aromatic hydrocarbons of the lead compounds mimic +4 aromatic a.a. when they bind TopBP 1-BRCT7 / 8. Calcein and Cpd 2H3 also compete with pS1159-TopBPl phosphopeptides for hydrogen bonding with the critical a.a. such as S1273, S1274 and K1317, and therefore block the phophoserine-binding pocket of TopBP 1-BRCT7 / 8 (FIG. 2A).

[0074] With the guide of docking, Applicant performed hit expansion through Pubchem and ZINC docking servers, and identified new analogs / derivatives of Cpd 2H3 with modified side chains on the desired positions. These compounds were tested by docking with TopBP 1-BRCT7 / 8 in mcule 1-Click Docking online server. Through this exercise, Applicant identified 28 novel derivatives of Cpd 2H3 which show improved docking scores and favorable drug-like properties as analyzed in FAF-Drugs3 server and Chembioserver. Applicant then performed experiments to characterize these derivatives.

[0075] Indeed, most derivatives exhibited more potent anticancer activities than the parental Cpd 2H3, particularly 5H3 and 5D4 (FIGS. 2A-2B). 5D4 bound TopBP 1-BRCT7 / 8 and blocked its binding to pS1159-TopBPl phosphopeptide, mutp53-R273H(DBD) and WT p53(DBD) with IC50 around 10 nM (FIGS. 2C-2D). Structure- Activity Relationship (SAR) analysis from totally 64 analogs of Cpd 2H3 (FIG. 2E) revealed that a naphthalene on the R1 position is preferred for their cytotoxic activities. Different side chains on the R2 position also affect their activities. The naphthalene on the R1 position corresponds to the +4 aromatic a.a. of the pT1133-BACH or pS1159-TopBPl phosphopeptide (FIG. 2F, yellow solid circles). On the other hand, the R2 side chain of the compounds corresponds to the phosphorylated threonine or serine residue in the docking structure (FIG. 2F, yellow dotted circles).

[0076] Based on the experimental results, Cpd 5H3 and 5D4 showed stronger cytotoxic activities than Cpd 2H3 in MDA-MB-468 cells, but barely had any effect in non-transformed MCF10A cells or AML12 mouse hepatocytes (FIG. 3A). Likewise, 5D4 showed strong cytotoxic activities against OVCAR8 ovarian carcinoma cells, MDAH-2774 and TOV-112D endometrioid ovarian cancer cells, and cisplatin-resistant A2780cis ovarian cancer cells (FIG. 3B).

[0077] The clonogenic cell survival assay showed that the IC50 of 5D4 and 5H3 in blocking MDA- MB-468 cell growth was within the nanomolar range (FIG. 3C). The 3D culture showed that 5D4 effectively inhibited tumor sphere formation of MDA-MB-468 (FIG. 3D), BT549, and T47D breast cancer cells as well as TOV-112D ovarian cancer cells. Consistently, 5D4 also decreased the viability of tumor spheres, which were established from TNBC patient-derived xenografts (PDX), BCM2665 (FIG. 3E).

[0078] To further validate if TopBPl is a specific target of 5D4, Applicant assessed the ability of 5D4 to bind endogenous TopBPl in MDA-MB-468 cells by performing a cellular thermal shift assay (CETSA). CETSA showed that 5D4 shifted the melting temperature (Tm) of TopBPl protein toward a higher temperature, providing evidence for the binding of 5D4 to TopBPl within the cells (FIG.3F).

[0079] On the other hand, depletion of TopBPl decreased the cell viability (FIGS. 3G and 3H) and blunted the effect of 5D4 on the growth inhibition in MDA-MB-468 cells as assayed by trypan blue exclusion (FIG. 3G) or CCK-8 cell viability assay (FIG. 3H). Moreover, depletion of TopBPl mildly elevated the basal caspase-3 / 7 activity in MDA-MB-468 cells, but attenuated 5D4-induced caspase- 3 / 7 activation (FIG. 31).

[0080] Considering that these experiments were performed in cells stably harboring a TopBP 1 shRNA, which might already undergo phenotypic changes due to the antiproliferative effect of TopBP 1 knockdown, Applicant also assessed the short-term impact of TopBP 1 depletion on 5D4- induced caspase-3 / 7 activation by transiently transfecting a TopBPl shRNA in H1299 cells stably expressing mutp53(R273H). Indeed, transient depletion of TopBPl increased the basal cacspase-3 / 7 activity and blocked the further response to 5D4 (FIG. 3J), suggesting that the anticancer activity of 5D4 is dependent on TopBPl expression. Cpd 5D4 and 5H3 have favorable predicted drug-like properties.

