WO2019232214A1

WO2019232214A1 - Methods of using napabucasin

Info

Publication number: WO2019232214A1
Application number: PCT/US2019/034658
Authority: WO
Inventors: Harry Rogoff
Original assignee: Boston Biomedical Inc
Current assignee: Sumitomo Pharma Oncology Inc
Priority date: 2018-05-31
Filing date: 2019-05-30
Publication date: 2019-12-05
Anticipated expiration: 2020-11-30

Abstract

Methods of using napabucasin to treat cancer that expresses NQO1, methods of detecting NQO1, and methods for determining a subject likely to respond to treatment with napabucasin are provided herein.

Description

METHODS OF USING NAPABUCASIN

There are many different genetic defects in mammalian or human cancer cells, and many have been studied in the quest to cure cancer. For example, many cancers have been shown to have increased reactive oxygen species (ROS) levels in comparison to normal tissues.

ROS are radicals, ions, or molecules that are highly reactive due to having single unpaired electrons. ROS can comprise a free oxygen radical group, including but not limited to superoxide and hydroxyl radicals, and a non-radical ROS group, including but not limited to hydrogen peroxide and highly reactive lipid- or carbohydrate- derived carbonyl groups. Different mechanisms in the cell can produce ROS, including but not limited to increased metabolic activity, mitochondrial dysfunction, increased cellular receptor signaling, increased activity of oxidases, and cross-talk with infiltrating immune cells. See Liou, G., et al., “Reactive oxygen species in cancer.” NIH Public Access Author Manuscript. 2010; 44(5): 1- 31.

Low levels of ROS can act as cellular signaling messengers, e.g., by oxidizing proteins; however, higher levels of ROS can disrupt cellular processes by attacking proteins, lipids, and DNA. See Schumacher, P.T.,“Reactive Oxygen Species in Cancer: A Dance with the Devil.” Cancer Cell. 2015; 27(2): 156-157. Under normal conditions, the intracellular levels of ROS are steadily maintained to prevent damage within the cell and systems are in place to facilitate the detoxification from ROS, including non-enzymatic molecules such flavonoids or gluthiones, or antioxidant enzymes which scavenge different kinds of ROS. For example, catalases facilitate the decomposition of hydrogen peroxide to water and oxygen. One explanation for why some cancers have increased ROS levels is that as cells become cancerous, they increase their metabolic activities to drive proliferation and survival, resulting in the production of high levels of ROS. Growth factors and cytokines may also drive the production of ROS in cancer cells. Additionally, as many cancers start at sites of inflammation, infiltrating leukocytes can induce the generation of ROS within tumor cells. See Liou, G., et al.

Although some studies have shown that ROS-driven oxidant stress promotes tumorigenesis, other studies have shown that oxidative stress inhibits tumorigenesis.

NAD(P)H-Quinone Oxidoreductase 1 ( NQQ1 )

NQOl is a xenobiotic metabolizing/ anti oxidant enzyme that uses NADH or NADPH as an electron donor for enzymatic activity and that detoxifies chemical stressors, providing cytoprotection in normal tissues. High levels of NQOl expression have been found in numerous human malignancies, suggesting a role in tumorigenesis and chemoresistance. One explanation is that this adaptation allows cancer cells to survive in a relatively high oxidative stress condition compared to normal cells, protecting these cancer cells from toxic action of chemotherapeutic agents. Thus, cancer cells may take advantage of the over-activation of an antioxidant defense, in particular antioxidant genes such as NQOl. See Srijiwangsa, P., et al,“Roles of NAD (P) H-Quinone Oxidoreductase 1 (NQOl) On Cancer Progression and Chemoresistance.” Journal of Clinical & Experimental Oncology. 2017, 6:4.

NQOl acts by catalyzing the reduction of quinones to their hydroquinone forms. There are a number of other enzymes that can catalyze reduction of quinones including cytochrome P450 reductase, cytochrome b5 reductase, NAD(P)H-quinone oxidoreductase 2 (NQ02), carbonyl reductases, and thioredoxin reductase. The two-electron reduction of quinones to hydroquinone by NQOl has been considered a detoxification mechanism because this reaction is thought to by-pass the formation of the highly reactive semiquinone. However, whether the formulation of the hydroquinone is a detoxification reaction or an activation reaction, depends upon the chemical reactivities of the quinone and hydroquinone. There are examples of naturally occurring and synthetic quinones that, following reduction to their corresponding hydroquinones, induce toxicity. Unstable hydroquinones can undergo chemical rearrangements leading to alkylation of essential biomolecules like DNA or undergo redox reactions leading to the formation of highly reactive oxygen species (ROS). For example, NQOl has been found to catalyze the redox cycling of b-lapachone, a naturally occurring ortho napthoquinone. This reaction generates an unstable hydroquinone, which under aerobic conditions is rapidly oxidized back to the parent quinone. Redox cycling of b- lapachone is characterized by the generation of ROS including superoxide and hydrogen peroxide. See Siegel, D., et al,“NAD(P)H: Quinone Oxidoreductase 1 (NQOl) in the Sensitivity and Resistance to Antitumor Quinones.” NIH Public Access Author Manuscript. Biochem Pharmacol. 2012; 83(8): 1033-1040.

Cytochrome P450 Oxidoreductase (POR)

Cytochrome P450 Oxidoreductase (POR) is an electron transfer protein that is involved in cytochrome P450-mediated drug metabolism. Recent studies with POR-null and cytochrome b5-null tissues have revealed the presence of additional microsomal redox enzymes that may substitute for or augment POR and b5- dependent pathways. See Porter, T.D.,“New insights into the role of cytochrome P450 reductase (POR) in microsomal redox biology.” Acta Pharmaceutica Sinica B. 20l2;2(2): 102-106. Although POR has been reported to act as a source of reactive oxygen species (ROS), it has not been implicated in pathophysiological conditions associated with oxidative stress. See Pillai, V.C., et al, “Effects of transient overexpression or knockdown of cytochrome P450 reductase on reactive oxygen species generation and hypoxia reoxygenation injury in liver cells.” Clinical and Experimental Pharmacology and Physiology. 2011; 38(l2):846-853.

STAT3

The p53 tumor suppressor has been found to be defective or altogether absent in more than half of the human cancers. The STAT (Signal Transducers and Activator of

Transcription) protein family are latent transcription factors activated in response to cytokines/growth factors to promote proliferation, survival, and other biological processes. Among them, STAT3 is activated by phosphorylation of a critical tyrosine residue mediated by growth factor receptor tyrosine kinases, Janus kinases, or the Src family kinases, etc.

These kinases include, but are not limited to EGFR, JAKs, Abl, KDR, c-Met, Src, and Her2. See Yu, H.“Stat3: Linking oncogenesis with tumor immune evasion. AACR 2008 Annual Meeting. 2008. San Diego, CA. Upon tyrosine phosphorylation, STAT3 forms homo-dimers, translocates to the nucleus, binds to specific DNA-response elements in the promoter regions of the target genes, and induces gene expression. See Pedranzini, L., A. Leitch, and J.

Bromberg,“Stat3 is required for the development of skin cancer.” J Clin Invest,

2004;l 14(5):619-22.

In normal cells, STAT3 activation is transient and tightly regulated, lasting from 30 minutes to several hours. However, STAT3 is found to be aberrantly active in a wide variety of human cancers, including all the major carcinomas as well as some hematologic tumors. STAT3 plays multiple roles in cancer progression. As a potent transcription regulator, it targets genes involved in many important cellular functions, such as Bcl-xl, c-Myc, cyclin Dl, Vegf, MMP-2, and survivin. See Catlett-Falcone, R., et al,“Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity.

1999; 10(1): 105-15. See also Bromberg, J.F., et al,“Stat3 as an oncogene.” Cell, 1999.

98(3): 295-303. See also Kanda, N., et al,“STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells.” Oncogene.

2004;23(28):492l-9. See also Schlette, E.J., et al,“Survivin expression predicts poorer prognosis in anaplastic large-cell lymphoma.” J Clin Oncol. 2004;22(9): 1682-8. See also Niu, G., et al,“Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis.” Oncogene. 2002;2l(l3):2000-8. See also Xie, T.X., et al,“Stat3 activation regulates the expression of matrix metalloproteinase-2 and tumor invasion and metastasis.” Oncogene. 2004;23(20):3550-60. STAT 3 has also been implicated in the regulation of oxidative stress in cells by an increase in antioxidant factors. See Poll, V., et al,“STAT3- mediated metabolic reprograming in cellular transformation and implications for drug resistance.” Frontiers in Oncology. 2015;5:121. Moreover, STAT3 is also a key negative regulator of tumor immune surveillance and immune cell recruitment. See Kortylewski, M., et al,“Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity.” Nat Med. 2005;l 1(12): 1314-21. See also Burdelya, L., et al,“Stat3 activity in melanoma cells affects migration of immune effector cells and nitric oxide- mediated antitumor effects,” J Immunol. 2005;l74(7):3925-3l. See also Wang, T., et al, “Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells.” Nat Med. 2004;l0(l):48-54.

In diseases other than cancer, over-activation of STAT3 by Interleukin 6 (IL6) has been demonstrated in a number of autoimmune and inflammatory diseases. See Campbell, I.L.,“Cytokine-mediated inflammation, tumorigenesis, and disease-associated

JAK/STAT/SOCS signaling circuits in the CNS.” Brain Res Rev. 2005;48(2): 166-77. The STAT3 pathway also promotes pathologic immune responses through its essential role in generating TH17 T cell responses. See Harris, T.J., et al,“Cutting edge: An in vivo requirement for STAT3 signaling in TH17 development and TH17-dependent

autoimmunity.” J Immunol. 2007;l79(7):43l3-7. In addition, IL6-STAT3 pathway mediated inflammation has been found to be the common causative origin for atherosclerosis, peripheral vascular disease, coronary artery disease, hypertension, osteoporosis, type 2 diabetes, and dementia.

Ablating Stat3 signaling by antisense, siRNA, a dominant-negative form of Stat3, and/or blockade of tyrosine kinases inhibits certain cancer cell lines or tumors in vitro and/or in vivo. See Pedranzini, L., A. Leitch, and J. Bromberg. See also Bromberg. J.F.. et al. See also Darnell, J.E.,“Validating Stat3 in cancer therapy. Nat Med. 2005;l l(6):595-6. See also Zhang, L., et al,“Intratumoral delivery and suppression of prostate tumor growth by attenuated Salmonella enterica serovar typhimurium carrying plasmid-based small interfering RNAs.” Cancer Res. 2007;67(l2):5859-64. STAT3 plays a role in both the survival and self renewal capacity of cancer stem cells (CSCs) across a broad spectrum of cancers. Napabucasin (2-acetylnaphtho[2,3-b]furan-4,9-dione), a quinone, is also capable of inhibiting the STAT3 pathway and of killing tumor cells across different types of cancer. See US Patent No. 9,745,278. Additionally, in a randomized phase III clinical trial of patients with advanced colorectal cancer, napabucasin demonstrated a beneficial effect in the overall survival of patients who expressed high levels of pSTAT3 in their tumor and stromal tissue in comparison to patients with low pSTAT3 levels in their tumors. See Jonker DJ, Nott L, Yoshino T, et al,“Napabucasin versus placebo in refractory advanced colorectal cancer: a randomized phase 3 trial.” Lancet Gastroenterol Hepatol. 20l8;3(4):263-270. The presence of activated STAT3 in a cancer may indicate that the cancer will be responsive to treatment with napabucasin.

However, even cancers with low or no pSTAT3 may be responsive to treatment with napabucasin. Thus, there is a need to provide methods that indicate that a cancer is responsive to treatment with napabucasin.

Provided herein are methods of treating cancer in a human subject in need thereof comprising administering to the human subject a composition comprising a therapeutically effective amount of napabucasin (2-acetylnaphtho[2,3-b]furan-4,9-dione), wherein the cancer expresses NQOl. Methods for detecting the presence of NQOl and diagnosing a cancer that is responsive to napabucasin are also provided herein. Also provided herein are methods for producing at least one reactive oxygen species in cancer cells comprising administering to a human subject in need thereof a composition comprising a therapeutically effective amount of napabucasin, wherein the cancer expresses NQOl.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A depicts enzymatic activity assays for mouse and human NAD(P)H- Quinone Oxidoreductase 1 (NQOl) in the presence of various substrates including napabucasin (BBI-608). Figure IB compares the catalytic efficiency and selectivity of human NQOl for napabucasin and b-lapachone.

Figure 2 depicts an assay for ROS generation and cell viability in NQOU cell lines BxPC3 and PACA2, and NQOl cell line PANC1 upon exposure to napabucasin (BBI608).

