Methods and compositions for treating pancreatic cancer

Abstract

Provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises an increased amount or expression of a GATA gene, a keratin 17 (KRT17) gene, a TP56 gene, a CYP3A gene, or a combination thereof.

Description

Attorney Docket No.52378-704.601 METHODS AND COMPOSITIONS FOR TREATING PANCREATIC CANCER CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 647,378 filed May 14, 2024, the disclosure of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] Pancreatic cancer is a one of the leading causes of cancer deaths, with around only about 12% of patients surviving 5 years after diagnosis. Pancreatic ductal adenocarcinoma (PDAC) is a form of pancreatic cancer that is particularly aggressive, with some reports indicating a five-year survival rate of as low as 5%, even in patients that have undergone tumor resection or have been treated with chemo- and radiotherapy. Systemic therapies for metastatic and locally advanced PDAC often include cytotoxic therapies; however, pancreatic tumors are often unresponsive to therapy, which may be due to the development of resistance. Many of the difficulties associated with treating pancreatic cancer, such as resistance to therapies, are believed to lie within the genetic and cellular levels. For example, it is believed that gene changes in pancreatic tumors may generate gene instability, which may play a role in PDAC tumor growth and resistance. As such, single targeted inhibitor therapy, even when combined with chemotherapy, has provided challenges due to complex redundant signaling. Accordingly, there is a need for new treatments for pancreatic cancer (e.g., PDAC) that increase the sensitivity of the cancer cells to therapy, and there is a need for therapies that target more than one key signaling pathway. SUMMARY

[0003] Provided herein, in some aspects, is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises an increased amount or expression of a GATA gene, a keratin 17 (KRT17) gene, a TP56 gene, a CYP3A gene, or a combination thereof. In some embodiments, the pharmaceutical composition comprises a dual inhibitor of GSK3-β and HDAC. In some embodiments, said cancer has previously been identified as comprising the increased amount or expression of the GATA gene, the keratin 17 (KRT17) gene, the TP56 gene, a CYP3A gene, or a combination thereof. In some embodiments, said GATA gene is a GATA6 gene. In some embodiments, said cancer comprises the increasedAttorney Docket No.52378-704.601 amount or expression of the GATA gene and the KRT17 gene. In some embodiments, said cancer comprises the increased amount or expression of the GATA gene. In some embodiments, said cancer comprises the increased amount or expression of the KRT17 gene. In some embodiments, said cancer comprises the increased amount or expression of the TP56 gene. In some embodiments, said cancer comprises the increased amount or expression of the CYP3A gene. In some embodiments, the CYP3A gene is a CYP3A5 gene. In some embodiments, said cancer comprises the increased amount or expression of said GATA gene, said KRT17 gene, and said TP56 gene. In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises an increased amount or function of a GATA protein, an increased amount or function of a KRT17 protein, an increased amount or function of p53, or a combination thereof (e.g., as compared to a non-cancerous cell). In some embodiments, said cancer has previously been identified as comprising the increased amount or function of the GATA protein, the increased amount or function of the KRT17 protein, the increased amount or function of p53, or a combination thereof (e.g., as compared to a non-cancerous cell). In some embodiments, said cancer comprises the increased amount or function of the GATA protein (e.g., as compared to a non-cancerous cell). In some embodiments, said cancer comprises the increased amount or function of the KRT17 protein. In some embodiments, said cancer comprises the increased amount or function of the p53 protein. In some embodiments, said cancer comprises the increased amount or function of the GATA protein and an increased amount or function of the KRT17 protein. In some embodiments, said subject has not previously received an anti- cancer agent. In some embodiments, said subject has previously received an anti-cancer agent. In some embodiments, said anti-cancer agent comprises gemcitabine. The expression of the genes (e.g., GATA, KRT17, TP56, and CYP3A) may be determined in any appropriate manner, such as, for example, with the use of RNA sequencing and / or ELISA.

[0004] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises a decreased amount or function of a GATA protein, an increased amount or function of a KRT17 protein, a decreased amount or function of p53, or a combination thereof (e.g., as compared to a non-cancerous cell). In some embodiments, said cancer has previously been identified as comprising the decreased amountAttorney Docket No.52378-704.601 or function of the GATA protein, the increased amount or function of the KRT17 protein, the decreased amount or function of p53, or a combination thereof (e.g., as compared to a non- cancerous cell). In some embodiments, said cancer comprises the decreased amount or function of the GATA protein (e.g., as compared to a non-cancerous cell). In some embodiments, said cancer comprises the increased amount or function of the KRT17 protein. In some embodiments, said cancer comprises the decreased amount or function of the p53 protein. In some embodiments, said cancer comprises the decreased amount or function of the GATA protein and an increased amount or function of the KRT17 protein.

[0005] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said subject has previously received an anti-cancer agent. In some embodiments, said anti-cancer agent is not said GSK3-β inhibitor and said HDAC inhibitor. In some embodiments, said anti-cancer agent comprises an autophagy inhibitor. In some embodiments, said autophagy inhibitor comprises a phosphatidylinositol 3-kinases (PI3K) inhibitor. In some embodiments, said PI3K inhibitor is a PI3Kү inhibitor. In some embodiments, said anti-cancer agent comprises gemcitabine, irinotecan, fluorouracil (5FU), oxaliplatin, paclitaxel, folinic acid, or a combination thereof. In some embodiments, said anti- cancer agent comprises folinic acid, 5FU, irinotecan, and oxaliplatin (FOLFIRINOX). In some embodiments, said cancer expresses an increased amount or expression of GATA and KRT17. In some embodiments, said cancer comprises classical-type cancer cells.

[0006] In some embodiments, said subject has previously received an anti-cancer agent for treatment of the cancer. In some embodiments, said subject has previously received an anti-cancer agent for treatment of a previous cancer.