[0081] Example 1.3. Cpd 5D4 treatment disrupts the interaction of TopBPl with E2F1, mutp53, MIZ1. PLK1 or CIP2A

[0082] TopBPl interacts with several proteins, such as E2F1, mutp53, MIZ1, and PLK1 in a BRCT7 / 8-dependent manner, but binds to other proteins, such as RPA2, Treslin or Rad9 through BRCT1 / 2 domains. Meanwhile, molecular docking showed that, among all of the available tandem BRCT structures, TopBP 1-BRCT7 / 8 domains show the best score for 5D4 docking. Applicant therefore investigated whether 5D4 treatment specifically disrupts the BRCT7 / 8-mediated proteinprotein interactions.

[0083] Indeed, 5D4 inhibited the TopBPl binding to E2F1 (FIG. 4A). 5D4 also blocked the interaction of TopBPl with mutp53, MIZ1 or PLK1 in MDA-MB-468 and MDAH-2774 cells (FIGS. 4B-4C and 4E-F). In contrast, 5D4 did not interfere with the TopBPl binding to RPA2, Rad9 or Treslin (FIGS. 4D-4E), and also did not affect the BRCT domain-mediated interaction of BRCA1 with BACH1. Together with the results shown in FIGS. 3F-3J, these data strongly suggest that TopBP 1-BRCT7 / 8 is a cellular target of 5D4.

[0084] Recently, CIP2A was identified as another TopBPl interacting protein and their interaction was proposed to serve as a synthetic lethal target in BRCA-mutated cancer. It was reported that CIP2A binds to a TopBPl peptide (a.a. 740-899) comprising the region between BRCT5 and 6. However, Applicant found that 5D4 could inhibit TopBP 1 / CIP2A interaction in both MDA-MB-468 and MDAH-2774 cells (FIGS. 4C, 4E and 4F). Applicant’ s result therefore suggests that either BRCT7 / 8 domains are also involved in the association with CIP2A, or the binding of 5D4 to TopBP 1-BRCT7 / 8 results in a conformational change of TopBPl, thereby hindering its binding to CIP2A.

[0085] Example 1.4. Transcriptome profiling reveals that TopBPl inhibitors possess the anti-MYC activity

[0086] To gain mechanistic insights into the anticanccr actions of TopBP 1 inhibitors, Applicant performed RNAseq transcriptome profiling of MDA-MB-468 cells treated with either DMSO vehicle, CalAM or 5D4 for 6 h. Consistent with prior results, both CalAM and 5D4 activated E2F1 target genes in apoptosis and autophagy as well as p53 / p63 / p73 target genes (FIG. 5A), supporting the notion that disruption of the TopBPl / E2Fl and TopBPl / mutp53 complex formation by these inhibitors can reactivate E2Fl-induced apoptosis and autophagy, and block mutp53 GOF. The transcriptome profile of CalAM or 5D4-treated MDA-MB-468 shares significant similarities with that of TopBP 1 -depleted MDA-MB-468, supporting that the changes of these gene expression are on-target effects of these compounds. The data also showed that 5D4 significantly inhibited the MYC gene signature (FIGS.5B1-5B2).

[0087] The anti-MYC activities of these compounds were further verified by immunoblotting, which showed the induction of p21Cipl by 5D4 and decreased protein expression of several MYC targets, including Cyclin A2, Cyclin DI, ACTL6A (Actin-like protein 6 A, also named BAF53A), PLK1 and Rad51, by 5D4 or CalAM in MDAMB-468, MDAH-2774 and A2780cis cells, respectively (FIGS. 5C-5E). Consistent with the RNAseq results, 5D4 also increased the protein levels of SQSTMl / p62 (FIGS. 5D and 5F).