Figure 3A depicts an assay measuring the generation of ROS in FaDu parental cells and NQOl -knockout cells incubated with napabucasin (BBI608). Figure 3B depicts an assay measuring the generation of ROS in A549-WT (NQOU) cells and A549-NQOl^CR (NQOU) cells incubated with napabucasin (BBI608). Figure 4A depicts an assay comparing cell viability between FaDu parental cells and NQOl knockout cells incubated with napabucasin (BBI608). Figure 4B depicts an assay comparing cell viability between A549-WT (NQOl⁺) cells and A549-NQOl^CR (NQOl ) cells incubated with napabucasin (BBI608).

Figure 5 depicts cell viability assays for different cell lines comparing the effects of napabucasin (BBI608) treatment in cells expressing high levels of NQOl (NQOl⁺/⁺) and cells expressing low levels of NQOl (NQOl / ). NQOl is overexpressed in NQOl^+/+ MDA- MB-231 and PANC1 cells, while NQOl has been silenced with shRNA in NQOl H596 and A549 cells.

Figure 6A depicts a cell viability assay comparing the effects of napabucasin (608) treatment in parental cells (Mock) in contrast to cells in which the expression of catalase is silenced (siCA). IC50 values are shown. Figure 6B depicts a cell viability assay for A549 cells treated with napabucasin (608) alone or in combination with EUK-134.

Figure 7A depicts a western blotting assay detecting the levels of pSTAT3, STAT3, NQOl and tubulin (control) for cells deficient in NQOl (MDAMB231) and cells

overexpressing NQOl (MDAMB231-NQ01) treated with DMSO or napabucasin (608) at a final concentration of 0.5 uM or 1 uM. Figure 7B depicts an electrophoretic mobility shift (EMSA) assay showing the binding of STAT3 to DNA in cells deficient in NQOl (MM231) and cells overexpressing NQOl (MM231-Q and HeLa) in the presence of DMSO (control), Oncostatin M (OSM), napabucasin (BBI-608), dicoumarol (Die), and combinations thereof. Figure 7C depicts an EMSA assay showing the binding of STAT3 to DNA in HeLa cells expressing NQOl and treated with DMSO (control), Oncostatin M (OSM), napabucasin (BBI-608), Dicoumarol (Die), EUK-134, beta-Lapachone (b-Lapachone) and combinations thereof.

Figure 8A depicts an enzymatic activity assay for Cytochrome P450 reductase (POR) in the presence of various substrates including napabucasin (BBI608). Figure 8B depicts an assay measuring the production of ROS in NQOl -deficient cells treated with Napabucasin (608) and silenced for the expression of various reductases including NQOl, NAD(P)H- Quinone Oxidoreductase 2 (NQ02), Ferredoxin reductase (FDXR), Cytochrome b5 reductase 1 (CYB5R1), Cytochrome b5 reductase 3 (CYB5R3), Cytochrome b5 reductase 4 (CYB5R4), Thioredoxin reductase 1 (TXNRD1), and Carbonyl Reductase 1 (CBR1). Figure 8C depicts a cell viability assay showing the effects of silencing the expression of various reductases in NQOl -deficient cells treated with napabucasin (608). IC50 values are shown. Figure 8D shows the napabucasin-induced death of cells in which POR expression has been silenced.

Figure 9A illustrates results from the CRC CO.23 monotherapy Phase III trial. The left plot shows overall survival rates in patients with pSTAT3 negative and pSTAT3 positive tumors, and suggests that pSTAT3 expression is a poor prognostic marker. The center and right plots show the results in patients whose tumor cells presented a pSTAT3 staining level > 5% or whose tumor microenvironments presented a pSTAT3 staining score of 2 or higher. Figure 9B shows napabucasin activity when STAT3 is knocked down or overexpressed.

Figure 10 depicts a human proteome cytokine profile array assay detecting secreted proteins present in the supernatant ofNQOl⁺ FaDu parental cells or NQOl^CR FaDu cells (koNQOl (Al); koNQOl(Dl6); and koNQOl (G6)) cultured alone or co-cultured with cancer associated fibroblasts (CAF) or Normal Fibroblasts (NF). Each pair of dots corresponds to each secreted protein being spotted twice on the array membrane. Rectangular boxes indicate NQOl -dependent secreted proteins.

Figure 11 depicts western blotting assays detecting the levels of pSTAT3, STAT3, NQOl and actin (control) in CAFs and NFs treated with the indicated recombinant proteins, and Osmostatin M as a positive control for pSTAT3. Diluent was used as a control for basal pSTAT3 levels. FaDu cell lysate was used as a positive control for pSTAT3.

Figure 12 illustrates a flow cytometry analysis of human peripheral blood mononuclear cells (PBMCs). Forward versus side scatter (FSC vs. SSC) gating identified distinct lymphocyte and monocyte populations.

Figures 13A-13C illustrate the levels of pSTAT3 measured by flow cytometry in PBMCs treated with the recombinant proteins indicated on the x-axis. Figure 13A illustrates the pSTAT3 levels in the total live cell populations. Figure 13B illustrates the pSTAT3 levels in the lymphocyte populations. Figure 13C illustrates the pSTAT3 levels in the monocyte populations and a flow cytometry analysis demonstrating the separation of the CD3⁺ monocyte population from human PBMCs using CD3 staining. Cells were co-stained for pSTAT3 and CD3 and stimulated with GM-CSF. Diluent was used as a control for the basal pSTAT3 levels. The pSTAT3-PE mean fluorescent intensity (MFI) was normalized to the MFI for diluent-only cells for each cell population.

Figure 14 illustrates a flow cytometry analysis demonstrating the separation of the CD3⁺ lymphocyte population from human PBMCs using CD3 staining. Cells were co-stained for pSTAT3 and CD3.

Figure 15A illustrates the levels of pSTAT3 measured by flow cytometry in CD3⁺ lymphocytes treated with the recombinant proteins indicated on the x-axis and flow cytometry analyses demonstrating the separation of recombinant protein stimulated CD3⁺ lymphocyte populations from human PBMCs using CD3 staining. Cells were co-stained for pSTAT3 and CD3 and stimulated with IL6 and CXCL10. Figure 15B illustrates the levels of pSTAT3 measured by flow cytometry in CD3 lymphocytes treated with the recombinant proteins indicated on the x-axis. Diluent was used as control for the basal pSTAT3 level in cells. The pSTAT3-PE mean fluorescent intensity (MFI) was normalized to the MFI for diluent-only cells for each population.

Figures 16A-16C depict the quantification of secreted IL6, GM-CSF and CXCL10 measured by a Luminex-based ELISA in NQOl⁺ FaDu parental cells or NQOl knockout cells (koNQOl Al, koNQOl D16, koNQOl G6) co-cultured with CAFs. Figure 16A depicts the quantification of secreted IL6. Figure 16B depicts the quantification of secreted GM- CSF. Figure 16C depicts the quantification of secreted CXCL10.

Figures 17A-17C depict the quantification of secreted IL6, GM-CSF and CXCL10 measured by a Luminex-based ELISA in NQOl⁺ A549 wild type cells or NQOl knockout A549 cells (koNQOl Al) co-cultured with CAFs. Figure 17A depicts the quantification of secreted IL6. Figure 17B depicts the quantification of secreted GM-CSF. Figure 17C depicts the quantification of secreted CXCL10.

Figure 18 illustrates the H-score distribution assigned to tumor specimens stained with an anti-NQOl antibody in an immunohistochemistry (IHC) assay.

Figure 19 depicts images from an NQOl IHC assay showing the range of NQOl staining in different tumor specimens.

Figure 20 depicts images of tumor single-cell type and multi-cell type spheroids consisting of fibroblasts (either CAF or NF) and FaDu cell lines (either the NQOl⁺ parental line or a NQOl^CR knockout line). Spheroids were generated using a U-bottom plate.

Figure 21 depicts cell viability and ROS generation assays performed on spheroids after incubation with varying amounts of BBI608 (Napabucasin). Single-cell type spheroids consisting of NF, CAF, parental FaDu (NQOl⁺) or FaDu-NQOl^CR or multi-cell type spheroids consisting of a cancer cell line and a fibroblast type were incubated with varying amounts of BBI608, and cell viability and ROS generation were determined at each concentration of BBI608.

Figures 22A-22C depict the quantification of IL6, GM-CSF, and CXCL10 proteins secreted from cocultures of a FaDu cell line (either FaDu parental cells (NQOl⁺) or FaDu- NQOl^CR Al (NQOl knockout)) and fibroblasts (either SMC or CAF), in either 2D or spheroid (3D) culture conditions at varying ratios of FaDu cells to fibroblast cells. Figure 22A depicts the quantification of secreted IL6. Figure 22B depicts the quantification of secreted GM-CSF. Figure 22C depicts the quantification of secreted CXCL10.

Figures 23A-23C depict the quantification of IL6, GM-CSF, and CXCL10 proteins secreted from cocultures of an A549 cell line (either A549-WT (NQOl⁺) or A549-NQOl^CR (NQOl knockout)) and fibroblasts (either SMC or CAF), in either 2D or spheroid (3D) conditions at varying ratios of A549 cells to Fibroblast cells. Figure 23A depicts the quantification of secreted IL6. Figure 23B depicts the quantification of secreted GM-CSF. Figure 23C depicts the quantification of secreted CXCL10.

Figure 24 depicts western blotting assays detecting the levels of pSTAT3, STAT3, NQOl and actin (control) in CAF and SMC (NF) cells treated with the indicated amounts of recombinant IL6 protein. Fibroblasts were grown in DMEM media supplemented with either 0.5 or 10% DMEM. Diluent was used as a control for basal pSTAT3 levels.

Figure 25 depicts western blotting assays detecting the levels of pSTAT3, STAT3, NQOl and actin (control) in tumor cells treated with the indicated amounts of recombinant IL6 protein. A549 cells with (A549-WT) and without (A549-NQOl^CR) NQOl were treated, as were FaDu cells with (FaDu) and without (FaDu-NQOl^CR) NQOl. Cells were grown in DMEM media supplemented with either 0.5 or 10% DMEM. Diluent was used as a control for basal pSTAT3 levels.

Figures 26A-26G depict representative immunofluorescence images and image analysis of multi-cell type spheroids probed for NQOl, Vimentin, and pSTAT3. Images were obtained of multi-cell type spheroids consisting of an A549 cell line (either A549-WT (NQOl⁺) or A549-NQOl^CR (NQOl knockout)) and a fibroblast type (either SMC or CAF) at varying ratios of A549 cells to fibroblast cells. Shown below each set of images in figures 26A-F are graphs quantifying the fluorescent intensity of cells screened under each condition. NQOl (high in A549-WT, null in A549-NQOl^CR) and Vimentin (high in fibroblast cells) signal intensities were used to identify tumor cells versus fibroblast cells by a K-mean clustering method. Nucleus pSTAT3 signal intensity was plotted for each cell in the spheroid. Figure 26A displays data obtained from CAF containing spheroids, at a ratio of A549:CAF cells of 1 :4. Figure 26B displays data obtained from SMC containing spheroids, at a ratio of A549:SMC cells of 1:4. Figure 26C displays data obtained from CAF containing spheroids, at a ratio of A549:CAF cells of 1 : 1. Figure 26D displays data obtained from SMC containing spheroids, at a ratio of A549:SMC cells of 1 : 1. Figure 26E displays data obtained from CAF containing spheroids, at a ratio of A549:CAF cells of 2: 1. Figure 26F displays data obtained from SMC containing spheroids, at a ratio of A549:SMC cells of 2: 1. Figure 26G displays representative immuno-fluorescence images of A549 and CAF 3D coculture spheroids, cocultured at a ratio of A549:CAF cells of 1 :4. The graph shows the combined data of at least 8 images, representing over 700 analyzed cells. The difference in pSTAT3 levels between spheroids containing NQOl⁺ cancer cells and spheroids containing NQOl cancer cells is statistically different with pO.OOOl.

Figure 27 depicts representative immunofluorescence images and image analysis of FaDu and CAF 3D coculture spheroids and a graph showing immunofluorescence image analysis results. NQOl (high in FaDu, null in FaDu-NQOl^CR) and Vimentin (high in CAF) signal intensities were used to identify tumor cells versus CAF by a K-mean clustering method. Nucleus pSTAT3 signal intensity was plotted for each cell in the spheroid. The graph shows the combined data of at least 3 images, representing over 700 analyzed cells.

The difference in pSTAT3 levels between spheroids containing NQOl⁺ cancer cells and spheroids containing NQOl cancer cells difference is statistically different with p<0.000l.

As used herein, the singular form“a”,“an”, and“the” include plural references unless the context clearly dictates otherwise. For example, the term“a cell” includes a plurality of cells including mixtures thereof.

As used herein, the term“self-renewal” refers to cancer stem cells’ ability to give rise to new tumorigenic cancer stem cells to replenish or increase their number.