[0007] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said subject has not previously received an anti-cancer agent. In some embodiments, said subject has not previously received an anti-cancer agent for treatment of the cancer (e.g., the present cancer being treated). In some embodiments, said subject has not previously received an anti-cancer agent for treatment of a previous cancer. In some embodiments, said cancer comprises classical-like cancer cells.

[0008] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical compositionAttorney Docket No.52378-704.601 comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises classical-like cancer cells. In some embodiments, said cancer was previously identified as comprising said classical-like cells. In some embodiments, said classical-like cells are identified using a gene classifier.

[0009] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises basal-like cancer cells. In some embodiments, the method further comprises administering an additional anti-cancer agent. In some embodiments, said additional anti-cancer agent comprises a chemotherapeutic agent. In some embodiments, wherein said chemotherapeutic agent comprises a topoisomerase inhibitor. In some embodiments, said chemotherapeutic agent comprises a topoisomerase I inhibitor. In some embodiments, said chemotherapeutic agent comprises a camptothecin derivative. In some embodiments, said chemotherapeutic agent comprises irinotecan or a derivative thereof. In some embodiments, the camptothecin derivative comprises irinotecan, SN38, topotecan, belotecan, silatecan, karenitecin, or a combination thereof. In some embodiments, said chemotherapeutic agent comprises irinotecan or a derivative thereof. In some embodiments, said chemotherapeutic agent comprises irinotecan. In some embodiments, said chemotherapeutic agent comprises SN38 and derivatives thereof. In some embodiments, said chemotherapeutic agent comprises SN38. In some embodiments, said chemotherapeutic agent comprises an antimetabolite. In some embodiments, said chemotherapeutic agent comprises gemcitabine. In some embodiments, said chemotherapeutic agent comprises 5FU. In some embodiments, said chemotherapeutic agent comprises an alkylating agent. In some embodiments, said chemotherapeutic agent comprises a platinum-containing agent. In some embodiments, said chemotherapeutic agent comprises oxaliplatin. In some embodiments, said chemotherapeutic agent comprises a taxane. In some embodiments, said chemotherapeutic agent comprises paclitaxel. In some embodiments, said chemotherapeutic agent comprises gemcitabine, irinotecan, 5FU, oxaliplatin, paclitaxel, folinic acid, or a combination thereof. In some embodiments, said chemotherapeutic agent comprises gemcitabine, irinotecan, paclitaxel, or a combination thereof. In some embodiments, chemotherapeutic agent comprises 5FU, irinotecan, and oxaliplatin. In some embodiments, the method further comprises administering folinic acid. In some embodiments, said GSK3-β inhibitor and said HDAC inhibitor comprises a dual inhibitor ofAttorney Docket No.52378-704.601 GSK3-β and HDAC (e.g., a single agent may provide inhibitory effects against GSK3-β and HDAC).

[0010] In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (IV):wherein: L1and L2are in dependently a linker; R1is an aromatic moiety, alkyl, acyl, cyclyl or heterocyclyl, each of which can be optionally substituted; R2is hydrogen, lower alkyl, cyclyl, heterocyclyl , aryl, or heteroaryl, each of which can be optionally substituted; R3is absent or an aromatic moiety, which can be optionally substituted ; p is 0, 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10; and wherein –L1R1is linked to one nitrogen of the thiadiazolidine ring and– (CH2)p-R3-L 2- C(O)NH OR2is linked to the other nitrogen of the thiadiazolidine ring.

[0011] In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound represented by the structure(also referred to as “Compound 1”). In some embodiments, the compositions or methods provided herein comprises Compound 1.

[0012] In some embodiments, said cancer comprises a sarcoma. In some embodiments, said pancreatic cancer comprises an epithelioid sarcoma. In some embodiments, said cancer comprises a carcinoma. In some embodiments, said carcinoma comprises an adenocarcinoma. In some embodiments, said cancer comprises pancreatic cancer. In some embodiments, said pancreatic cancer comprises pancreatic ductal adenocarcinoma.

[0013] In some aspects, further provided herein is a kit for treating a cancer, comprising a test for determining whether a cancer comprises an increased amount or expression of a GATA gene, a keratin 17 (KRT17) gene, a TP56 gene, a CYP3A gene, or a combination thereof and a GSK3-β inhibitor and a HDAC inhibitor. In some embodiments, said GSK3-βAttorney Docket No.52378-704.601 inhibitor and said HDAC inhibitor comprises a dual inhibitor of GSK3-β and HDAC. In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (IV). In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (III-1). In some embodiments, the kit further comprises an additional anti-cancer agent. In some embodiments, said additional anti-cancer agent comprises a chemotherapeutic agent. In some embodiments, said chemotherapeutic agent comprises a topoisomerase I inhibitor. In some embodiments, said chemotherapeutic agent comprises irinotecan. In some embodiments, said chemotherapeutic agent comprises an antimetabolite. In some embodiments, said chemotherapeutic agent comprises gemcitabine. In some embodiments, said chemotherapeutic agent comprises 5FU. In some embodiments, said chemotherapeutic agent comprises an alkylating agent. In some embodiments, said chemotherapeutic agent comprises a platinum-containing agent. In some embodiments, said chemotherapeutic agent comprises oxaliplatin. In some embodiments, said chemotherapeutic agent comprises a taxane. In some embodiments, said chemotherapeutic agent comprises paclitaxel. In some embodiments, said chemotherapeutic agent comprises gemcitabine, irinotecan, 5FU, oxaliplatin, paclitaxel, folinic acid, or a combination thereof. In some embodiments, said chemotherapeutic agent comprises gemcitabine, irinotecan, paclitaxel, or a combination thereof. In some embodiments, said chemotherapeutic agent comprises 5FU, irinotecan, oxaliplatin, and folinic acid (FOLFIRINOX). In some embodiments, said cancer comprises a sarcoma. In some embodiments, said pancreatic cancer comprises an epithelioid sarcoma. In some embodiments, said cancer comprises a carcinoma. In some embodiments, said carcinoma comprises an adenocarcinoma. In some embodiments, said cancer comprises pancreatic cancer. In some embodiments, said pancreatic cancer comprises pancreatic ductal adenocarcinoma. The contents of Jingyu An, et al.., “Metavert synergises with standard cytotoxics in human PDAC organoids and is associated with transcriptomic signatures of therapeutic response,” Translational Oncology, Volume 49, 2024, 102109,ISSN 1936-5233 are incorporated by reference in its entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:Attorney Docket No.52378-704.601