[0088] Applicant next performed immunoblotting to determine the effect of TopBP 1 depletion on the expression of these proteins in MDA-MB-468 cells. As expected, depletion of TopBPl decreased the basal levels of PLK1 , Cyclin DI and Rad51 (FIG. 5F). Since the TopBPl -depleted cells still expressed low levels of TopBPl, they were able to respond, albeit at a lower degree, to 5D4. As a result, 5D4 could further attenuate the expression of these proteins in TopBPl -depleted cells. On the other hand, depletion of TopBPl elevated the basal levels of p21Cipl, but did not further induce its expression after 5D4 treatment. Depletion of TopBPl also slightly increased the basal levels of p62, but significantly attenuated 5D4-induced p62 expression. (FIG. 5F). Together, these results demonstrate that 5D4 effectively inhibits the TopBPl target gene expression and exhibits anti-MYC activity.

[0089] Example 1,5. Treatment with 5D4 promotes MIZ1 binding to the p21Cipl promoter

[0090] TopBPl binds MIZ1 and inhibits its function in p21Cipl transactivation. Thus, TopBPl- BRCT7 / 8 inhibitors may release MIZ1 from TopBPl (FIGS. 4A-4F), thereby promoting its activity.To investigate this potential mechanism of action, Applicant performed chromatin immunoprecipitation (ChIP) assay, and found that indeed, 5D4 induced the MIZ1 chromatin binding to the p21Cipl promoter in MDA-MB-468, MDAH-2774 and two lung cancer cell lines, HCC95 and NCI-H2170 (harboring MYC gene amplification) (FIGS. 6A-6D).

[0091] MIZ1 accumulates on MYC target genes globally and contributes to the repression of MYC- activated target genes, particularly when HUWE1, an E3 ubiquitin ligase that targets MIZ1 for degradation, is inhibited. Thus, the release of MIZ1 from TopBPl by BRCT7 / 8 inhibitors may contribute to the observed inhibition of MYC activity by 5D4. To determine a role for MIZ1 in the anticancer activity of 5D4, Applicant depleted MIZ1 in MDA-MB-468 and MDAH-2774 cells. Indeed, MIZ1 depletion dampened the effect of 5D4 on the inhibition of cell viability in both cell lines (FIGS. 6E-6F) and the induction of p21Cipl in MDA-MB-468 and H2170 cells (FIGS. 6G-6H).

[0092] Example 1.6. Cpd 5D4 inhibits the growth of ovarian cancer and breast cancer xenografts

[0093] To investigate the in vivo activity of 5D4, Applicant next established MDAH-2774 ovarian cancer and MDA-MB-468 breast cancer xenografts, respectively, in NSG mice, and injected intraperitoneally (i.p.) with 5D4 (40 mg / kg) or vehicle (DMSO) every three days for three doses. Indeed, 5D4 significantly reduced tumor growth in both models (FIGS. 7A-7B).

[0094] Applicant next implanted BCM2665 breast cancer PDX into NSG mice. BCM2665 is a TNBC PDX harboring mutp53-R249S, MYC amplification (4 copies) and WT BRCA1 / 2 (BCM PDX Portal). Consistently, 5D4 also significantly reduced tumor growth in the PDX model (FIG. 7C). These mice tolerated 5D4 well without changes of body weight or apparent side effects.

[0095] Example 1 ,7. Synergism between TopBPl inhibitors and PARP inhibitors

[0096] PARP inhibitors (PARPi) arc the first synthetic lethal therapy for the patients with homologous recombination (HR) deficiency. The interaction between TopBPl and PLK1 is important for Rad51 chromatin recruitment and HR DNA repair. As such, CalAM, the first TopBP 1-BRCT7 / 8 inhibitor that Applicant identified, has been shown to block TopBPl / PLKl interaction and sensitize U2OS cells to PARPi olaparib. Likewise, 5D4 can also block TopBPl / PLKl interaction (FIGS. 4A-4F).

[0097] Therefore, Applicant next investigated whether 5D4 and PARPi have synergistic anticancer effect. The HR-proficient MDA-MB-468, MDAH-2774 and A2780cis cells, as expected, responded to 5D4 or CalAM, but not PARPi rucaparib. Nonetheless, combined treatment with rucaparib and either 5D4 or CalAM rendered these cells even more responsive than individual drug treatment (FIGS.8A-8D). The combination index (CI) analysis showed that indeed, both 5D4 and CalAM synergized (CI < 1) with rucaparib in MDAMB-468, MDAH-2774 and A2780cis cells. Likewise, this synergy was also observed in HCC95 and HCC2814 lung cancer cells. In addition to rucaparib, another PARPi, talazoparib, also showed similar synergy when combined with 5D4 or CalAM.