As used herein, the terms“cancer” and“cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. “Cancer cells” and“tumor cells” as used herein refer to the total population of cells derived from a tumor including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). Some cancer cells, in part, are characterized by undergoing aberrant cell division, or as being derived from cells that undergo aberrant cell division. In some embodiments, cancer cells can be differentiated by morphology, examination of cell surface markers,

transcriptional profile, and drug response. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, gastroesophageal junction cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, thymic cancer, hepatic carcinoma and various types of head and neck cancer.

As used herein, the term“tumor microenvironment” or“TME” refers to the cellular and non-cellular components that exist within and around the tumor mass. In some embodiments, the TME may comprise one or more of blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix. In some embodiments, the TME is in close proximity to the tumor. In some embodiments, the tumor and the TME interact with one another, for example, through protein factors secreted by the tumor.

As used herein, the terms“cancer stem cell(s)” and“CSC(s)” are interchangeable. CSCs are mammalian, and in preferred embodiments, these CSCs are of human origin, but they are not intended to be limited thereto. Cancer stem cells are defined and functionally characterized as a population of cells originating from a solid tumor that: (1) have extensive proliferative capacity; 2) are capable of asymmetric cell division to generate one or more kinds of differentiated progeny with reduced proliferative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance. Other common approaches to characterize CSCs involve morphology and examination of cell surface markers, transcriptional profile, and drug response. CSCs are also called in the research literature tumor/cancer initiating cells, cancer stem-like cells, stem-like cancer cells, highly tumorigenic cells, tumor stem cells, solid tumor stem cells, drug survival cells (DSC), drug resistant cells (DRCs) or super malignant cells. The existence of cancer stem cells has fundamental implications for future cancer treatments and therapies. These implications are manifested in disease identification, selective drug targeting, prevention of cancer metastasis and recurrence, and development of new strategies in fighting cancer.

“Tumor” as used herein refers to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including precancerous lesions.

“Biological sample” as used herein refers to a blood or tissue sample collected from a subject. Examples of a biological sample may include but are not limited to a cancer tissue sample, a tumor tissue sample, or a noncancerous tissue sample. In some embodiments, a biological sample from a cancer is compared to a biological cancer from a control, e.g., noncancerous tissue from the subject or from a healthy subject.

“Metastasis” as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A“metastatic” or“metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

“Reactive oxygen species” and“ROS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal-catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H2O2), organic peroxide (ROOH), hydroxyl ion (OH ), hydroxyl radical (•OH), superoxide or superoxide anion (·02 ), singlet oxygen ( Ό2). ozone (O3), carbonate radical, peroxide or peroxyl radical (·02 ²), hypochlorous acid (HOC1), hypochlorite ion (OC1 ). sodium hypochlorite (NaOCl), nitric oxide (NO·), and peroxynitrite or peroxynitrite anion (ONOO ) (unpaired electrons denoted by ·).

An“antioxidant” as used herein refers to a substance or molecule that inhibits oxidation. An antioxidant can include an antioxidant enzyme. Antioxidant enzymes can include, but are not limited to superoxide dismutases, glutathione peroxidises, glutathione reductases, and catalases.

“Biomarker” or“marker” are used interchangeably to refer to a measurable or detectable molecule whose presence is indicative of some phenomenon such as a disease, disorder, or sensitivity to a specific treatment. The measuring or detecting of a biomarker can be done by known methods in the art, or by any of the methods disclosed herein. For example, the biomarker can be measured by detecting its presence as a DNA molecule, an RNA molecule, or a polypeptide.

As used herein, the term“subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms“subject” and“patient” are used interchangeably herein in reference to a human subject.

Terms such as“treating” or“treatment” or“to treat” or“alleviating” or“to alleviate” as used herein refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. A subject is successfully“treated” according to the methods disclosed herein if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; and improvement in quality of life.

As used herein, the term“inhibiting”,“to inhibit” and their grammatical equivalents, when used in the context of a bioactivity, refer to a down-regulation of the bioactivity, which may reduce or eliminate the targeted function, such as the production of a protein or the phosphorylation of a molecule. In particular embodiments, inhibition may refer to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted activity. When used in the context of a disorder or disease, the terms refer to success at preventing the onset of symptoms, alleviating symptoms, or eliminating the disease, condition or disorder.

Napabucasin refers to 2-acetylnaphtho[2,3-b]furan-4,9-dione also known as“BBI- 608” and includes pharmaceutically acceptable solvate(s) thereof. Formula (I) shows the structure of napabucasin.

In some embodiments, napabucasin is synthetic. Napabucasin may form salts which are also within the scope of this disclosure. As used herein, reference to napabucasin is understood to include reference to salts thereof, unless otherwise indicated. The term“salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when a compound contains both a basic moiety, such as but not limited to a pyridine or imidazole, and an acidic moiety such as but not limited to a carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the term“salt(s)” as used herein. Pharmaceutically acceptable (i.e., non- toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts may be formed, for example, by reacting a compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. Solvates of napabucasin are also contemplated herein. Solvates include, for example, hydrates.

The term“pharmaceutically-acceptable excipient, carrier, or diluent” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Methods of Treatment Using Napabucasin

The present disclosure provides a method of treating a subject with napabucasin (2- acetylnaphtho[2,3-b]furan-4,9-dione). In some embodiments, the method comprises administering to the subject a composition comprising napabucasin, wherein the cancer expresses NQOl. In some embodiments, the method of treatment produces at least one reactive oxygen species in cancer cells and comprises administering napabucasin to a subject, wherein the cancer expresses NQOl. In some embodiments, NQOl expression is relative to levels in a control, e.g., noncancerous tissue from the subject with cancer or a healthy subject, as described herein. For example, the cancer expresses higher levels of NQOl as compared to a control, e.g., noncancerous tissue from the subject or from a healthy subject. In some embodiments, the cancer also expresses activated STAT3, e.g., pSTAT3. For example, the cancer expresses higher levels of activated STAT3 in comparison to a control, e.g., noncancerous tissue from the subject or from a healthy subject. In alternate embodiments, the cancer does not express activated STAT3 and/or expresses lower levels of activated STAT3 in comparison to a control, e.g., noncancerous tissue from the subject or from a healthy subject. In some embodiments, the cancer also expresses POR. For example, the cancer expresses higher levels of POR in comparison to a control, e.g., noncancerous tissue from the subject or from a healthy subject. In alternate embodiments, the cancer does not express POR or expresses lower levels of POR in comparison to a control, e.g., noncancerous tissue from the subject or from a healthy subject. In some embodiments, the cancer also expresses one or more antioxidases. For example, the cancer expresses higher levels of an antioxidase in comparison to a control, e.g., noncancerous tissue from the subject or from a healthy subject. In alternate embodiments, the cancer does not express one or more antioxidases or expresses lower levels of antioxidase(s) in comparison to a control, e.g., noncancerous tissue from the subject or from a healthy subject.

In some embodiments, the method comprises administering to the subject a composition comprising napabucasin, wherein the cancer expresses NQOl and does not express or expresses lower levels of POR, e.g., relative to control. In some embodiments, concomitant expression of POR may counteract the effect of NQOl expression. In some embodiments, the method comprises administering to the subject a composition comprising napabucasin, wherein the cancer expresses NQOl, does not express or expresses lower levels of activated STAT3, and does not express or expresses lower levels of POR, e.g., relative to control. In some embodiments, the method comprises administering to the subject a composition comprising napabucasin, wherein the cancer expresses NQOl and does not express or expresses lower levels of antioxidase, e.g., relative to control. In some embodiments, concomitant expression of antioxidase may counteract the effect of NQOl expression. In some embodiments, the method comprises administering to the subject a composition comprising napabucasin, wherein the cancer expresses NQOl, does not express or expresses lower levels of activated STAT3, and does not express or expresses lower levels of antioxidase, e.g., relative to control.

As used herein, expression refers to the detection of a nucleic acid encoding a polypeptide, e.g., NQOl, STAT3, pSTAT3, POR, or the polypeptide itself by methods described herein as well as methods known in the art. Expression of the polypeptide may refer to the detection of the polypeptide and/or polypeptide activity, e.g., enzymatic activity, by methods described herein as well as methods known in the art.

In some embodiments, the cancer expresses lower levels of at least one antioxidant in comparison to a control, e.g., noncancerous tissue from the subject or from a healthy subject. The antioxidant can include an antioxidant enzyme, e.g., an anti oxidase. Examples of antioxidant enzymes may include but are not limited to a superoxide dismutase, a flutathione peroxidase, a glutathione reductase, a catalase, or any other antioxidant enzyme.

In some embodiments, the methods disclosed herein treat cancer that expresses high levels of NQOl and low levels of a catalase, e.g., relative to control. In some embodiments, the methods disclosed herein treat cancer that expresses NQOl and any one of STAT3 (e.g., pSTAT3), POR, NAD(P)H-Quinone Oxidoreductase 2 (NQ02), Ferredoxin reductase (FDXR), Cytochrome b5 reductase 1 (CYB5R1), Cytochrome b5 reductase 3 (CYB5R3), Cytochrome b5 reductase 4 (CYB5R4), Thioredoxin reductase 1 (TXNRD1), Carbonyl Reductase 1 (CBR1), any antioxidant enzyme, or any combination thereof. Biomarkers provided herein include but are not limited to NQOl, STAT3 (including pSTAT3), POR, NQOl, NAD(P)H-Quinone Oxidoreductase 2 (NQ02), Ferredoxin reductase (FDXR), Cytochrome b5 reductase 1 (CYB5R1), Cytochrome b5 reductase 3 (CYB5R3), Cytochrome b5 reductase 4 (CYB5R4), Thioredoxin reductase 1 (TXNRD1), Carbonyl Reductase 1 (CBR1), any antioxidant enzyme, or any combinations thereof. Additional biomarkers provided herein include but are not limited to CCL2, CCL5, IL6, CXCL1, CXCL8, CXCL10, CXCL11, CXCL12, GM-CSF or sVCAMl. In some embodiments, the methods of the present disclosure treat cancer that expresses NQOl and/or any other biomarker provided herein. In some embodiments, the methods of the present disclosure treat cancer that expresses activated STAT3. In some embodiments, the methods of the present disclosure treat cancer that expresses activated STAT3 and/or any other biomarker provided herein. In some embodiments, ROS generated by napabucasin functions as a mediator of cell death and has functional consequences on cellular signaling pathways, including the STAT3 pathway. For example, data suggest that NQOl positive cells that are sensitive to napabucasin interact with the TME in a different manner from NQOl negative cells that are insensitive to napabucasin, in part through the secretion of soluble factors that can act on both the cells of the TME and on the tumor cells themselves. These observations suggest a hypothesis that pSTAT3 in the tumor/TME may serve as a marker of a tumor redox balance that is favorable to the activity of napabucasin. Accordingly, in some embodiments, disclosed herein is a method of predicting whether a human subject suffering from cancer will be responsive to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione, comprising determining the presence or absence of expression and/or activity of NQOl and at least one other marker for 2- acetylnaphtho[2,3-b]furan-4,9-dione in a biological sample from the cancer of the human subject, wherein the presence of NQOl or the presence of NQOl and the at least one other marker indicates that the human subject will be responsive to treatment with 2- acetylnaphtho[2,3-b]furan-4,9-dione. Also disclosed herein is a method of predicting whether a human subject suffering from cancer will be responsive to treatment with 2- acetylnaphtho[2,3-b]furan-4,9-dione, comprising determining expression and/or activity of NQOl and at least one other marker for 2-acetylnaphtho[2,3-b]furan-4,9-dione in a biological sample from the cancer of the human subject, wherein NQOl expression and/or activity or the presence of NQOl and the at least one other marker indicates that the human subject will be responsive to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione. In some

embodiments, the detection of NQOl (e.g., NQOl expression and/or activity or the presence of NQOl) in the biological sample is relative to NQOl (e.g., NQOl expression and/or activity or the presence of NQOl, respectively) in a control sample. In some embodiments, the detection of NQOl (e.g., NQOl expression and/or activity or the presence of NQOl) is higher as compared to levels in a control sample. In some embodiments, the control sample comprises noncancerous tissue from the subject or from a healthy subject.