[0015] FIGS.1A-1N demonstrate the cytotoxic effects of Compound 1 on classical-like versus basal-like selected cell lines and organoids. FIGS.1A-1D show the protein levels of serine-9 phosphorylation of GSK-3β (p-GSK3β) and acetylation of H3K9 (ace-H3K9) in PDAC cell lines and hPDOs via western blot analysis after 72 hours of treatment with Compound 1. FIGS.1E-1F show the protein levels of p-GSK3β and ace-H3K9 by immunofluorescence localization in the PDAC cell lines treated with Compound 1 or the positive controls tideglusib (TDG) and SAHA (inhibitors of GSK-3β and HDAC) (scale bar=20 µm). Cells were stained for p-GSK-3β (green), ace-H3K9 (red), and DAPI (blue). FIG.1G shows dose response cell viability curves using MTT assay of Basal-like cell lines (BxPC3, MiaPaCa2 and PANC1) and Classical-like cell lines (AsPC1, PXC) after treatment with Compound 1. FIGS.1H-1I show dose response cell viability curves using MTT assay in Classical-like and Basal-like cell lines to mono-inhibitor treatment for 72 hours. FIG.1J shows specific IC50 values in response to 72h treatment with Compound 1, tideglusib and SAHA in PDAC cell lines. FIG.1K shows IC50 values (mean ± SEM) of Compound 1 in Basal-like organoids, and Classical-like organoids, chemo-naive group, and post-treatment group). FIG.1L shows western blot analysis of protein GSK-3β expression of PDAC cell lines re-probed for GAPDH. FIGS.1M-1N show representative immunofluorescent co- localization images stained for GSK-3β (green) and DAPI (blue) also showing greater expression of GSK-3β in Basal-like compared to Classical-like organoids (scale bar=100 µm).

[0016] FIGS.2A-2G show Compound 1 increases the sensitivity to chemotherapy. FIG. 2A: Different IC50 distribution for the 36 hPDOs treated with Compound 1 and five standard cytotoxics. FIGS.2B-2G: Individual drug organoid sensitivities, comparing Basal versus Classical-like hPDOs and organoids derived from chemo-naive versus post-chemotherapy PDAC tissues. Compound 1 (FIG.2B), gemcitabine (FIG.2C), irinotecan (FIG.2D), 5-FU (FIG.2E), oxaliplatin (FIG.2F), and paclitaxel (FIG.2G) were tested.

[0017] FIGS.3A-3H show synergistic effects of Compound 1 with cytotoxics in cell lines and organoids. FIG.3A shows a synergy map of Basal-like cells (AsPC1, PXC) and the Classical-like cells (BxPC3, MIA PaCa2, PANC1), treated with Compound 1 in combination with chemotherapeutics. The strongest synergy across all cell lines was the combination of Compound 1+IR. FIG.3B shows synergistic scores of Compound 1 combined with other drugs (gemcitabine, irinotecan, 5-FU, oxaliplatin, paclitaxel). FIG.3C shows the synergy score of Classical-like organoids and basal-like hPDOs treat with Compound 1. FIGS.3D- 3H: Synergistic scores for individual cytotoxic pairings with Compound 1 and GemcitabineAttorney Docket No.52378-704.601 (FIG.3D), irinotecan (FIG.3E), 5-FU (FIG.3F), oxaliplatin (FIG.3G), and paclitaxel (FIG.3H) were tested.

[0018] FIGS.4A-4C show Compound 1 transcriptomic signature and network analysis. FIG.4A displays heatmap showing the relative expression of signature genes between Compound 1 (“Cmpd1”) High and Low IC50 values. FIG.4B displays network of signature genes representing the maximum scoring subgraph obtained from the set of genes differentially expressed between high and low Cmpd1 IC50 groups. FIG.4C displays reactome pathway enrichment analysis showing significantly enriched pathways associated with the set of Cmpd1 signature genes.

[0019] FIGS.5A-5E show Compound 1 transcriptomic signature associations in organoid transcriptomic subtypes and treatment groups. FIG.5A Top panel, Bar charts showing Compound 1 (Cmpd1) score and percent tumor enrichment of GATA6 / CYP3A / KRT17 cell populations as determined by multiplexed IF; bottom panel displays a heatmap showing the relative mRNA expression of Cmpd1 signature genes. FIG. 5B displays boxplots showing Cmpd1 signature scores stratified according to treatment as indicated. FIG.5C displays boxplots showing Cmpd1 signature scores stratified according to the Moffitt subtype in chemo-naive patient samples. FIG.5D displays boxplots showing Cmpd1 signature scores stratified according to treatment type in post-CTX patient samples. FIG.5E displays boxplots showing Cmpd1 signature scores stratified according to the Moffitt subtype in post-CTX patient samples.

[0020] FIGS.6A-6C show Compound 1+irinotecan signature and network analysis. FIG.6A displays Heatmap showing the relative expression of signature genes between Compound 1 (Cmpd1) plus irinotecan High and Low synergy scores. FIG.6B displays network of signature genes representing the maximum scoring subgraph obtained from the set of genes differentially expressed between samples exhibiting high and low Cmpd1plus irinotecan synergy scores. FIG.6C displays reactome pathway enrichment analysis showing significantly enriched pathways associated with the set of Cmpd1 plus irinotecan signature genes.