[0098] Accordingly, CalAM and 5D4 also synergized with PARPi veliparib. On the contrary, treatment with 5D4, rucaparib or both did not elicit any effect in non-transformed AML12 mouse hepatocytes. The binding of TopBP 1-BRCT7 / 8 to PLK1 is required for Rad51 recruitment and foci formation, a key step for HR.

[0099] Applicant’s data showed that 5D4 or CalAM treatment decreased the expression of Rad51 and PLK1 (FIGS. 5C-5F). Moreover, treatment with CalAM or 5D4 greatly prevented rucaparib-induced Rad51 foci formation in MDAH-2774 cells. Together, these data demonstrate that TopBPl inhibitors dampen HR and show synergistic effect with PARPi in HR-proficient cancer cells.

[0100] Example 1.8. Synergism between TopBPl inhibitors and a PARP14 inhibitor

[0101] Using a genome-wide CRISPR dropout screen to identify synthetic lethal targets in ADPribosyltransferase P ARP 14 knockout cells, TopBPl was identified to have a synthetic lethal interaction with PARPI 4. Therefore, Applicant next tested the combinational effect between 5D4 and a PARP14 inhibitor GeA-69. Indeed, 5D4 and GeA-69 synergistically inhibited cell viability (FIGS. 9A-9B) and promoted apoptosis (FIGS. 9C-9D) in both MDA-MB-468 and MDAH-2774 cells. The immmunoblotting also showed that 5D4 and GeA-69 synergistically repressed the expression of MYC and its targets, such as ACTL6A, Cyclin DI and Rad51.

[0102] Example 1 .9. Synergism between Cpd 5D4 and talazoparib in two breast cancer PDX models

[0103] To further determine the in vivo efficacy of the combination between TopBPl inhibitors and PARPi, Applicant implanted BCM2665 PDX into NSG mice. Indeed, combination of 5D4 and talazoparib synergistically inhibited the tumor growth of BCM2665 PDX without apparent toxicity or changes in mouse body weight (FIG. 10A).

[0104] Applicant performed linear mixed model analysis using tumor size as the dependent variable and adding fixed effects of time of measurement, treatment, and the interaction between time and treatment. The biological replicates were treated as random effects. Indeed, there was a significant effect of treatment (P <0.001) and an interaction between time and treatment (P < 0.001). Thecomparison between two treatments also showed statistically significant difference (P < 0.001) (FIG. 10A).

[0105] Applicant further performed similar experiments in another TNBC PDX BCM3107, which harbors mutp53-T155N, MYC amplification (5 copies) and BRCA1-Q1395* mutation (BCM PDX Portal). BCM3107 PDX also shows a loss of one allele of 53BP1 and DYNLL1, which may confer resistance to PARPi in BRCA1 -mutated cancers. Applicant’s data showed that BCM3107 was resistant to talazoparib, but responded well to 5D4 (FIG. 10B). Moreover, combination of 5D4 and talzoparibe further inhibited tumor growth without apparent toxicity or changes in mouse body weight (FIG. 10B).

[0106] Applicant next performed immunohistochemistry in BCM2665 PDXs (FIGS. 10C andIOD). As expected, talazoparib only had mild proapoptotic effect. 5D4 induced apoptosis in breast cancer PDXs, as indicated by elevated PARP-1 cleavage; and when combined with talazoparib, this further enhanced apoptosis (FIG. 10C).

[0107] In contrast, treatment with 5D4, talazoparib or both neither induced apoptosis nor affected Ki67 staining in mouse intestinal epithelium (FIGS. 10C-10D). Applicant then performed CETSA in the BCM2665 xenograft tumor tissues. Consistent with the results shown in FIG. 3F, 5D4 shifted the Tm of TopBP 1 protein, providing evidence for the binding of 5D4 to TopBP 1 in vivo (FIG.IOE).

[0108] Taken together, 5D4 targets TopBP 1-BRCT7 / 8 and impedes its interactions with E2F1, mutp53, MIZ1, CIP2A, and PLK1. As a result, the E2F1 -mediated pro-apoptotic function is enhanced and the mutp53 GOF, Rad51 recruitment and MYC activities are decreased by 5D4 treatment. Through these mechanisms of action, 5D4 exerts antitumor activities and shows synergy with PARPi (FIG. 10F).