In some embodiments, the disclosure provides a method for treating a cancer that expresses activated STAT3, wherein the method comprises administering a therapeutically effective amount of 2-acetylnaphtho[2,3-b]furan-4,9-dione, or a pharmaceutically acceptable solvate thereof to a patient, and the cancer has been determined to express activated STAT3 by a method comprising; a) obtaining a biological sample from a human subject; and b) contacting the biological sample with a reagent that detects activated STAT3 expression. In some embodiments, the disclosure provides a method for treating a human subject suffering from cancer, wherein the method comprises; 1) determining whether the cancer is expressing activated STAT3 by obtaining a biological sample from the patient and contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity; and 2) if the cancer is determined to express activated STAT3, then administering 2- acetylnaphtho[2,3-b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof to the patient. In some embodiments, the disclosure provides a method for treating a human subject suffering from cancer, wherein the method comprises; 1) determining whether the cancer is expressing activated STAT3 by obtaining a biological sample from the patient and contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity; and 2) if the cancer is determined to express activated STAT3, then administering 2-acetylnaphtho[2,3-b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof to the patient; and if the cancer does not express activated STAT3, then not administering 2-acetylnaphtho[2,3-b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof. In some embodiments, the cancer killing activity of acetylnaphtho[2,3- b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof is greater in a patient who suffers from a cancer that expresses activated STAT3 then in a patient who suffers from a cancer that does not express activated STAT3. In some embodiments, the patient is suffering from thymic cancer. In some embodiments the patient is suffering from pancreatic cancer. In some embodiments the patient is suffering from colorectal cancer. In some embodiments, the activated STAT3 expression is higher as compared to levels in a control, e.g., noncancerous tissue from the subject and/or a healthy control.

In some embodiments, the method of treatment described above further comprises administering a therapeutically effective amount of another therapeutic or combination of other therapeutics, e.g., pembrolizumab, FOLFIRI, bevacizumab, gemcitabine, paclitaxel, and nab-paclitaxel. According to PCT Patent Application No. PCT/US2017/028178, Example 3, the safety, tolerability, and anti-tumor activity of napabucasin in combination with FOLFIRI, with and without bevacizumab, were assessed in a clinical study of patients having advanced gastrointestinal cancer, including colorectal and gastric cancer. The disclosure of Patent Application No. PCT/US2016/028178, including the dosing regimens therein, is incorporated herein by reference in its entirety. According to PCT Patent Application No. PCT/US2017/014163, Example 4, the safety, tolerability, PK profile, and anti-tumor activity of napabucasin in combination with nab-paclitaxel and gemcitabine were assessed in a clinical study of patients with metastatic pancreatic cancer. The disclosure of Patent Application No. PCT/US2017/014163, including the dosing regimens described therein, is incorporated herein by reference in its entirety. According to PCT Patent Application No. PCT/US2018/032937, Example 8, the safety and anti-tumor activity of napabucasin in combination with pacbtaxel were assessed in a clinical study of patents with advanced thymoma or thymic carcinoma. The disclosure of Patent Application No.

PCT/US2018/032927, including the dosing regimens described therein, is incorporated herein by reference in its entirety. In some embodiments, napabucasin is formulated in a pharmaceutical composition as described herein.

The methods of treatment described herein may be used to treat cancers. Examples of cancers include but are not limited to various types of breast cancers, head and neck cancers, lung cancers, ovarian cancers, pancreatic cancers, colorectal cancers, colorectal carcinoma, prostate cancers, renal cell carcinoma, melanoma, hepatocellular carcinomas, cervical cancers, sarcomas, brain tumors, gastric cancers, thymic cancers, multiple myeloma, leukemia, and lymphomas. In some embodiments, the cancer is a colorectal cancer that does not express activated STAT3. In some embodiments, the cancer is a gastroesophageal junction cancer that does not express activated STAT3. In some embodiments, the cancer is a pancreatic cancer that does not express activated STAT3. In some embodiments, the cancer is a thymic cancer that does not express activated STAT3. Other disorders that may be treated by the methods using napabucasin described herein may include but are not limited to: autoimmune diseases, inflammatory diseases, inflammatory bowel diseases, arthritis, autoimmune demyelination disorder, Alzheimer’s disease, stroke, ischemia reperfusion injury, and multiple sclerosis.

In some embodiments, the method of treating cancer using napabucasin results in the selective killing of cancer cells in a subject. For example, in some embodiments, treatment with napabucasin results in cancer cell cytotoxicity. A pharmaceutical composition comprising napabucasin may be administered to the subject such that the napabucasin concentration in the subject’s plasma is not maintained above a critical concentration for more than 24 hours after each dose. This method can be used to treat cancers as disclosed herein. Alternatively, the duration can be further restricted to 12, 16, and 20 hours after each dose. The critical concentration for each compound may vary. In various embodiments, the critical concentration is about 100 mM, about 50 pM, about 30 pM, or about 20 pM.

The cancer may be metastatic. In some embodiments, the cancer is refractory to chemotherapy or radiotherapy. In some embodiments, the cancer is resistant to

chemotherapy. In some embodiments, the cancer has relapsed. The subject may be a mammal, e.g., a human being.

For any of the methods of treating a subject described herein, effective dosing ranges, dosing frequencies, and plasma concentrations are provided. In some embodiments, the pharmaceutical composition is administered at a dosage: (a) from about 1 mg/m² to about 5,000 mg/m² (I.V.) or from about 1 mg/m² to about 50,000 mg/m² (PO); (b) from about 2 mg/m² to about 3,000 mg/m² (I.V.) or from about 10 mg/m² to about 50,000 mg/m² (PO). In some embodiments the pharmaceutical composition is administered at a dosage from about 1 mg/m² to about 4,000 mg/m² (PO); or from about 10 mg/m² to about 4,000 mg/m² (PO). In some embodiments, napabucasin may be administered in an amount ranging from about 80 mg to about 1500 mg. In some embodiments, napabucasin may be administered in an amount ranging from about 160 mg to about 1000 mg. In some embodiments, napabucasin may be administered in an amount ranging from about 300 mg to about 700 mg. In some embodiments, napabucasin may be administered in an amount ranging from about 700 mg to about 1200 mg. In some embodiments, napabucasin may be administered in an amount ranging from about 800 to about 1100 mg. In some embodiments, napabucasin may be administered in an amount ranging from about 850 mg to about 1050 mg. In some embodiments, napabucasin may be administered in an amount ranging from about 960 mg to about 1000 mg. In some embodiments, napabucasin is administered every other day (Q2D), once daily (QD), or twice a day (BID). In some embodiments, the pharmaceutical composition is administered orally and no more than four times a day (QID). In some embodiments, napabucasin is administered in a dose of about 480 mg daily. In some embodiments, napabucasin is administered in a dose of about 960 mg daily. In some embodiments, napabucasin is administered in a dose of about 1000 mg daily. In some embodiments, napabucasin may be administered in an amount ranging from about 80 mg to about 750 mg twice daily. In some embodiments, napabucasin may be administered in an amount ranging from about 80 mg to about 500 mg twice daily. In some embodiments, napabucasin is administered in a dose of about 240 mg twice daily. In some embodiments, napabucasin is administered in a dose of about 480 mg twice daily. In some embodiments, napabucasin is administered in a dose of about 500 mg twice daily. In some embodiments, napabucasin is administered in combination with a therapeutically effective regimen of FOLFIRI and/or a therapeutically effective amount of bevacizumab. In some embodiments, napabucasin is administered in combination with a therapeutically effective amount of gemcitabine and/or a therapeutically effective amount of nab-pacbtaxel. In some embodiments, napabucasin is administer in combination with a therapeutically effective amount of paclitaxel.

In some embodiments, the pharmaceutical composition is administered to the subject such that the compound concentration in the subject’s plasma is not maintained above a critical concentration for more than 24 hours (or 12, 16, and 20 hours) after each dose. In some embodiments, the plasma concentration of the compound does not exceed the critical concentration at a certain time point after each does, e.g., 12, 16, 20, or 24 hours, as a regimen that avoids non-selective toxicity. In some embodiments, the critical concentration is about 100 mM, about 50 mM, about 30 pM, or about 20 pM. The compositions, in certain cases, are isolated, purified or synthesized.

Also provided herein are a pharmaceutical composition that comprises napabucasin, and a pharmaceutically-acceptable excipient, carrier, or diluent for the treatment of a cancer that expresses NQOl. In some embodiments, the composition is suitable for oral, nasal, topical, rectal, vaginal or parenteral administration, or intravenous, subcutaneous or intramuscular injection.

Detection and Diagnostic Methods

Provided herein are methods for detecting the presence of NQOl in a biological sample, comprising: obtaining a biological sample from a human subject and contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample, wherein the detection of NQOl expression and/or activity indicates the presence of NQOl in the biological sample. In some embodiments, the NQOl expression and/or activity is higher as compared to levels in a control, e.g., noncancerous tissue from the subject and/or a healthy control. The methods for detecting may further comprise detecting the presence, absence, expression, and/or activity of STAT3, pSTAT3, POR, NQOl, NAD(P)H-Quinone Oxidoreductase 2 (NQ02), Ferredoxin reductase (FDXR), Cytochrome b5 reductase 1 (CYB5R1), Cytochrome b5 reductase 3 (CYB5R3), Cytochrome b5 reductase 4 (CYB5R4), Thioredoxin reductase 1 (TXNRD1), Carbonyl Reductase 1 (CBR1), an antioxidant, and an antioxidant enzyme, e.g., relative to control.

Also provided herein are methods for detecting the presence of activated STAT3 in a biological sample, comprising: obtaining a biological sample from a human subject and contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity to determine whether activated STAT3 is present in the biological sample, wherein the detection of activated STAT3 expression and/or activity indicates the presence of activated STAT3 in the biological sample. In some embodiments, the NQOl expression and/or activity is higher as compared to levels in a control, e.g., noncancerous tissue from the subject and/or a healthy control.

Also provided herein are methods for diagnosing cancer in a human subject, comprising: obtaining a biological sample from the human subject and contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample, wherein the detection of NQOl expression and/or activity indicates susceptibility or presence of cancer. Also provided herein are methods for diagnosing cancer in a human subject, comprising: obtaining a biological sample from the human subject and contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample, wherein the detection of NQOl expression and/or activity indicates presence of cancer. In some embodiments, the NQOl expression and/or activity is higher as compared to levels in a control, e.g., noncancerous tissue from the subject and/or a healthy control. The methods for diagnosing may further comprise detecting the presence, absence, expression, and/or activity of STAT3, pSTAT3, POR, NQOl, NAD(P)H-Quinone

Oxidoreductase 2 (NQ02), Ferredoxin reductase (FDXR), Cytochrome b5 reductase 1 (CYB5R1), Cytochrome b5 reductase 3 (CYB5R3), Cytochrome b5 reductase 4 (CYB5R4), Thioredoxin reductase 1 (TXNRD1), Carbonyl Reductase 1 (CBR1), an antioxidant, and an antioxidant enzyme, e.g., relative to control. In some embodiments, pSTAT3 expression in the cancer is also higher as compared to levels in a control. In alternate embodiments, pSTAT3 expression in the cancer is absent or lower as compared to levels in a control. In some embodiments, POR expression in the cancer is also higher as compared to levels in a control. In alternate embodiments, POR expression in the cancer is absent or lower as compared to levels in a control. In some embodiments, antioxidase expression in the cancer is also higher as compared to levels in a control. In alternate embodiments, antioxidase expression in the cancer is absent or lower as compared to levels in a control.

Also provided herein are methods for diagnosing cancer in a human subject, comprising: obtaining a biological sample from the human subject and contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity to determine whether activated STAT3 is present in the biological sample, wherein the detection of activated STAT3 expression and/or activity indicates susceptibility or presence of cancer. Also provided herein are methods for diagnosing cancer in a human subject, comprising: obtaining a biological sample from the human subject and contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity to determine whether activated STAT3 is present in the biological sample, wherein the detection of activated STAT3 expression and/or activity indicates presence of cancer. In some embodiments, the activated STAT3 expression and/or activity is higher as compared to levels in a control, e.g., noncancerous tissue from the subject and/or a healthy control.

Also provided herein are methods for determining a human subject likely to respond to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione, comprising: obtaining a biological sample from the human subject; contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample; and administering a therapeutically effective amount of 2-acetylnaphtho[2,3-b]furan- 4,9-dione and/or a pharmaceutically acceptable solvate thereof if NQOl is present in the biological sample. Also provided herein are methods for determining a human subject likely to respond to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione, comprising: obtaining a biological sample from the human subject; contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample; determining the human subject likely to respond to treatment with 2- acetylnaphtho[2,3-b]furan-4,9-dione if NQOl expression and/or activity is detected in the biological sample; and, optionally, administering a therapeutically effective amount of 2- acetylnaphtho[2,3-b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof if NQOl is present in the biological sample. In some embodiments, the presence of NQOl in the biological sample is relative to presence of NQOl in a control sample. In some embodiments, the presence of NQOl in the biological samples is higher as compared to levels in a control sample. In some embodiments, the control sample comprises

noncancerous tissue from the subject or from a healthy subject. The methods for determining a subject likely to respond to treatment with napabucasin may further comprise detecting the presence, absence, expression, and/or activity of STAT3, pSTAT3, POR, NQOl, NAD(P)H- Quinone Oxidoreductase 2 (NQ02), Ferredoxin reductase (FDXR), Cytochrome b5 reductase 1 (CYB5R1), Cytochrome b5 reductase 3 (CYB5R3), Cytochrome b5 reductase 4 (CYB5R4), Thioredoxin reductase 1 (TXNRD1), Carbonyl Reductase 1 (CBR1), an antioxidant, and an antioxidant enzyme, e.g., relative to control. In some embodiments, pSTAT3 expression in the cancer is also higher as compared to levels in a control. In alternate embodiments, pSTAT3 expression in the cancer is absent or lower as compared to levels in a control. In some embodiments, POR expression in the cancer is also higher as compared to levels in a control. In alternate embodiments, POR expression in the cancer is absent or lower as compared to levels in a control. In some embodiments, antioxidase expression in the cancer is also higher as compared to levels in a control. In alternate embodiments, antioxidase expression in the cancer is absent or lower as compared to levels in a control.