[0021] FIGS.7A-7E show Compound 1+irinotecan transcriptomic signature associations in organoid transcriptomic subtypes and treatment groups. FIG.7A displays bar graphs (top panel) showing Compound 1 plus irinotecan synergy scores and the percent tumor enrichment of GATA6 / CYP3A / KRT17 cells. Bottom panel displays a heatmap showing the relative mRNA expression of Compound 1 plus irinotecan signature genes. FIG.7B displays boxplots showing Cmpd1 plus irinotecan synergy scores stratified according to treatment asAttorney Docket No.52378-704.601 indicated. FIG.7C displays boxplots showing Cmpd1 plus irinotecan synergy scores stratified according to the Moffitt subtype in chemo-naive patient samples. FIG.7D displays boxplots showing Cmpd1 plus irinotecan synergy scores stratified according to treatment type in post-CTX patient samples. FIG.7E displays boxplots showing Cmpd1 plus irinotecan synergy scores stratified according to the Moffitt subtype in post-CTX patient samples.

[0022] FIGS.8A-8E show clinical and molecular details of the 36 human derived pancreatic cancer derived organoids. FIG.8A shows pie charts of number derived by biopsy versus from resection specimen, chemo-naive versus post-chemotherapy, and PurIST Classical- like versus Basal-like organoids. FIG.8B shows results of whole exome sequencing in 29 organoids showing the tumor mutational burden (TMB), and specific gene alterations. FIG.8C shows a chromosomal analysis heat map showing the log2 of the copy number (CN) ratio. FIG. 8D shows gene loss and gain plots between chemo-naive versus post-chemotherapy organoids. FIG.8E shows a heat map of RNASeq of all of the organoids showing the PurIST classification based on the continuous score and the expression levels of the eight most common Basal-like genes (including KRT5), and the eight most common Classical-like genes (including GATA6).

[0023] FIGS.9A-9B show mRNA expression levels of HDACs and GSK-3β (FIG.9A), and HADC-9 (FIG.9B) levels following Compound 1 treatment in organoids.

[0024] FIG.10 shows western blot analysis of CD44, SOX2, and ace-H3K9, and CD44 and SOX2 mRNA levels before and after Compound 1 treatment.

[0025] FIGS.11A-11K show induction of autophagy and apoptosis contributes to the anticancer activity of Compound 1. FIG.11A shows images for h02 wherein apoptosis-like morphology was observed in hPDOs following treatment with Compound 1. FIGS.11B-11E show western blot analysis of the corresponding cell death proteins in BxPC3 and PANC1 cells after treatment with Compound 1 versus the control group. FIGS.11F-11H show multiplexed IF images of representative BxPC3 cells after Compound 1 treatment and positive controls (irinotecan; TNF-α) stained with c-PARP (green), cleaved-Caspase 3 (red), p-MLKL (yellow) and DAPI (blue). Scale bars indicated in the figures are 20 µm. FIG.11I shows fluorescent images of BxPC3 cells transfected with the mCherry-GFP-LC3 plasmid which were subsequently treated with Compound 1. Chloroquine (CQ) was used as a control. FIG.11J shows a western blot of BxPC3 and PANC1 cells treated with Compound 1 in the presence or absence of 24 hours of CQ pre-treatment. The expression levels of LC3-II and p62 are shown. FIG.11K show bar graphs of cell viability (MTT assay) of BxPC3 andAttorney Docket No.52378-704.601 PANC1 cells treated with Compound 1 in the absence or presence of cell death inhibitors necrostatin-1 (targets RIP1), ZVAD-FMK (pan-caspase inhibitor), CQ, and 3-Methyladenine (3-MA, inhibits blocks autophagosome formation via class III PI3K complex inhibition). Scale bars indicated in the figures are 20 µm.

[0026] FIGS.12A-12D show Compound 1 synergistically increased apoptosis and autophagy when combined with irinotecan. FIGS.12A-12B show western blot analysis of different cell death proteins (c-PARP, p-MLKL and LC3-I / II) in BxPC3 cells after treatment with Compound 1 and cytotoxics as shown, re-probed for GAPDH to confirm equal loading. FIGS.12C-12D show representative IF localization images c-PARP (red), LC3-II (green), and DAPI (blue) after treatment of Compound 1, irinotecan and gemcitabine or combined (scale bar =100 µm). DETAILED DESCRIPTION

[0027] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0028] Disclosed herein are treatments of cancers by inhibiting GSK3-β, as well as blocking histone deacetylase (HDAC). Inhibition of GSK3-β driven tumor-promotion may be achieved via NF-κB activation, and HDAC inhibition may interfere with epithelial to mesenchymal transition (EMT), which otherwise would be enhanced by GSK3-β inhibition. Compound 1, a dual inhibitor of GSK3-β and HDAC, may be useful for achieving synergistic effects with certain chemotherapeutic treatment regimens and for uses in particular cancers.

[0029] Compound 1 is a dual inhibitor that was developed to inhibit GSK3-β driven tumor-promotion via NF-κB activation, as well as blocking histone deacetylase (HDAC) 75 classes to interfere with epithelial to mesenchymal transition (EMT), which otherwise would be enhanced by GSK3-β inhibition.

[0030] So far, studies involving Compound 1 have utilized two dimensional cell lines and KPC genetically engineered mouse models. Further investigations of Compound 1 using human PDAC derived three dimensional organoids (hPDO) that successfully model the genetic, morphological and biological properties of human tumor tissues have now provided further insights into particularly useful treatment utilizing Compound 1. The present disclosure introduces unique treatments for cancer that could be useful for increasing cancer cell sensitivity to chemotherapeutic agents by utilizing a glycogen synthase kinase-3 betaAttorney Docket No.52378-704.601 (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor (e.g., Compound 1). For example, incorporating Compound 1 into specific patient populations and / or with certain combinations of chemotherapeutics may be useful for obtaining synergies that could increase the effectiveness of these treatments.