[0109] Example 1.10, Discussion

[0110] Through multiple rounds of compound screening, Applicant identified a novel compound 5D4 that targets TopBP 1-BRCT7 / 8 and exhibits anticancer activity in vivo. Applicant further demonstrated that both TopBPl-BRCT7 / 8 inhibitors, CalAM and 5D4, exert anti-MYC activity and can synergize with inhibitors of PARP1 / 2 or PARP14 in a number of cancer types. To verify the on-target effects of 5D4, Applicant provide multiple independent evidences.

[0111] First, 5D4 can block the TopBP 1-BRCT7 / 8 binding to pS1159-TopBPl phosphopeptide or mutp53(DBD) in vitro. Second, the CETSA results confirm the 5D4 binding to TopBP 1 in breast cancer cells and PDX tumors. Third, 5D4 treatment inhibits the interactions of TopBPlwith several BRCT7 / 8-interacting partners, such as E2F1, mutp53, PLK1, MIZ1, and CIP2A. This leads to the induction of E2F1 pro-apoptotic targets and p53 / p63 / p73 targets, as well as the inhibition of MYC targets. Fourth, depletion of TopBPl or MIZ1 blunts the anticancer effect of 5D4. Fifth, there is a correlation between the response to 5D4 and the levels of TopBPl mRNA and protein in the cancer cell lines that we examined. Sixth, 5D4 can synergize with inhibitors of PARP1 / 2 or PARP14, which is likely attributed to the disruption of TopBPl-BRCT7 / 8 interaction with PLK1 by 5D4.

[0112] It is worth noting that both CalAM and 5D4 do not affect the epithelial cell proliferation of mouse intestines, indicating a specific anticancer effect of TopBP 1-BRCT7 / 8 inhibitors. MYC is the most frequently amplified gene in human cancers. However, direct inhibition of MYC is widely recognized as a challenge due to its disordered structure and lack of targetable pockets.

[0113] Applicant’s study uncovers an alternative approach to target MYC with small molecule inhibitors against TopBP 1-BRCT7 / 8. Applicant’s SAR analysis and molecular docking identify a pS / TxxxQ motifbinding pocket in TopBP 1-BRCT7 / 8 as a targetable site. Both MYC and TopBPl compete for MIZ1 binding.

[0114] Thus, overexpressed TopBPl might sequester MIZ1 and tip the balance toward MYC activation. In fact, TopBPl expression is highly correlated with MYC target gene signature in many types of cancer (TCGA datasets). Accordingly, small molecule inhibitors that bind to TopBPl - BRCT7 / 8 might release MIZ1 from TopBPl complex, allowing it to bind and repress MYC activity in cancer.

[0115] Together with the effects of TopBPl-BRCT7 / 8 inhibitors on the activation of E2F1- mediated apoptosis and inhibition of mutp53 GOF, these events contribute to the anticancer activities of TopBP 1-BRCT7 / 8 inhibitors. Without being bound by theory, the synergy between TopBPl- BRCT7 / 8 inhibitors and PARPi may be attributed to several mechanisms. First, given a role of TopBP 1-BRCT7 / 8 in Rad51 recruitment, TopBP 1-BRCT7 / 8 inhibitors are able to prevent Rad51 foci formation and thus synergize with PARPi through the inhibition of HR. Second, many proteins involved in DNA repair are MYC targets, such as Rad51, PLK1, and ACTL6A. Indeed, TopBPl-BRCT7 / 8 inhibitors can down-regulate the expression of these proteins, thereby enhancing the drug synergism with PARPi. Moreover, 5D4 can overcome acquired PARPi resistance in BRCA1 -mutated breast cancer harboring DYNLL1 loss. DYNLL1 suppresses the activity of several components of the endresection machinery involved in HR, including the MRN complex. Thus, loss of DYNLL1 in B / ?C4 / -mutated cells enhances end-resection, and restores Rad51 foci formation and HRmediated DSB repair.

[0116] Since 5D4 inhibits Rad51 expression and recruitment, it can prevent DYNLL1 loss- mediated HR restoration and overcome PARPi resistance in BRCA1 -mutated cancer. Applicant’s study further extends the synergy of TopBP 1-BRCT7 / 8 inhibitors with PARP14 inhibitors. It has been shown that defective DNA replication is responsible for the synthetic lethal interaction between PARP14 and either ATR or TopBPl. PARP14 is involved in the processing of the stalled replication forks. Unlike PARP1 / 2, PARP14 is a mono-ADPribosyltransferase. While PARPI, 2 and 14 are all involved in the repair of DNA damage, especially on the replication forks, PARP14 also plays a role in cell structure, adhesion, motility and innate immunity. In addition, PARP14 is required for Cyclin DI 3’UTR mRNA stability and Cyclin DI expression.