Also provided herein are methods for determining a human subject likely to respond to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione, comprising: obtaining a biological sample from the human subject; contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity to determine whether activated STAT3 is present in the biological sample; and administering a therapeutically effective amount of 2- acetylnaphtho[2,3-b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof if activated STAT3 is present in the biological sample. In some embodiments, the activated STAT3 expression is higher as compared to levels in a control, e.g., noncancerous tissue from the subject and/or a healthy control.

In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of 2-acetylnaphtho[2,3-b]furan-4,9-dione for use in treating a human subject suffering from cancer that expresses NQOl is provided herein.

In some embodiments, the detection disclosed herein may be accomplished using one or more diagnostic agents. The diagnostic agent can be any suitable reagent and may include agents needed to draw a blood sample, take a biopsy, screen for a biomolecule (e.g., antigen or antibody) or extract genetic information from a sample. The agent may include a solvent, a detergent, an anticoagulant, an antigen, an antibody, an enzyme, a PCR primer, and so on.

Diagnostic agents, tools, and/or instructions for performing the methods for the detection of any of the biomarkers disclosed herein can be provided separately or in a kit. Diagnostic agents separately or as a kit can include reagents used in the collection of a tissue sample from a subject, such as by biopsy, and reagents for tissue processing. Diagnostic agents separately or as a kit can include one or more reagents for detecting or measuring the expression level or activity of any one of the biomarkers of the disclosure, such as reagents used for nucleic acid amplification, including RT-PCR and qPCR, Next Generation

Sequencing (NGS), Southern blot, northern blot, enzymatic activity assays, any form of proteomic analysis, western blot, or an immunohistochemistry (IHC) assay. Non-limiting examples of these reagents, which are used for detecting or measuring the expression level or activity of any of the biomarkers disclosed herein, include primers for performing PCR, RT- PCR, NGS, probes for performing northern blot analyses, and/or antibodies or aptamers for performing proteomic analysis such as western blot, immunohistochemistry (IHC) and ELISA assays, or substrates enzymatic activity assays. Any of these reagents can be provided separately or in a kit. Appropriate buffers for the assays can also be provided separately or in a kit. Additional agents, such as for example visualization agents, required for detecting or measuring expression level or activity of any of the biomarkers of the disclosure in the assays described can also be provided separately or in a kit. The kits may be array- or PCR-based kits for example and may include additional agents, such as a polymerase and/or dNTPs for instance. The kits may be protein detection-based kits for example and may include reagents such as antibodies, and buffers, and visualization agents for detecting a signal. The kits may be enzymatic activity-based kits. The kits can also include an instruction sheet describing how to perform the assays for detecting or measuring the expression level or activity of any of the biomarkers of the disclosure.

In some embodiments, the methods disclosed herein comprise detecting NQOl polypeptide expression and/or activity. In some embodiments, the methods disclosed herein comprise detecting NQOl nucleotide expression or activity. In some embodiments, NQOl, pSTAT3, POR, catalase, an antioxidase, and/or any of the other biomarkers disclosed herein may be detected by immunohistochemistry. In some embodiments, NQOl, pSTAT3, POR, catalase, an antioxidase, and/or any of the other biomarkers disclosed herein may be detected by western blot. In some embodiments, NQOl, pSTAT3, POR, catalase, an anti oxidase, and/or any of the other biomarkers disclosed herein may be detected by PCR, in situ hybridization, microarray, enzymatic assay, and/or colorimetric assay, e.g., MTT.

In some embodiments, detecting one or more of the biomarkers disclosed herein by immunohistochemistry comprises: obtaining a tissue sample from a patient; contacting the tissue with an antibody to detect the expression of one of the biomarkers disclosed herein; further contacting the sample with a reagent that generates a signal when the antibody binds to its target; and scoring the staining for biomarker positivity.

In some embodiments, detecting one or more of the biomarkers disclosed herein by immunohistochemistry comprises: obtaining a tissue sample from a patient, e.g., tumor tissue; optionally fixing the sample with a fixative reagent, e.g., formalin; optionally embedding the sample to prepare tissue blocks, e.g., in paraffin; optionally cutting the tissue blocks into tissue sections; contacting the tissue with an antibody to detect the expression of one of the biomarkers disclosed herein; further contacting the sample with a reagent that generates a signal when the antibody binds to its target; and scoring the staining for biomarker positivity.

In some embodiments, the antibody used in the immunohistochemistry assay detects NQOl. In some embodiments, the antibody used in the immunohistochemistry assay detects STAT3. In some embodiments, the antibody used in the immunohistochemistry assay detects pSTAT3. In some embodiments, the antibody used in the immunohistochemistry assay detects POR. In some embodiments, the antibody used in the immunohistochemistry assay detects an antioxidase. In some embodiments, the antibody used in the

immunohistochemistry assay detects catalase.

In some embodiments, the staining score is determined by the percentage of cells stained in comparison to a control. In some embodiments, the staining score, e.g., for NQOl, is determined by the staining intensity in a cell (e.g., 0 = negative, 1 = weak, 2 =

intermediate, and 3 = strong). In some embodiments, the staining score, e.g., for pSTAT3, is determined by the nuclear staining intensity in a cell (e.g., 0 = nuclear staining not detectable, 1+ = nuclear straining translucent, 2+ = nuclear staining opaque, and 3 = nuclear staining solid). In some embodiments, the staining score is determined by the percentage of cells stained at least 1 in comparison to a control. In some embodiments, the staining score is determined by the percentage of cells stained at least 2 in comparison to a control. In some embodiments, the staining score is determined by the percentage of cells stained 3 in comparison to a control. In some embodiments the immunohistochemistry assay is tested for repeatability and/or reproducibility. In some embodiments, the immunohistochemistry assay is tested for accuracy, sensitivity and/or specificity.

The disclosure also provides a method for studying a condition characterized by the presence of NQOl. In some embodiments, this method comprises coculturing cancer cells with or without NQOl, e.g., with cancer associated fibroblasts. In some embodiments, the cancer cells are cocultured with non-cancer associated fibroblasts. In some embodiments the ratio of the fibroblast cells to cancer cells is at least 100: 1. In some embodiments the ratio of the fibroblast cells to cancer cells is at least about 4: 1. In some embodiments the ratio of the fibroblast cells to cancer cells is at least about 3: 1. In some embodiments the ratio of the fibroblast cells to cancer cells is at least about 2: 1. In some embodiments the ratio of the fibroblast cells to cancer cells is at least about 1 : 1. In some embodiments the ratio of the fibroblast cells to cancer cells is at least about 1 :2. In some embodiments the cells are cultured in 2D conditions, e.g., with both cell types adhered to the surface of a culture container. In other embodiments, the cells are cocultured in 3D conditions, e.g., with multi cell type spheroids.

In some embodiments, the method may be used for predicting whether a human subject suffering from cancer will be responsive to treatment with napabucasin. In some embodiments, the method comprises coculturing cancer cells obtained from the human subject with fibroblast cells, and quantifying the amount of a biomarker secreted in the coculture, wherein the presence of the biomarker in excess of a predetermined amount indicates that the patient will respond to treatment with napabucasin. In some embodiments, the secreted biomarker(s) that are quantified are CCL2, CCL5, IL6, CXCL1, CXCL8, CXCL10, CXCL11, CXCL12, GM-CSF and/or sVCAMl.

In other embodiments, the method may be used for identifying new biomarkers, which may be used to determine whether a human subject suffering from cancer will be responsive to treatment with napabucasin. In some embodiments, the method comprises comparing the proteins secreted in cocultures of NQOl⁺ cancer cells and fibroblasts to the proteins secreted in cocultures of NQOl cancer cells and fibroblasts, wherein a protein that is present in higher concentrations in cocultures of NQOl⁺ cancer cells than cocultures of NQOl cells indicates that the protein may be used as a biomarker to determine the responsiveness of a cancer to treatment with napabucasin. In some embodiments, the secreted protein is present at higher concentrations in cocultures of cancer cells expressing NQOl. In some embodiments, the amount of secreted protein is at least 1.2 times greater in cocultures containing cancer cells expressing NQOl. In some embodiments, the amount of secreted protein is at least 1.5 times greater in cocultures containing cancer cells expressing NQOl. In some embodiments, the amount of secreted protein is at least 2 times greater in cocultures containing cancer cells expressing NQOl. In some embodiments, the amount of secreted protein is at least 5 times greater in cocultures containing cancer cells expressing NQOl. In some embodiments, the amount of secreted protein is at least 10 times greater in cocultures containing cancer cells expressing NQOl. In some embodiments, the biomarkers identified using this method may be used to determine whether a human subject suffering from cancer is likely to respond to treatment with napabucasin. In some embodiments, the biomarkers identified using this method may be used to detect the presence of a tumor that is likely to respond to treatment with napabucasin. In other embodiments, the method may be used for identifying agents that are useful for treating a condition characterized by the presence of NQOl, for example, by

administering the agent to cocultures of cells. Exemplary parameters to monitor in order to determine efficacy include, but are not limited to, one or more of the following:

(i) a change in kinase activity, e.g., phosphorylation levels of STAT3 (e.g., decreased phosphorylation or autophosphorylation);

(ii) a change in an activity of a cell containing NQOl, e.g., a change in proliferation, morphology, or tumorigenicity of the cell;

(iii) a change in the level, e.g., expression, transcription, and/or translation level, of a secreted factor, e.g., CCL2, CCL5, IL6, CXCL1, CXCL8, CXCL10, CXCL11, CXCL12, GM-CSF and/or sVCAMl; or

(iv) a change in an activity of a signaling pathway involving STAT3, e.g.,

phosphorylation or activity of an interacting or downstream target, or expression level of a target gene.

Formulations of Nanabucasin

Formulations of napabucasin include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the mammal being treated and the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form, will generally be that amount of the napabucasin which produces a therapeutic effect.

Generally, out of 100%, this amount will range, for example, from about 1% to about 99% of active ingredient, from about 5% to about 70%, from about 10% to about 30%.

Therapeutic compositions or formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of napabucasin as an active ingredient. The napabucasin may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the napabucasin is mixed with one or more

pharmaceutically-acceptable carriers, such as sodium citrate or di calcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, and sodium starch glycolate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and polyethylene oxide-polypropylene oxide copolymer; absorbents, such as kaolin and bentonite clay;

lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration of the napabucasin include

pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Additionally, cyclodextrins, e.g., hydroxypropyl-beta-cyclodextrin, may be used to solubilize compounds.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain napabucasin as well as one or more suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing napabucasin with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room

temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active pharmaceutical agents. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of napabucasin include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.

The active compound may be mixed under sterile conditions with a pharmaceutically - acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to the napabucasin, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain napabucasin and excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated.

Pharmaceutical compositions suitable for parenteral administration comprise napabucasin in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

In some cases, in order to prolong the effect of the napabucasin, it is desirable to slow its absorption by the body from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered composition is accomplished by dissolving or suspending the compound in an oil vehicle. One strategy for depot injections includes the use of polyethylene oxide-polypropylene oxide copolymers wherein the vehicle is fluid at room temperature and solidifies at body temperature.

The pharmaceutical compounds comprising napabucasin may be administered alone or in combination with other pharmaceutical agents, or with other anti-cancer therapies as described herein, as well as in combination with a pharmaceutically-acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutically acceptable excipient, carrier, or diluent is chosen from Gelucire®.

In some embodiments, the pharmaceutically acceptable excipient, carrier, or diluent comprises a lipid for intravenous delivery. The lipid can be: phospholipids, synthetic phophatidylcholines, natural phophatidylcholines, sphingomyelin, ceramides,

phophatidylethanolamines, phosphatidylglycerols, phosphatidic acids, cholesterol, cholesterol sulfate, and hapten and PEG conjugated lipids. The lipid may be in the form of

nanoemulsion, micelles, emulsions, suspension, nanosuspension, niosomes, or liposomes. In some embodiments, the pharmaceutically acceptable excipient, carrier, or diluent is in a form of micellar emulsion, suspension, or nanoparticle suspension, and it further comprises an intravenously acceptable protein, e.g., human albumin or a derivative thereof, for intravenous delivery.