[0031] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Methods of treating

[0032] In some aspects, provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor. In some embodiments, said cancer comprises an increased amount or expression of a GATA gene, a keratin 17 (KRT17) gene, a TP56 gene, a CYP3A gene, or a combination thereof (e.g., as compared to a non-cancerous cell). In some embodiments, said cancer comprises a decreased amount or function of a GATA protein, an increased amount or function of a KRT17 protein, a decreased amount or function of p53, or a combination thereof. In some embodiments, said subject has previously received an anti-cancer agent. In some embodiments, said subject has not previously received an anti-cancer agent. In some embodiments, said cancer comprises classical-like cancer cells. In some embodiments, said cancer comprises basal-like cancer cells.

[0033] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises an increased amount or expression of a GATA gene, a keratin 17 (KRT17) gene, a TP56 gene, a CYP3A gene, or a combination thereof.

[0034] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises a decreased amount or function of a GATA protein, an increased amount or function of a KRT17 protein, a decreased amount or function of p53, or a combination thereof (e.g., as compared to a non-cancerous cell).

[0035] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical compositionAttorney Docket No.52378-704.601 comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said subject has previously received an anti-cancer agent.

[0036] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said subject has not previously received an anti-cancer agent.

[0037] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises classical-like cancer cells.

[0038] In some aspects, further provided herein is a method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises basal-like cancer cells.

[0039] In some embodiments, said cancer has previously been identified as comprising the increased amount or expression of the GATA gene, the keratin 17 (KRT17) gene, the TP56 gene, a CYP3A gene, or a combination thereof. In some embodiments, said GATA gene is a GATA6 gene. In some embodiments, said cancer comprises the increased amount or expression of the GATA gene and the KRT17 gene. In some embodiments, said cancer comprises the increased amount or expression of the GATA gene. In some embodiments, said cancer comprises the increased amount or expression of the KRT17 gene. In some embodiments, said cancer comprises the increased amount or expression of the TP56 gene. In some embodiments, said cancer comprises the increased amount or expression of the CYP3A gene. In some embodiments, the CYP3A gene is a CYP3A5 gene. In some embodiments, said cancer comprises the increased amount or expression of said GATA gene, said KRT17 gene, and said TP56 gene.

[0040] The expression of the genes (e.g., GATA, KRT17, TP56, and CYP3A) may be determined in any appropriate manner, such as, for example, with the use of RNA sequencing and / or ELISA.

[0041] In some embodiments, said cancer comprises the increased amount or expression of the GATA protein and the KRT17 protein. In some embodiments, said cancer comprises the increased amount or expression of the GATA protein. In some embodiments, said cancer comprises the increased amount or expression of the KRT17 protein. In some embodiments, said cancer comprises the increased amount or expression of the TP56 protein. In someAttorney Docket No.52378-704.601 embodiments, said cancer comprises the increased amount or expression of the CYP3A protein. In some embodiments, the CYP3A protein is a CYP3A5 protein. In some embodiments, said cancer comprises the increased amount or expression of said GATA protein, said KRT17 protein, and said TP56 protein.

[0042] In some embodiments, said subject has not previously received an anti-cancer agent. In some embodiments, said subject has previously received an anti-cancer agent. In some embodiments, said anti-cancer agent comprises gemcitabine.

[0043] In some embodiments, said cancer has previously been identified as comprising the decreased amount or function of the GATA protein, the increased amount or function of the KRT17 protein, the decreased amount or function of p53, or a combination thereof .

[0044] In some embodiments, said cancer comprises the decreased amount or function of the GATA protein. In some embodiments, said cancer comprises the increased amount or function of the KRT17 protein. In some embodiments, said cancer comprises the decreased amount or function of the p53 protein. In some embodiments, said cancer comprises the decreased amount or function of the GATA protein and an increased amount or function of the KRT17 protein.

[0045] In some embodiments, said anti-cancer agent is not said GSK3-β inhibitor and said HDAC inhibitor. In some embodiments, said anti-cancer agent comprises an autophagy inhibitor. In some embodiments, said autophagy inhibitor comprises a phosphatidylinositol 3- kinases (PI3K) inhibitor. In some embodiments, said PI3K inhibitor is a PI3Kү inhibitor. In some embodiments, said autophagy inhibitor is selected from chloroquine, hydroxychloroquine, 3-methyladenine (3-MA), Verteporfin, NSC185058, SAR405, Spautin- 1, SBI-0206965, LY294002, and Wortmannin. In some embodiments, said anti-cancer agent comprises gemcitabine, irinotecan, fluorouracil (5FU), oxaliplatin, paclitaxel, folinic acid, or a combination thereof. In some embodiments, said anti-cancer agent comprises folinic acid, 5FU, irinotecan, and oxaliplatin (FOLFIRINOX).

[0046] In some embodiments, the method of treating a cancer in a subject in need thereof comprises administering a pharmaceutical composition comprising a compound represented by Formulas (I), (II), (III), (III-b), (IV), (V), (VI), or (VII), wherein said subject has previously received FOLFIRINOX as an anti-cancer agent. In some embodiments, said cancer expresses an increased amount or expression of GATA and KRT17. In some embodiments, said cancer comprises classical-type cancer cells.Attorney Docket No.52378-704.601

[0047] In some embodiments, said subject has previously received an anti-cancer agent for treatment of the cancer. In some embodiments, said subject has previously received an anti-cancer agent for treatment of a previous cancer.

[0048] In some embodiments, said subject has not previously received an anti-cancer agent for treatment of the cancer (e.g., the present cancer being treated). In some embodiments, said subject has not previously received an anti-cancer agent for treatment of a previous cancer. In some embodiments, said cancer comprises classical-like cancer cells.

[0049] In some embodiments, the method of treating a cancer in a subject in need thereof comprises administering a pharmaceutical composition comprising Compound 1 and Irinotecan, wherein said cancer comprises classical-like cancer cells.