[0117] Indeed, combined treatment with 5D4 and PARPI 4 inhibitor GeA-69 synergistically decreased the levels of c-MYC, Cyclin DI, ACTL6A and Rad51. Thus, it is possible that the synergy between TopBP 1-BRCT7 / 8 inhibitors and PARP14 inhibitors also involves multiple mechanisms.

[0118] In summary, this Example identifies a novel inhibitor of TopBP 1-BRCT7 / 8 and demonstrates its anticancer activity and synergy with PARP inhibitors in HR-proficient cancer cells. These data support the potential use of TopBPl -BRCT7 / 8 inhibitors for targeted cancer therapy.

[0119] Example 2. Pharmacokinetic studies of 5D4 in mice

[0120] This Example provides various pharmacokinetic (PK) studies of 5D4 in mice. The goal of these studies were to (1) develop a specific, sensitive, and reproducible liquid chromatography (LC) and double mass spectrometry (MS) (LC-MS / MS) method to quantitate 5D4 concentrations in mouse blood; (2) quantitate concentrations of 5D4 in more than 76 mouse blood samples and in the dosing solutions for PK studies; and (3) perform PK animal experiments following intraperitoneal (IP) administration of 5D4 in 2 mice (preliminary tests) and oral (PO) administration in 4 mice (C57 BL / 6).

[0121] Example 2.1. LC-MS / MS Quantification of 5D4 in Mouse Blood

[0122] Example 2.1.1. Preparation of standard samples. A stock solution of 5D4 was prepared by dissolving 50 mg of 5D4 in 2 mL of DMSO to achieve a concentration at 25 mg / mL. Working solutions were prepared by dilution with acetonitrile from the stock to the concentrations of 5, 10, 50, 100, 500, 1000, and 2500 ng / mL. Each standard sample was prepared by spiking respective standard working solutions into blank mouse blood at a volume ratio of 1 to 10 (2 pL of working solution and 18 pL of blank mouse blood were mixed). Accordingly, the effective standard sample concentrations in the blood were 0.5, 1, 5, 10, 50, 100, and 250 ng / mL.

[0123] Example 2.1.2. Blood sample extractions. The blood samples were extracted by protein precipitation using 7X volume of acetonitrile containing IS (2.5 ng / mL of warfarin). After mixing, the samples were vortexed for 3 minutes followed by 20 minutes centrifugation at 14,000 rpm, 40C. An aliquot of the supernatant was injected onto the LC-MS / MS for analysis. For standard curve samples, 20 pl of each spiked standard blood sample was precipitated with 140 pL of acetonitrile containing IS. For unknown blood samples, 10 pL of each unknown blood sample was precipitated with 70 pL of acetonitrile containing IS.

[0124] Example 2.1.3. LC-MS / MS Methods. The assay was performed on a 6500+ Triple Quad LC-MS / MS System (SCIEX) with a KINETEX® PS C18 column (100 x 2.1 mm, 2.6pm, 100A, Phenomenex Inc.). The optimized method used binary gradient mobile phases with water containing 2 mM ammonium formate and 0.01% formic acid as mobile phase A and acetonitrile as mobile phase B. A flow rate of 0.5 mL / min was used with 2 pL of injection volume. Running time was 7.5 min. The time program of the gradient was as follows: Phase B was initially kept at 20% for 0.5 min, increased from 20% to 80% in the following 2.5 min for drug elution, and then increased from 80% to 95% in 0.1 min to wash for 2.5 min; finally reduced to 20% in 0.1 min to equilibrate for 2 min.