In some embodiments, the pharmaceutically acceptable excipient, carrier, or diluent comprises a waxy material for oral delivery. The waxy material may be mono-, di-, or tri glycerides, mono-, di-fatty acid esters of PEG, PEG conjugated vitamin E (vitamin E TPGs), and/or Gelucire. The Gelucire can be selected from Gelucire 44/14, Gelucire 43/01, Gelucire 50/02, Gelucire 50/13, Gelucire 37/02, Gelucire 33/01, Gelucire 46/07, and Gelucire 35/10.

In some embodiments, the pharmaceutically acceptable excipient, carrier, or diluent is selected from capryol, transcutol hp, labrafil M, labrasol, triacetin, pharmasolv, ethanol, poly vinyl pyrrolidine, carboxymethyl cellulose, tween 20, and tween 80. In some embodiments, the pharmaceutically acceptable excipient, e.g., Gelucire 44/14, is mixed with a surfactant, which can be Tween 80 or Tween 20. These embodiments of pharmaceutical compositions can be further formulated for oral administration. The napabucasin can be synthesized using commercially available starting materials and processes well known to one skilled in the art of organic chemistry. In some embodiments, napabucasin is synthetic.

EXAMPLES

Example 1: Identification of napabucasin binding in NQOl-expressing cells

An MTT colorimetric assay was performed comparing NQOl -deficient MDA-MB- 231 cells and MDA-MB-231 cells transfected with NQOl. Napabucasin activity (EC50) was determined as shown below and is higher in NQOl-expressing cells than in NQOl -deficient cells.

6002 0.743 0.051

A cell-free enzyme kinetic assay was performed comparing NQOl enzymatic activity in the presence of substrates including napabucasin (compound BBI608), compound BBI608- Ml (the primary human metabolite of BBI608), and b-lapachone, another quinone compound. Concentration ranges for each substrate were mixed with 20 nM purified recombinant NQOl protein in 50 mM potassium phosphate pH 7.4. The reactions were started with the addition of 0.8 mM NAD(P)H. Conversion of NAD(P)H to NAD(P)+ was measured by reading the absorbance at 340 nm every 3 seconds for 10 min. Enzymatic activity of NQOl was also determined with respect to BBI608 in the presence of dicoumarol, a reductase inhibitor, as a control. As demonstrated in FIG. 1, the catalytic efficiency of NQOl was higher for napabucasin in comparison to b-lapachone and BBI608-M1.

Example 2: Role of NQOl in napabucasin-induced ROS generation and cytotoxicity

Three cell lines (BxPC3, PACA2, and PANC1) were incubated with increasing concentrations of napabucasin to assess napabucasin-induced generation of reactive oxygen species (ROS). ROS generation and cell viability were measured simultaneously using luminescence-based assays. FIG. 2 shows that treatment with napabucasin induces the production of ROS, which correlates with the cytotoxic effect of napabucasin.

To generate NQOl knockout lines, parental cells were transfected with the guide RNA (designed using Desk Genetics, synthesized by IDT) and Cas9 protein complex (RNP complex) by Lipofectamine™ CRISPRMAX™ transfection reagent (Thermo Fisher) following manufacturer’s protocol. In brief, functional guide RNA (gRNA) was generated by annealing tracrRNA and crRNA prior to RNP complex formation. A 1 : 1 ratio of gRNA and Cas9-NLS protein was mixed with LipoCas9 plus reagent followed by 5 minutes incubation at room temperature to produce RNP complex. RNP complex was mixed with transfection regent and added to the parental cells. After overnight incubation, cells were incubated in new culture medium and allowed to expand until enough genomic DNA could be extracted. Successful gene editing was verified by heteroduplex analysis. Potential NQOl knockout single clones were selected and complete NQOl knockout was verified by western blot analysis.

Napabucasin activity was further characterized in FaDu (a human epithelial cell line derived from squamous cell carcinoma of the hypopharynx) and A549 cells (a human epithelial lung cancer cell line). Cell viability was determined using CellTiter-Glo® (Promega, Madison, Wisconsin), and ROS levels were quantified using ROS-Glo™

(Promega, Madison, Wisconsin) at 3 hours post dosing with napabucasin. In FaDu and A549 cells in which NQOl was genetically deleted using CRISPR, ROS were not generated in the presence of napabucasin (FIGS. 3 A and 3B) and the cell-killing activity of napabucasin was reduced (FIGS. 4A and 4B). Additionally, the cytotoxic effect of napabucasin was also altered in other cell lines when NQOl was not present (FIG. 5), suggesting that napabucasin activity is dependent on the presence of NQOl in cells.

A cell assay assessing the role of anti-oxidants in the effects of napabucasin was performed. A549 cells were mock transfected or transfected with 2 different siRNAs targeting the expression of anti-oxidant enzyme catalase. Twenty-four hours after transfection, cells were treated with a dose curve of napabucasin, and cell viability determined 24 hours following treatment using a luminescence-based cell viability assay. Results demonstrated that silencing catalase improved the cytotoxic effect of napabucasin (FIG. 6A). Additionally, A549 cells were plated at 1000 cells per well in 96 well plates, and 24 hours after plating, cells were pre-treated with 50 mM EUK-134 overnight, prior to treatment with a dose curve of napabucasin. Viability was determined using luminescence- based cell viability assay 6 hours after napabucasin treatment. Inhibition of ROS

accumulation using the ROS scavenger EUK-134 was shown to reduce the cell-kill activity of napabucasin (FIG. 6B), further suggesting that napabucasin activity is related to its ability to generate ROS.

Example 3: Role of NQOl in the napabucasin-dependent inhibition of STAT3

Treating cells with napabucasin has been shown to inhibit STAT3 activity, resulting in cell death and inhibition of spherogenesis. See Patent No. 9,745,278. Knocking down STAT3 in cancer stem cells (CSCs) induced apoptosis and inhibited CSC spherogenesis in vitro. See id., Examples 1-3.

MDA-MB-231 cells (deficient in NQOl) and MDA-MB-231-NQ01 cells

(overexpressing NQOl) were treated with DMSO, or 0.5 mM or 1 pM napabucasin for 2 hours before being harvested for western blotting analysis. pSTAT3, total STAT3, NQOl and tubulin (control) protein levels were measured. (FIG. 7A). Napabucasin was more effective in reducing levels of pSTAT3 when cells expressed higher levels of NQOl.

An electrophoretic mobility shift assay (EMSA) was performed also using MDA-MB- 231 cells and MDA-MB-231-NQ01 cells (FIG. 7B). Cells were treated with DMSO, Oncostatin M (OSM) to stimulate the STAT3 pathway, or napabucasin, and the binding of STAT3 was assessed. Gel shift analysis was performed with nuclear extracts and a radiolabeled STAT3 DNA binding oligonucleotide. In cells overexpressing NQOl, napabucasin inhibited STAT3 DNA binding, in contrast to cells deficient for NQOl in which DNA binding was observed in the presence of napabucasin. In some embodiments, napabucasin inhibits the DNA binding capability of STAT3 in cells that express higher levels ofNQOl.

Cells were also treated either with napabucasin alone, with napabucasin in combination with reductase inhibitor dicoumarol that inhibits NQOl, or with napabucasin in combination with anti-oxidant EUK-134 (FIG. 7C). When cells were treated with napabucasin, DNA binding was inhibited, but treatment of cells with napabucasin in the presence of dicoumarol reduced the inhibitory effect and restored STAT3 DNA binding. Treatment of cells with napabucasin in combination with EUK-134 also resulted in a reduced inhibitory effect of napabucasin, as STAT3 DNA binding was detected. In some embodiments, napabucasin-mediated generation of ROS inhibits STAT3 DNA binding. Example 4: Napabucasin is a substrate for POR

A cell-free enzyme kinetic assay was performed comparing Cytochrome P450 Oxidoreductase (POR) enzymatic activity in the presence of napabucasin, b-lapachone, and Menadione. FIG. 8 shows that the catalytic efficiency of POR was higher for napabucasin than for b-lapachone or Menadione (FIGS. 8A-C).

Example 5: STAT3 expression in the tumor microenvironment (TME)

Napabucasin was studied in a double-blind randomized phase III clinical trial directed to patients with advanced colorectal cancer. See Jonker DJ, Nott L, Yoshino T, et al, “Napabucasin versus placebo in refractory advanced colorectal cancer: a randomised phase 3 trial” Lancet Gastroenterol Hepatol. 20l8;3(4):263-270. Although no overall survival benefit was observed when napabucasin was compared with placebo, patients who expressed pSTAT3 in tumor and stromal tissue had an overall survival benefit when treated with napabucasin. Elevated pSTAT3 is associated with poor prognosis in colorectal cancer because of its effects on tumor cells (promotion of proliferation, cell survival, angiogenesis, and invasion) and tumor stromal cells (suppression of anti-tumor responses of the innate and adaptive immune systems).

In further analyses of samples from patients in the clinical trial, pSTAT3-positivity was defined as cancer cell nuclear staining of 5% or greater or by a staining intensity score (0 = negative, 1= weak, 2= intermediate, and 3 = strong). Patients with higher pSTAT3 levels in the tumor microenvironment, not just in tumor cells, had an even higher overall survival benefit when treated with napabucasin (FIG. 9).

Example 6: Expression of NQOl

In order to assess how the expression of NQOl in the tumor cells can affect the TME, FaDu parental cells, which express endogenous NQOl (NQOl⁺), and three independent clones, which had NQOl genetically deleted using CRISPR (NQOl^CR), were either cultured alone or co-cultured with cancer associated fibroblasts (CAFs) or normal fibroblasts (NFs) for 2 days. Proteome human cytokine profiler arrays were used to examine secreted protein levels in the culture and co-culture supernatants.

The cytokines secreted into the supernatant of 2D monolayer co-cultures of FaDu or FaDu-NQOl^CR tumor cells and CAFs was determined by proteome profiler human XL cytokine array (R&D Systems, Minneapolis, Minnesota). Cytokine levels in the supernatant of 3D spheroid co-cultures of FaDu or FaDu-NQOl^CR tumor cells and CAFs were quantified by ProcartaPlex assay (Thermo Fisher Scientific, Waltham, Massachusetts) using Luminex® technology.

Under co-culture conditions, the tumor-CAF and tumor-NF secretome profiles revealed a number of proteins which were differently secreted, based on the expression level of NQOl in the tumor cells (FIG. 10). Proteins which were differently secreted included: CCL2, CCL5, IL6, CXCL1, CXCL8, CXCL10, CXCL11, CXCL12, GM-CSF and sVCAMl.

Proteins which were secreted at higher levels in the co-cultures of NQOU cells with CAFs and NFs were tested for their effects on the phosphorylation of STAT3 in peripheral blood mononuclear cells (PBMCs), CAFs and NFs. CAFs and NFs were serum-starved for 2 hours before being treated with each recombinant protein at a concentration of 100 ng/ml for 20 minutes prior to the cells being collected and lysed for western blotting analysis (FIG. 11). Treatment with Oncostatin M and FaDu cell lysate were used as pSTAT3 positive controls. These results showed that IL6 treatment induced increased levels of STAT3 phosphorylation in CAFs and NFs. Dose response curves show that pSTAT3 levels increase with IL6 treatment in CAFs and NFs (FIG. 24), and A549 and FaDu cell lines (FIG. 25) regardless of the presence of NQOl. PBMCs contain both myeloid and lymphoid cells; and as myeloid cells in human PBMCs are mostly monocytes, they can be distinguished from lymphocytes by flow cytometry based on their size difference (FIG. 12A). Similar to the treatment of CAFs, PBMCs were treated with the same recombinant proteins being tested (FIG. 13A- 13C). Rested human PBMCs were serum starved for 2 hours before being treated with each recombinant protein at a concentration of 100 ng/ml for 20 min. After treatment, the PBMCs were fixed and permeabilized prior to intracellular staining for pSTAT3, followed by flow cytometry analysis. The pSTAT3 levels were determined in the specific cell population by gating on each unique cell type within the PBMCs. These results demonstrated that IL6 increased pSTAT3 levels in the unsorted, total PBMC population (FIG. 13 A), with the effect more pronounced in the gated lymphocyte population (FIG. 13B). In the monocyte population, the addition of IL6 or GM-CSF induced an increase also pSTAT3 levels (FIG. 13C). Additional staining of human PBMC with T-cell marker CD3 can separate T cells from the rest of the lymphocyte population, which consists mostly of B cells and NK cells (FIG. 14). PBMCs treated, processed and stained in the same way as just described, and additionally stained for CD3, showed that IL6 treatment induced an increase in the pSTAT3 levels in the T cell population (FIG. 15 A); and that IL6 or CXCL10 treatments induced an increase in pSTAT3 levels in the non-T lymphocyte population (FIG. 15B). These results demonstrated that IL6, GM-CSF, and CXCL10 are secreted at higher levels in co-cultures of NQOl⁺ cells with CAFs and that these secreted proteins can increase STAT3

phosphorylation in other cells present in the TME, including fibroblasts, human PBMC, or both.