[0050] The pancreatic cancer may contain two subtypes of pancreatic cancer that show high concordance in their gene expression profiles. In some embodiments, one subtype of pancreatic cancer is a classical-like subtype. In some embodiments, the classical-like subtype comprises classical-like cancer cells. In some embodiments, the classical-like subtype and the classical-like cancer cells are characterized by overlapping signature with the genes described in the Collisson classification, including GATA6. Generally, pancreatic cells categorized as classical-like cells demonstrate an overall better prognosis than cells categorized as basal-like cells. In some embodiments, the Collisson classification describes the classical-like subtype and the classical-like cancer cells as demonstrating high expression of adhesion-associated and epithelial genes, and epithelial cell terminal differentiation genes, notably GATA6, elevated KRAS mRNA levels relative to the other subtypes, and / or Classical subtype cell lines are more sensitive to erlotinib. The Collisson classification will be readily apparent to a person of skill in the art.

[0051] In some embodiments, the subtype of pancreatic cancer can be determined using RNA sequencing. The subtype of pancreatic cancer can be determined using gene expression microarray, whole-genome DNA microarrays, virtual microdissection, deep-exome sequencing, custom targeted gene panel sequencing, immunohistochemistry, DNA panel sequencing, single cell analysis, or a combination thereof. The subtype analysis may comprise structural rearrangements, tumor transcriptional profiles, stroma transcriptional profiles, or a combination thereof. Additionally, the Purity Independent Subtyping of Tumors (PurIST) classifier may be utilized to categorize cell types. The PurIST involves analysis of the expression of 16 genes to predict the previously established Moffitt basal-like and classical subtypes for each sample individually. In some embodiments, common basal- like subtype genes comprise BCAR3, ITGA3, PTGES, C16orf74, GPR87, S100A2, KRT6A,Attorney Docket No.52378-704.601 and KRT5 (FIG.8E). In some embodiments, common classical-like subtype genes comprise SLC40A1, DDC, CLRN3, CLDN18, REG4, GATA6, LGALS4, and ANXA10 (FIG.8E). The Moffitt classification will be readily apparent to a person of skill in the art.

[0052] The Basal-like cancer subtype and the basal-like cancer cells are often associated with worse survival than the classical-like cancer subtype or classical-like cancer cells and are characterized by high expression of genes related to epithelial to mesenchymal transition, a process by which tumor cells gain migratory and invasive properties. Concepts provided herein may be useful for treating a cancer (e.g., pancreatic cancer) comprising basal-like cells. In some embodiments, the basal-like subtype and the basal-like cancer cells may demonstrate an improved response to adjuvant therapy. In some embodiments, one subtype of pancreatic cancer is a basal-like subtype. In some embodiments, the cancer (e.g., pancreatic cancer) comprises basal-like cells.

[0053] In some embodiments the method of treating a cancer in a subject in need thereof comprises administering a pharmaceutical composition comprising Compound 1, wherein said cancer comprises basal-like cancer cells. In some embodiments, treatment of basal-like cells demonstrated increased sensitivity to Compound 1 than classical-like cells. In some embodiments, Compound 1 increases the sensitivity of basal-like subtype cells (e.g., compared to treatment of classical-like subtype cell lines).

[0054] In some embodiments, said cancer was previously identified as comprising said classical-like cells. In some embodiments, said cancer was previously identified as comprising said basal-like cells. In some embodiments, said classical-like cells are identified using a gene classifier. In some embodiments, said basal-like cells are identified using a gene classifier.

[0055] In some embodiments, the method further comprises administering an additional anti-cancer agent. In some embodiments, said additional anti-cancer agent comprises a chemotherapeutic agent.

[0056] In some embodiments, said chemotherapeutic agent comprises a topoisomerase I inhibitor. In some embodiments, the topoisomerase inhibitor is selected from topotecan, irinotecan, etoposide, and teniposide. In some embodiments, said chemotherapeutic agent comprises irinotecan or a derivative thereof. In some embodiments, said chemotherapeutic agent comprises irinotecan.

[0057] In some embodiments, said chemotherapeutic agent comprises a camptothecin derivative. In some embodiments, the camptothecin derivative comprises irinotecan, SN38, topotecan, belotecan, silatecan, karenitecin, or a combination thereof. In some embodiments,Attorney Docket No.52378-704.601 said chemotherapeutic agent comprises SN38 and derivatives thereof. In some embodiments, said chemotherapeutic agent comprises SN38. In some embodiments, said chemotherapeutic agent comprises an antimetabolite. In some embodiments, the antimetabolite is selected from 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), cytarabine, capecitabine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, and thioguanine. In some embodiments, the antimetabolite is 5-fluorouracil (5-FU). In some embodiments, the antimetabolite is 6- mercaptopurine (6-MP). In some embodiments, the antimetabolite is cytarabine. In some embodiments, the antimetabolite is capecitabine. In some embodiments, the antimetabolite is fludarabine. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the antimetabolite is methotrexate. In some embodiments, the antimetabolite is pemetrexed. In some embodiments, the antimetabolite is pentostatin. In some embodiments, the antimetabolite is thioguanine. In some embodiments, said chemotherapeutic agent comprises gemcitabine. In some embodiments, said chemotherapeutic agent comprises 5FU. In some embodiments, said chemotherapeutic agent comprises an alkylating agent. In some embodiments, the alkylating agent is selected from a nitrogen mustard, alkylsulfonate, nitrosourea, triazine, ethylenimine, and platinum drug. In some embodiments, the nitrogen mustard is selected from chlorambucil, cyclophosphamide, ifosfamide, and melphalan. In some embodiments, the alkylsulfonate is busulfan. In some embodiments, the nitrosourea is selected from streptozotocin, carmustine, and lomustine. In some embodiments, the triazine is dacarbazine. In some embodiments, the ethylenimine is thiotepa or altretamine. In some embodiments, the platinum drug is selected from cisplatin, carboplatin and oxaliplatin. In some embodiments, said chemotherapeutic agent comprises a platinum-containing agent. In some embodiments, said chemotherapeutic agent comprises oxaliplatin. In some embodiments, said chemotherapeutic agent comprises an anthracycline. In some embodiments, the anthracycline is selected from doxorubicin, daunorubicin, idarubicin and epirubicin. In some embodiments, said chemotherapeutic agent comprises a plant alkaloid. In some embodiments, the plant alkaloid is a taxane or a vinca alkaloid. In some embodiments, the taxane is paclitaxel or docetaxel. In some embodiments, the vinca alkaloid is selected from vinblastine, vincristine and vinorelbine. In some embodiments, said chemotherapeutic agent comprises a taxane. In some embodiments, said chemotherapeutic agent comprises paclitaxel. In some embodiments, said chemotherapeutic agent comprises a corticosteroid. In some embodiments, the corticosteroid is selected from prednisone, methylprednisolone and dexamethasone. In some embodiments, said chemotherapeutic agent comprises gemcitabine, irinotecan, 5FU, oxaliplatin, paclitaxel, folinic acid, or a combination thereof. In someAttorney Docket No.52378-704.601 embodiments, said chemotherapeutic agent comprises gemcitabine, irinotecan, paclitaxel, or a combination thereof. In some embodiments, chemotherapeutic agent comprises 5FU, irinotecan, and oxaliplatin. In some embodiments, the method further comprises administering folinic acid.