[0125] Mass spectrometry data were recorded using ESI, positive ion detection, and MRM scanning. The ion spray voltage and temperature were set at 5500 V and 450°C, respectively. The curtain gas was set at a flow rate of 45 psi; gas 1 and gas 2 were set at 70 and 50 psi. CAD was set at 9. Data was acquired by Analyst software 1.6.3. The MS / MS parameters for 5D4 and internal standard (IS) warfarin (WFN) are shown in Table 1.QI (m / Z) Q3 (m / Z) DP EP CE CXP5D4 467.200 121.140 41 10 61 891.000* 41 10 103 10WFN 309.100 251.000 41 10 29 10Table 1. Compound dependent parameters for 5D4 and IS. QI = Parent ion, Q3 = Product ion, DP = Declustering Potential, EP = Entrance Potential, CE = Collision Energy, and CXP = Collision Cell Exit Potential. WFN: warferin as internal standard *: transition used for quantification.

[0126] A typical calibration curve is shown in FIG. 11. MRM chromatograms are shown in FIGS. 12A1-C2.

[0127] Example 2,2, Pharmacokinetic (PK) Studies

[0128] Pharmacokinetic studies were conducted following intraperitoneal (IP) and oral administration of 5D4 in mice. The results are summarized in Tables 2 and 3, respectively.Table 2. Pharmacokinetic study data following IP administration of 5D4 in mice. *Mouse A and Mouse B were preliminary studies. An apparent sustained release / absorption was observed in both mouse A and B following the IP drug administration. There was a faster and increased absorption of 5D4 following the administration of the co-solvent drug formulation in mouse B.Table 3. Pharmacokinetic study data following PO (oral) administration of 5D4 in C57BL / 6 mice.* Mouse C was preliminary study.

[0129] Example 2.3. Pharmacokinetic Analysis

[0130] Table 4 summarizes PK parameters of 5D4 (dissolved in 100% DMSO) after 40 mg / kg IP administration of 5D4 in a mouse. Following 40 mg / kg IP administration (10 mg / mL of 5D4 dissolved in DMSO), 5D4 was continuously absorbed with a maximum blood drug concentration of 1255 ng / mL at 24 hours post dose. The blood drug concentration levels sustained for almost two (2) weeks with an observed 27.6 ng / mL at 312 hours post dose. It was suspected that the administered drug might have precipitated at the administration site after DMSO was absorbed and the drug particles might have continued to release to the systemic circulation over a long period which had generated significant areas under the curve (AUC). Thus, the observed terminal elimination half-life was not considered the true half-life of 5D4, and rather the absorption half-life of the precipitated drug particles.Table 4. PK parameters of 5D4 (dissolved in 100% DMSO) after 40 mg / kg IP administration of 5D4 in a mouse.

[0131] Table 5 summarizes PK parameters of 5D4 (dissolved in a co-solvent formulation) after 40 mg / kg IP administration of 5D4 in a mouse. Following 40 mg / kg IP administration (5 mg / mL of 5D4 dissolved in a co-solvent formulation containing 20% DMSO), 5D4 was rapidly absorbed witha maximum blood drug concentration of 4538 ng / mL at 30 min post dose. Similar to the 100% DMSO formulation, there were sustained high blood 5D4 levels at 48 hours post dose, but at a lesser extent as compared with that in mouse A (Table 4). The observed AUC level in mouse B (Table 5) was lower than that in mouse A. This was because of continued drug absorption. However, sampling stopped at 48 hours in mouse B versus 312 hours in mouse A.Table 5. PK parameters of 5D4 (dissolved in a co-solvcnt formulation) after 40 mg / kg IP administration of 5D4 in a mouse.

[0132] Table 6 summarizes PK parameters of 5D4 after 40 mg / kg oral administration of 5D4 in mice. Following 40 mg / kg oral administration (5 mg / mL of 5D4 dissolved in a co-solvent formulation containing 20% DMSO), 5D4 was continuously absorbed with a maximum blood drug concentration of 1266 ng / mL at 5.5 hours post dose. The mean terminal elimination half-life of the drug appeared to be of 26.1 hours. The mean AUC was 61.5 hr*pg / mL. An estimated relative bioavailability of the oral versus IP (in a co- solvent formulation containing 20% DMSO) was about 62.8%.Table 6. PK parameters of 5D4 after 40 mg / kg oral administration of 5D4 in mice.[001331 Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein arc hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