To further confirm the differences in the secretome profiles of NQOl⁺ cells co cultured with CAFs and NQOl cells co-cultured with CAFs, a Luminex based enzyme- linked immunosorbent assay (ELISA) was performed. NQOl⁺ FaDu parental cells and three independent clones which had NQOl genetically deleted through CRISPR (NQOl^CR or koNQOl clones) were individually co-cultured with CAFs for 2 days. Cell culture supernatant was collected and subjected to Luminex-based ELISA assays. For all three koNQOl clones co-cultured with CAFs, secretion levels of IL6 (FIG. 16A), GM-CSF (FIG. 16B) and CXCL10 (FIG. 16C) were found to be reduced in comparison to the those measured for NQOl⁺ cells co-cultured with CAFs. Similar results were obtained using A549 cells, a human lung adenocarcinoma cell line which has endogenous expression of NQOl. A549 cells which had NQOl genetically deleted through CRISPR (A549-NQOl^CR or A549- koNQOl clones) were generated. NQOl⁺ A549 wild type cells and A549-koNQOl cells were individually co-cultured with CAFs for 2 days, before collecting supernatants to be subjected to a Luminex-based ELISA assay. For the A549-koNQOl cells co-cultured with CAFs secretion levels of IL6 (FIG. 17A), GM-CSF (FIG 17B) and CXCL10 (FIG 17C) were found to be reduced in comparison to the cultures of NQOl⁺ A549 wild type cells with CAFs.

Example 7: Detecting pSTAT3 expression by immunohistochemistry (IHC)

pSTAT3 immunohistochemistry was performed on formalin-fixed paraffin-embedded (FFPE) normal and neoplastic colorectal cancer (CRC) specimens. FFPE tissue blocks were stored at room temperature while FFPE cut tissue sections were stored at -20 °C throughout the duration of the study. pSTAT3 IHC staining accuracy, sensitivity and specificity were determined.

A cutoff of > 1% of tumor cells staining 1+, 2+, or 3+ intensity was used to define positivity. The microenvironment was assessed for the presence of infiltrating inflammatory cells. The threshold for scoring a pSTAT3 microenvironment was the presence of at least 10% (by area of tumor) infiltrating immune cells. If the threshold was met, the percentage and average intensity of pSTAT3 positive inflammatory and stromal cells were recorded. Where possible, the score of the inflammatory cell population was recorded separately from the stromal cell population.

The pSTAT3 staining protocol used Dako Target Retrieval Solution with a high pH for 20 minutes in the PT Link (antigen retrieval chamber) at 97 °C, Cell Signaling Phospho- Stat3 (Y705) clone D3A7 antibody at a 0.79 pg/mL concentration for 60 minutes, and Dako EnVision™ FLEX HRP (visualization reagent) for 30 minutes.

Seven CRC FFPE specimens and a 40-core CRC multi-tissue micro array were stained using the optimized CRC pSTAT3 IHC assay. Stained slides were evaluated for pSTAT3 nuclear staining of neoplastic and infiltrating inflammatory cells (when present) using the scoring system as follows: 0 = nuclear staining not detectable; 1+ = nuclear straining translucent; 2+ = nuclear staining opaque; 3+ = nuclear staining solid. These results were compared to results found in literature to determine pSTAT3 assay accuracy.

Two of the CRC TMA scores did not contain tumor cells and were excluded from analysis. Six of seven CRC FFPE specimens (86%) and fifteen of 38 CRC TMA cores (39%) were positive for pSTAT3 IHC expression. Originally only ten CRC TMA cores were scored as positive. Reevaluation identified five additional cores as positive for pSTAT3 IHC expression. Original evaluation looked for positivity in 5% increments. For low expressing specimens, the detection threshold was reset to positivity in 1% increments. In total 21 of 45 evaluable CRC specimens (47%) were positive for pSTAT3 IHC expression. References have shown positivity rates of 52-55%. See, e.g., Morikawa et al.“STAT3 Expression, Molecular Features, Inflammation Patterns, and Prognosis in a Database of 724 Colorectal Cancers,” Clinical Cancer Research 17.6 (2011): 1452-62; Lin et al.“STAT3 Is Necessary for Proliferation and Survival in Colon Cancer-Initiating Cells,” Cancer Research 71.23 (2011): 7226-37.

Combining the results of the two studies yields an overall positivity rate of 52% (435/833). This combined value was used to calculate the accuracy of the optimized CRC pSTAT3 IHC assay. The results from the tested CRC specimens compared to findings in the literature are shown in the contingency table below (Table 1).

Table 1.

Results from Table 1 were used to determine pSTAT3 IHC assay accuracy:

Accuracy = [(% True Positives+% True Negatives) / (% True Positives+% False Positives+% False Negatives+% True Negatives)] x 100%

Accuracy = [(47%+ 48% I (47% + 0% + 5% + 48%)] x 100% = 95%

Results from Table 1 were also used to determine pSTAT3 IHC assay sensitivity:

Sensitivity = [% True Positives / (% True Positives + % False Negatives)) x 100

Sensitivity = [47% I (47% + 5%)) x 100 = 90%

Results from Table 1 were also used to determine pSTAT3 IHC assay specificity:

Specificity= [%True Negatives / (% True Negatives+% False Positives)) x 100

Specificity= [48% I (48% + 0%)) x 100 = 100%

The pSTAT3 IHC assay was tested for repeatability. Three CRC specimens with a range of pSTAT3 expression were selected for this study. Serial sections from each tissue block were stained on one run, with the same reagents, on one instrument using the optimized CRC pSTAT3 IHC assay. An additional slide per specimen was stained using an isotype control reagent instead of pSTAT3 antibody. The slides were scored for percent positive tumor cell nuclear staining at each intensity level as previously described. The H-score was measured as follows:

H-score = l(% of cells staining 1+) + 2(% of cells staining 2+) + 3(% of cells staining 3+)

The repeatability study with the optimized pSTAT3 CRC IHC assay yielded consistent results between slides within one run. All three specimens had 100% consistency in H-Score results, with %CV = 0%. The repeatability of the pSTAT3 CRC IHC assay met the acceptability criteria.

The pSTAT3 IHC assay was also tested for reproducibility. Three CRC specimens were used. Five sections from each tissue block were cut. One slide from each specimen was stained on five separate runs using the optimized pSTAT3 IHC assay. Different instruments and operators were used. The same procedure was performed using an isotype control reagent per specimen per staining run. The slides were scored for percent positive cell nuclear staining at each intensity level as previously described and the H-score was using the same formula as before.

The reproducibility study with the optimized pSTAT3 CRC IHC assay yielded consistent results across four staining runs, which included using different instruments. All three specimens had 100% consistency in H-Score results, with %CV = 0%. The repeatability of the pSTAT3 CRC IHC assay met the acceptability criteria.

Example 8: Detecting NQOl expression by IHC

An NQOl immunohistochemistry assay was performed using formalin-fixed paraffin- embedded gastric cancer specimens. Specimens were evaluated only if they contained at least 100 tumor cells. For NQOl IHC scoring, the stained sections were evaluated for

identification of staining in positive control tissue (xenograft or cell lines) and tumor cells in tumor specimens. During optimization of the assay the staining intensity range of positive staining as well as the intensity of any background staining were evaluated. Pathology evaluation included the percent of tumor cell positivity at each intensity (0-3+) for all scored specimens.

The NQOl staining protocol used Dako Target Retrieval Solution with a high pH for 20 minutes in the PT Link (antigen retrieval chamber) at 97 °C, a primary anti-NQOl antibody (A180) (Cell Signaling) at a 0.0.82 pg/mL concentration for 30 minutes, and Dako EnVision™ FLEX HRP (visualization reagent) for 30 minutes.

In order to evaluate the accuracy of the NQOl IHC assay, thirty -three gastric specimens were stained using the optimized assay, wherein stained slides were evaluated according to the scoring criteria previously described. Results were compared to overall NQOl prevalence reported in the literature. See, e.g., Srijiwangsa et al.“Roles of NAD (P) H-Guinone Oxidoreductase 1 (NQOl) On Cancer Progression and Chemoresistance,” Journal of Clinical & Experimental Oncology 6:4 (2017).

The accuracy and H-score were determined according to the following formulas:

Accuracy = [(% True Positives + % True Negatives) / (% True Positives + % False Positives + % False Negatives + % True Negatives)] x 100%

H-score = (%l+) + (2 * %2+) + (3 * %3+)

Two specimens did not have sufficient tumor for evaluation. H-scores ranged from 0- 295 with 23/31 (74.2%) specimens having an H-score > 50. The H-score distribution is displayed in FIG. 18. NQOl IHC staining was detected in the cytoplasm and/or membrane of cells; cytoplasmic and membranous staining was observed. Results obtained were compared to those reported in the literature in a study of NQOl gastric cancer specimens showing a 75.9% prevalence (Table 2). This comparison used an H-score cutoff of > 50. Table 2.

Accuracy was determined to be:

Accuracy = [(74.2%+ 24.1% / (74.2% + 0% + 1.7% + 24.1%)] x 100% = 98.3%

Results from Table 2 were also used to determine NQOl IHC assay sensitivity:

Sensitivity = [% True Positives / (% True Positives + % False Negatives)] x 100 Sensitivity = [74.2% / (74.2% + 1.7%)] x 100% = 97.9%

Images representing the dynamic range of NQ01 staining are shown in FIG. 19. The sensitivity of the optimized NQOl IHC assay is 97.9% using an H-score cutoff of >50.

Using alternate cutoffs of >25 and >100 yielded sensitivity values of 100% and 93.5%, respectfully. All values met the acceptability criteria.

Results from Table 1 were also used to determine NQOl IHC assay specificity:

Specificity= [%True Negatives / (% True Negatives+% False Positives)] x 100

Specificity = [24.1% / (24.1% + 0%)] x 100 = 100%

The napabucasin can be synthesized using commercially available starting materials and processes well known to one skilled in the art of organic chemistry. In some

embodiments, napabucasin is synthetic.

The specificity of the NQOl IHC assay is 100% using an H-score cutoff of >50.

Using alternate cutoffs of >25 and >100 yielded specificity values of 80.5% and 100%, respectfully. H-score cutoffs of >50 and >100 met the acceptability criteria.

The NQOl IHC assay was tested for repeatability. Four gastric cancer specimens, representing a range of NQOl IHC staining, were selected for this study. Five sections from each specimen were cut and stained on one run using the NQOl IHC assay. An additional slide per specimen was stained using a matched isotype control. Stained slides were evaluated using the scoring criteria described herein. Variance was calculated based on percent CV :

%CV = (standard deviation of data set) / (average of data set)* 100.

The percent CVs of H-score ranged from 0% - 22.1%, with an overall average of 9.1%, meeting the acceptability criterial.

The NQOl IHC assay was also tested for reproducibility. Four gastric cancer specimens, representing a range of NQOl IHC staining, were selected for this study. These corresponded to the same specimens from the repeatability test. Ten sections from each specimen were cut. Two slides from each specimen were stained on five independent runs using the NQOl IHC assay: one slide with NQOl antibody and the other with a matched negative isotype control. Slides from repeatability (slide 5 for each case) were used as one of the inter-runs. A second operator was used to perform one of the inter-runs (inter-operator).

A second instrument was used to perform another of the inter-runs (inter-instrument).

Stained slides were evaluated using the scoring criteria previously described. Variance was calculated based on percent CV as described above. The percent CVs of H-score ranged from 0% - 20.7%, with an overall average of 10.9%, meeting the acceptability criteria.

Nevertheless, a staining variance was observed, and further investigation was done on the repeatability and reproducibility tests. Variance was thought to be caused by slight variation in tissue section thickness. Two specimens were re-tested, with an emphasis on applying consistent tissue sectioning techniques. Repeatability and reproducibility studies were repeated as previously described. The overall percent CVs improved from 9.1% to 3.9% and 10.9% to 5.0% for repeatability and reproducibility, respectively.

Example 9: Evaluation of Tumor/Tumor Microenvironment Interactions in 3D sphere cultures.

To better understand the complex signals within the TME, A549 and FaDu cancer cells, or cells deleted for NQOl using CRISPR (NQOl^CR), were cultured in 2D monolayer cultures or in 3D sphere cultures (FIG. 20) alone or in combination with normal or cancer- associated fibroblasts.

To generate sphere cultures, cells in the exponential growth phase were trypsinized, resuspended in 10% DMEM and filtered to ensure single cell suspension. Live cells were identified by trypan blue staining and counted twice. Tumor cells and fibroblasts were mixed at the desired ratio and seeded into an ultra-low attachment (ULA) U-bottom plate (96 or 384 wells). Cells were concentrated at the bottom of the plate by a brief centrifugation at 250 rpm for 5 min. Cells were incubated at 37° C for at least 2 days to allow spheroid formation.