[0058] In some embodiments, said GSK3-β inhibitor and said HDAC inhibitor comprises a dual inhibitor of GSK3-β and HDAC. In some embodiments, said dual inhibitor of GSK3-β and HDAC is Compound 1.

[0059] In some embodiments, said cancer comprises a sarcoma. In some embodiments, said cancer comprises a carcinoma. In some embodiments, said carcinoma comprises an adenocarcinoma. In some embodiments, said cancer comprises pancreatic cancer. In some embodiments, said pancreatic cancer comprises an epithelioid sarcoma. In some embodiments, said pancreatic cancer comprises pancreatic ductal adenocarcinoma. Compounds

[0060] In some embodiments, said dual inhibitor of GSK3-β and HDAC is a compound represented by Formulas (I), (II), (III), (III-b), (IV), (V), (VI), or (VII).

[0061] In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (IV):wherein: L1 and L2 are in dependently a linker; R1is an aromatic moiety, alkyl, acyl, cyclyl or heterocyclyl, each of which can be optionally substituted; R2is hydrogen, lower alkyl, cyclyl, heterocyclyl, aryl, or heteroaryl, each of which can be optionally substituted; R3is absent or an aromatic moiety, which can be optionally substituted; p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and wherein –L1R1is linked to one nitrogen of the thiadiazolidine ring and– (CH2)p-R3-L 2- C(O)NH OR2is linked to the other nitrogen of the thiadiazolidine ring.Attorney Docket No.52378-704.601

[0062] In some embodiments for a compound of Formula (IV), the compound is further represented by Formula (VI):

[0063] In some embodiments for a compound of Formula (VI), the compound is further represented by Formula (I):wherein: X is a linker group; and Y is absent or an aromatic substituent.

[0064] In some embodiments for a compound of Formula (IV), the compound is further represented by Formula (VII):

[0065] In some embodiments for a compound of Formula (VII), the compound is further represented by Formula (II):wherein: X is a linker group, and R is -L1R1.Attorney Docket No.52378-704.601

[0066] In some embodiments for a compound of Formula (II), the compound is.

[0067] In some embodiments for a compound of Formula (IV), the compound is ,

[0068] In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound represented by the structure of:some embodiments, Compound 1 comprises a compound represented by.Attorney Docket No.52378-704.601

[0069] In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (V):L1 and L2 are independently a linker, wherein L1 is not a bond; R1is an aromatic moiety, alkyl, acyl, cyclyl or heterocyclyl, each of which is optionally substituted; R2is lower alkyl, cyclyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted, or hydrogen; R3is an aromatic moiety, which is optionally substituted; and p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0070] In some embodiments for a compound of Formula (V), L1 is NH, substituted amino, or alkyl.

[0071] In some embodiments for a compound of Formula (V), L2 is a bond.

[0072] In some embodiments for a compound of Formula (V), p is 0 or 1.

[0073] In some embodiments for a compound of Formula (V), R1is C1-C10alkyl, aryl, or heteroaryl, each of which is optionally substituted.

[0074] In some embodiments for a compound of Formula (V), R1is phenyl, which is optionally substituted.

[0075] In some embodiments for a compound of Formula (V), R2is hydrogen or lower alkyl.

[0076] In some embodiments for a compound of Formula (V), R3is phenyl, which is optionally substituted.

[0077] In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (III):wherein: X is a linker group; andAttorney Docket No.52378-704.601 Y is absent or selected from the group consisting of alkyl, CF3, NO2, CO2H, SO2H, cyano, hydroxyl, thiol, alkylthio, alkoxy, acyl, halogen, amino, alkyl amino, and dialkylamino.

[0078] In some embodiments for a compound of Formula (III), Y is alkoxy and X is a bond.

[0079] In some embodiments for a compound of Formula (III), the compound is.

[0080] In some embodiments, the compound has the structure of Formula (III-b):wherein: X is a linker group; and Y is selected from the group consisting of alkyl, CF3, NO2, CO2H, SO2H, cyano, hydroxyl, thiol, alkylthio, alkoxy, acyl, halogen, amino, alkyl amino, and dialkylamino.