CLAIMS1. A method of treating or preventing a cancer in a subject, said method comprising: administering to the subject a therapeutically effective amount of a composition, wherein the composition comprises a topoisomerase II[3-binding protein 1 (TopBPl ) inhibitor, wherein the TopBPl inhibitor comprises a structure selected from the group consisting of:derivatives thereof, or combinations thereof, wherein Ri is selected from the group consisting of polycyclic aromatic hydrocarbons, naphthalene, biphenyl, fluorene, anthracene, phenanthrene, phcnalcnc, derivatives thereof, or combinations thereof, wherein R2 is selected from the group consisting of furan groups, phenyl groups, benzoyl groups, methoxybenzene, methoxybenzene amine, 4-methoxylphenyl, 4-(dimethylamino) benzamide, pyridine, 4-methoxyaniline, thiophene, methylamine, dimethylamine, propylamine, anisole, aniline, toluene, morpholine, phenetole, 4-methoxyacetophenone, tert-butylamine, 1 ,2- dimethoxybenzene, 2-methylformanilide, derivatives thereof, or combinations thereof, wherein X comprises NH or NH2, wherein Y comprises NH, NH2, or O, and wherein Z comprises O or S.

2. The method of claim 1, wherein Ri comprises naphthalene.

3. The method of claim 1, wherein the TopBP 1 inhibitor is selected from the group consisting ofderivatives thereof, or combinations thereof.

4. The method of claim 1, wherein the TopBP 1 inhibitor comprises the following structure:

5. The method of claim 1, wherein the composition further comprises a poly (ADP-ribose) polymerase (PARP) inhibitor.

6. The method of claim 5, wherein the PARP inhibitor is selected from the group consisting of talazoparib, rucaparib, olaparib, veliparib, niraparib, pamiparib, GeA-69, RBN-3143, RBN012759, or combinations thereof.

7. The method of claim 1, wherein the administering occurs by a method selected from the group consisting of oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, intrathecal injection, intra-articular administration, topical administration, central administration, peripheral administration, aerosol-based administration, nasal administration, transmucosal administration, transdermal administration, parenteral administration, or combinations thereof.

8. The method of claim 1, wherein the administering occurs by intraperitoneal administration.

9. The method of claim 1, wherein the administering occurs by oral administration.

10. The method of claim 1, wherein the subject is a human being.11 . The method of claim 1 , wherein the subject is suffering from the cancer.

12. The method of claim 1, wherein the subject is vulnerable to the cancer.

13. The method of claim 1, wherein the cancer comprises breast cancer, ovarian cancer, lung cancer, a cancer associated with TopBPl over-expression, or combinations thereof.

14. A composition comprising a topoisomerase lip-binding protein 1 (TopBPl) inhibitor, wherein the TopBPl inhibitor comprises a structure selected from the group consisting of:derivatives thereof, or combinations thereof, wherein Ri is selected from the group consisting of polycyclic aromatic hydrocarbons, naphthalene, biphenyl, fluorene, anthracene, phenanthrene, phenalene, derivatives thereof, or combinations thereof, wherein R2 is selected from the group consisting of furan groups, phenyl groups, benzoyl groups, methoxybenzene, methoxybenzene amine, 4-methoxylphenyl, 4-(dimethylamino) benzamide, pyridine, 4-methoxyaniline, thiophene, methylamine, dimethylamine, propylamine, anisole, aniline, toluene, morpholine, phenetole, 4-methoxyacetophenone, tert-butylamine, 1,2- dimethoxybenzene, 2-methylformanilide, derivatives thereof, or combinations thereof, and wherein X comprises NH or NH2, wherein Y comprises NH, NH2 or O, and wherein Z comprises O or S.

15. The composition of claim 14, wherein Ri comprises naphthalene.

16. The composition of claim 14, wherein the TopBPl inhibitor is selected from the group consisting ofderivatives thereof, or combinations thereof.

17. The composition of claim 14, wherein the TopBPl inhibitor comprises the following structure:

18. The composition of claim 14, wherein the composition further comprises a poly (ADP-ribose) polymerase (PARP) inhibitor.

19. The composition of claim 18, wherein the PARP inhibitor is selected from the group consisting of talazoparib, rucaparib, olaparib, veliparib, niraparib, pamiparib, GeA-69, RBN-3143, RBN012759, or combinations thereof.

20. The composition of claim 14, wherein the composition is suitable for use in treating or preventing a cancer in a subject.

21. The composition of claim 20, wherein the subject is a human being.

22. The composition of claim 20, wherein the cancer comprises breast cancer, ovarian cancer, lung cancer, a cancer associated with TopBP 1 over-expression, or combinations thereof.

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