Cell viability assays found that FaDu NQOl positive cancer cells are highly sensitive to treatment to napabucasin, while FaDu NQOl negative cancer cells are resistant in 3D sphere cultures (FIG. 21). In FaDu NQOl positive cancer cells, administration of napabucasin was associated with an increase in ROS levels (FIG. 21). In contrast, ROS levels in FaDu-NQOl^CR cancer cells did not become elevated in the presence of napabucasin. Similar results were obtained in A549/A549-NQOl^CR co-cultures (data not shown).

Collectively, these data suggest that the anti-tumor activity of napabucasin is dependent on the generation of ROS in general and the presence of NQOl in particular, with NQOl positive cancer cells being highly sensitive to napabucasin and NQOl^CR cancer cells being comparatively resistant.

Luminex-based ELISA assays were used to quantify the amount of IL6, GM-CSF, and CXCL10 secreted in 2D and 3D cultures of either cancer cells or fibroblast cells alone, or in cocultures of fibroblast and cancer cell lines. NQOl positive cancer cells co-cultured with fibroblasts were shown to secrete different levels of factors than NQOl negative cancer cell co-cultures (FIG. 22A-C and FIG. 23A-C) suggesting that their secretion is dependent on NQOl.

Immunofluorescence was used to identify phosphorylated STAT3 (pSTAT3) positive populations in tumor/fibroblast spheroid co-cultures (FIG. 26A-G and FIG. 27). At least 48 spheroids were collected for immunofluorescence staining. Harvested spheroids were incubated in 10% neutral buffered formalin over night at 4° C, washed in IX PBS, and then resuspended in 20-30 pL of 2% agarose. These samples then underwent dehydration and paraffmization. Afterwards, the spheroids were embedded in paraffin and sectioned into 4 pm slices. Spheroids were baked at 60° C for 1 hour, deparaffmized, and underwent antigen retrieval in pH 6.0 citrate buffer at 98° C for 30 minutes. After washing with water and permeabilization with TBS buffer with 0.3% Triton-X, samples were then blocked with 10% normal goat serum for 1 hour at room temperature, and then probed with pSTAT3 (CST 9145, 1: 100), NQOl (CST 3187, 1 :400), and vimentin (R&D Systems MAB2105, 1 :50) antibodies overnight at 4° C. Samples were then incubated with secondary antibodies for 1 hour at room temperature, and mounted with ProLong Gold antifade mountant with DAPI prior to imaging. Fluorescent microscopic images of spheroids were analyzed and quantified using the CellProfiler tool. CellProfiler is a software tool developed by the Broad Institute for quantitative analysis of biological images.

NQOl (high in FaDu and A549-WT, null in A549-NQOl^CR and FaDu-NQOl^CR) and Vimentin (high in CAF and NF) signal intensities were used to identify tumor cells versus fibroblasts by a K-mean clustering method. Nucleus pSTAT3 signal intensity was plotted for each cell in the spheroid. FIG. 26G shows the combined data of at least 3 images of A549 and CAF containing spheroids and FIG. 27 shows the combined data of at least 8 images of FaDu and CAF containing spheroids, with over 700 cells analyzed in each figure. pSTAT3 levels were found to be significantly higher in both cancer cells and fibroblast cells in cocultures with NQOl⁺ cancer cell lines as compared to cocultures with NQOl cancer cell lines, with pO.OOOl.

Claims

1. A method for treating cancer in a human subject in need thereof comprising administering to the human subject a composition comprising a therapeutically effective amount of 2- acetylnaphtho[2,3-b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof, wherein the cancer expresses NQOl.

2. The method of claim 1, wherein the cancer does not express activated STAT3.

3. The method of claim 1, wherein the cancer expresses activated STAT3.

4. The method of any one of claims 1-3, wherein the cancer expresses POR.

5. The method of any one of claims 1-3, wherein the cancer does not express POR.

6. A method for producing at least one reactive oxygen species in cancer cells comprising administering to a human subject in need thereof a composition comprising a therapeutically effective amount of 2-acetylnaphtho[2,3-b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof,

wherein the cancer expresses NQOl.

7. The method of claim 6, wherein the cancer does not express activated STAT3.

8. The method of claim 6, wherein the cancer expresses activated STAT3.

9. The method of any one of claims 6-8, wherein the cancer expresses POR.

10. The method of any one of claims 6-8, wherein the cancer does not express POR.

11. The method of any of the preceding claims, further comprising administering a therapeutically effective amount of pembrolizumab.

12. The method of any one of the preceding claims, wherein the cancer is metastatic.

13. The method of any one of the preceding claims, wherein the cancer is refractory to chemotherapy or radiotherapy.

14. The method of any one of the preceding claims, wherein the cancer is resistant to chemotherapy.

15. The method of any one of the preceding claims, wherein the cancer has relapsed.

16. The method of any one of the preceding claims, wherein the cancer is colorectal cancer that does not express activated STAT3.

17. The method of any one of the preceding claims, wherein the cancer is gastroesophageal junction cancer that does not express activated STAT3.

18. The method of any one of the preceding claims, wherein the cancer is pancreatic cancer that does not express activated STAT3.

19. The method of any one of the preceding claims, wherein the cancer is thymic cancer that does not express activated STAT3.

20. The method of any one of the preceding claims, wherein the composition consists of a therapeutically effective amount of 2-acetylnaphtho[2,3-b]furan-4,9-dione and/or a pharmaceutically acceptable solvate thereof.

21. The method of any one of the preceding claims, wherein the composition further comprises at least one pharmaceutically acceptable excipient, carrier, or diluent.

22. The method of claim 21, wherein the at least one pharmaceutically acceptable excipient, carrier, or diluent is chosen from Gelucire®.

23. The method of any one of the preceding claims, wherein the composition is formulated for oral administration.

24. A method for detecting the presence of NQOl in a biological sample, comprising:

a) obtaining a biological sample from a human subject; and

b) contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample, wherein the detection of NQOl expression and/or activity indicates the presence of NQOl in the biological sample.

25. A method for diagnosing cancer in a human subject, comprising:

a) obtaining a biological sample from the human subject; and

b) contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample, wherein the detection of NQOl expression and/or activity indicates susceptibility or presence of cancer.

26. A method for determining a human subject likely to respond to treatment with 2- acetylnaphtho[2,3-b]furan-4,9-dione, comprising:

a) obtaining a biological sample from the human subject;

b) contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample; and c) administering a therapeutically effective amount of 2-acetylnaphtho[2,3-b]furan- 4,9-dione and/or a pharmaceutically acceptable solvate thereof if NQOl is present in the biological sample.

27. A method for determining a human subject likely to respond to treatment with 2- acetylnaphtho[2,3-b]furan-4,9-dione, comprising:

a) obtaining a biological sample from the human subject;

b) contacting the biological sample with a reagent that detects NQOl expression and/or activity to determine whether NQOl is present in the biological sample; and c) determining the human subject likely to respond to treatment with 2- acetylnaphtho[2,3-b]furan-4,9-dione if NQOl expression and/or activity is detected in the biological sample.

28. The method of claim 27, further comprising:

d) administering to the human subject a therapeutically effective amount of 2- acetylnaphtho[2,3-b]furan-4,9-dione.

29. A method of predicting whether a human subject suffering from cancer will be responsive to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione, comprising determining the presence or absence of NQOl expression and/or activity in a biological sample from the cancer of the human subject, wherein the presence of NQOl indicates that the human subject will be responsive to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione.

30. A method of predicting whether a human subject suffering from cancer will be responsive to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione, comprising determining NQOl expression and/or activity in a biological sample from the cancer of the human subject, wherein NQOl expression and/or activity indicates that the human subject will be responsive to treatment with 2-acetylnaphtho[2,3-b]furan-4,9-dione.

31. The method of any one of claims 24-30, wherein the detection of NQOl in the biological sample comprises detecting NQOl polypeptide expression or activity.

32. The method of any one of claims 24-30, wherein the detection of NQOl in the biological sample comprises detecting NQOl nucleotide expression or activity.

33. The method of any one of claims 24-32, wherein the detection of NQOl in the biological sample comprises detecting NQOl expression and/or activity in one or more assays selected from: immunohistochemistry, western blot, PCR, in situ hybridization, microarray, enzymatic assay, and colorimetric assay.

34. The method of any one of claims 24-33, wherein detection of NQOl in the biological sample is relative to NQOl in a control sample.

35. The method of claim 34, wherein the control sample comprises noncancerous tissue from the subject or from a healthy subject.

36. A pharmaceutical composition comprising a therapeutically effective amount of 2- acetylnaphtho[2,3-b]furan-4,9-dione for use in treating a human subject suffering from cancer that expresses NQOl.

37. A method of treating a human subject having cancer, comprising administering a therapeutically effective amount of a compound having formula (I)

(i) and/or a pharmaceutically acceptable solvate thereof, wherein the cancer expresses activated STAT3 as determined by:

a) obtaining a biological sample from a human subject; and

b) contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity to determine whether activated STAT3 is present in the biological sample.

38. A method for treating a patient with a therapeutically effective amount of a compound having formula (I)

( and/or a pharmaceutically acceptable solvate thereof, wherein the patient is suffering from cancer, the method comprising the steps of: 1) determining whether the cancer is expressing activated STAT3 by:

a) obtaining a biological sample from the patient; and

b) contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity to determine whether activated STAT3 is present in the biological sample; and

2) if the cancer expresses activated STAT3, then administering the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof to the patient.

39. A method for treating a patient with a therapeutically effective amount of a compound having formula (I)

(l) and/or a pharmaceutically acceptable solvate thereof, wherein the patient is suffering from cancer, the method comprising the steps of:

1) determining whether the cancer is expressing activated STAT3 by:

a) obtaining a biological sample from the patient; and

b) contacting the biological sample with a reagent that detects activated STAT3 expression and/or activity to determine whether activated STAT3 is present in the biological sample,

2) if the cancer expresses activated STAT3, then administering the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof to the patient; and if the cancer does not express activated STAT3, then not administering the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof.

40. A method for treating a patient with a therapeutically effective amount of a compound having formula (I)

(1), and/or a pharmaceutically acceptable solvate thereof, wherein the patient is suffering from cancer, the method comprising the steps of:

1) determining whether the cancer is expressing activated STAT3 by:

a) obtaining a biological sample from the patient; and

2) if the cancer expresses activated STAT3, then administering the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof to the patient; and if the cancer does not express activated STAT3, then not administering the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof,

wherein the cancer cell killing activity of the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof is greater when the compound having formula (I) is administered to a patient suffering from a cancer that expresses activated STAT3 than it would be if the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof were administered to a patient suffering from a cancer that does not express activated STAT3.

41. The method of any one of claims 37-40, wherein the cancer is selected from thymic cancer, pancreatic cancer, and colorectal cancer.

42. The method of claim 41, wherein the cancer is colorectal cancer.

43. The method of claim 42, wherein the compound having formula (I) is administered at a dose of about 480 mg per day.

44. The method of any one of claims 42 and 43, wherein the compound having formula (I) is administered at a dose of about 240 mg twice daily.

45. The method of any one of claims 42-44, further comprising administering a

therapeutically effective regimen of FOLFIRI if the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof is administered.

46. The method of claim 41, wherein the cancer is pancreatic cancer.

47. The method of claim 46, wherein the compound having formula (I) is administered at a dose of about 480 mg per day.

48. The method of any one of claims 46 and 47, wherein the compound having formula (I) is administered at a dose of about 240 mg twice daily.

49. The method of any one of claims 46-48, further comprising administering (i) a therapeutically effective amount of gemcitabine and (ii) a therapeutically effective amount of nab-paclitaxel if the compound having formula (I) and/or a pharmaceutically acceptable solvate thereof is administered.

50. A method for detecting the presence of activated STAT3 in a biological sample, comprising:

a) obtaining a biological sample from a human subject; and

wherein the detection of activated STAT3 expression and/or activity indicates the presence of activated STAT3 in the biological sample.

51. A method for diagnosing cancer in a human subject, comprising:

a) obtaining a biological sample from the human subject; and

wherein the detection of activated STAT3 expression and/or activity indicates susceptibility or presence of cancer.

52. The method of any one of claims 2-5, 7-23, and 37-51, wherein the presence or absence of activated STAT3 is determined by immunohistochemical staining.

53. The method of any one of claim 2-5, 7-23, and 37-52, wherein the presence or absence of activated STAT3 is determined in Formalin Fixed Paraffin Embedded (FFPE) tumor tissue.

54. The method of any one of claims 37 to 53, wherein the presence of activated STAT3 in the biological sample is relative to presence of activated STAT3 in a control sample.

55. The method of claim 54, wherein the control sample comprises noncancerous tissue from the subject or from a healthy subject.