[0081] In some embodiments for a compound of Formula (III-b), the compound is. Pharmaceutical Compositions

[0082] In certain embodiments, the pharmaceutical composition is administered to a mammal or human. In various embodiments, the pharmaceutical compositions according to the invention are formulated for intravenous, intratumoral, intramuscular, subcutaneous, or oral administration. In various embodiments, the pharmaceutical compositions according to the invention are formulated for intravenous or oral administration. In various embodiments, the pharmaceutical compositions according to the invention are formulated for intravenous administration. In various embodiments, the pharmaceutical compositions according to the invention are formulated for oral administration.Attorney Docket No.52378-704.601

[0083] In various embodiments, the pharmaceutical compositions according to the invention can be formulated for delivery via any route of administration. Kits

[0084] In some aspects, further provided herein is a kit for treating a cancer, comprising a test for determining whether a cancer comprises an increased amount or expression of a GATA gene, a keratin 17 (KRT17) gene, a TP56 gene, a CYP3A gene, or a combination thereof and a GSK3-β inhibitor and a HDAC inhibitor.

[0085] In some embodiments, said GSK3-β inhibitor and said HDAC inhibitor comprises a dual inhibitor of GSK3-β and HDAC.

[0086] In some embodiments, said dual inhibitor of GSK3-β and HDAC is a compound represented by Formulas (I), (II), (III), (III-b), (IV), (V), (VI), or (VII). In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (IV).

[0087] In some embodiments, said dual inhibitor of GSK3-β and HDAC comprises a compound represented by the structure

[0088] In some embodiments, the kit further comprises an additional anti-cancer agent.

[0089] In some embodiments, said additional anti-cancer agent comprises a chemotherapeutic agent. In some embodiments, said chemotherapeutic agent comprises a topoisomerase I inhibitor. In some embodiments, said chemotherapeutic agent comprises irinotecan. In some embodiments, said chemotherapeutic agent comprises an antimetabolite. In some embodiments, said chemotherapeutic agent comprises gemcitabine. In some embodiments, said chemotherapeutic agent comprises 5FU. In some embodiments, said chemotherapeutic agent comprises an alkylating agent. In some embodiments, said chemotherapeutic agent comprises a platinum-containing agent. In some embodiments, said chemotherapeutic agent comprises oxaliplatin. In some embodiments, said chemotherapeutic agent comprises a taxane. In some embodiments, said chemotherapeutic agent comprises paclitaxel. In some embodiments, said chemotherapeutic agent comprises a chemotherapeutic agent disclosed elsewhere herein.

[0090] In some embodiments, said chemotherapeutic agent comprises gemcitabine, irinotecan, 5FU, oxaliplatin, paclitaxel, folinic acid, or a combination thereof. In some embodiments, said chemotherapeutic agent comprises gemcitabine, irinotecan, paclitaxel, orAttorney Docket No.52378-704.601 a combination thereof. In some embodiments, said chemotherapeutic agent comprises 5FU, irinotecan, oxaliplatin, and folinic acid (FOLFIRINOX).

[0091] In some embodiments, said cancer comprises a sarcoma. In some embodiments, said cancer comprises a carcinoma. In some embodiments, said carcinoma comprises an adenocarcinoma. In some embodiments, said cancer comprises pancreatic cancer. In some embodiments, said pancreatic cancer comprises an epithelioid sarcoma. In some embodiments, said pancreatic cancer comprises pancreatic ductal adenocarcinoma. Definitions

[0092] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, certain preferred methods, devices, and materials are now described. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0093] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “an excipient” is a reference to one or more excipients and equivalents thereof known to those skilled in the art, and so forth.

[0094] The term “about” is used to indicate that a value includes 10% level of error for the device or method being employed to determine the value.

[0095] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and to “and / or.”

[0096] As used herein, the term “alkyl” means a straight or branched, saturated aliphatic radical having a chain of carbon atoms. Cxalkyl and Cx-Cyalkyl are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C1-C6alkyl includes alkyls that have a chain of between 1 and 6 carbons (e.g., methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and the like). Alkyl represented along with another radical (e.g., as in arylalkyl) means a straight or branched, saturated alkyl divalent radical having the number of atoms indicated or when no atoms are indicated means a bond, e.g., (C6-C10)aryl(C0-C3)alkyl includes phenyl, benzyl, phenethyl, 1-phenylethyl 3-Attorney Docket No.52378-704.601 phenylpropyl, and the like. Backbone of the alkyl can be optionally inserted with one or more heteroatoms, such as N, O, or S. The term “alkyl” includes heteroalkyl.

[0097] The term “heteroalkyl”, as used herein, refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0098] In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The term “alkyl” (or “lower alkyl”) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.

[0099] Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls.

[0100] The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl or heterocyclyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, n-propyloxy, iso-propyloxy, n-butyloxy, iso-butyloxy, and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O-cyclyl, —O-heterocyclyl, —O-aryl and —O- heteroaryl. The terms “alkoxyl” or “alkoxy” includes aroxy and aryloxy. Aroxy can be represented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined below. The alkoxy and aroxy groups can be substituted as described above for alkyl.

[0101] The term “aryl” refers to monocyclic, bicyclic, or tricyclic fused aromatic ring system. Cx aryl and Cx-Cyaryl are typically used where X and Y indicate the number of carbon atoms in the ring system. Exemplary aryl groups include, but are not limited to,Attorney Docket No.52378-704.601 pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, phenyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4- oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4- thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can be substituted by a substituent.

[0102] The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively. Cxheteroaryl and Cx- Cyheteroaryl are typically used where X and Y indicate the number of carbon atoms in the ring system. Heteroaryls include, but are not limited to, those derived from benzo[b]furan, benzo[b] thiophene, benzimidazole, imidazo[4,5-c]pyridine, quinazoline, thieno[2,3- c]pyridine, thieno[3,2-b]pyridine, thieno[2,3-b]pyridine, indolizine, imidazo[1,2a]pyridine, quinoline, isoquinoline, phthalazine, quinoxaline, naphthyridine, quinolizine, indole, isoindole, indazole, indoline, benzoxazole, benzopyrazole, benzothiazole, imidazo[1,5- a]pyridine, pyrazolo[1,5-a]pyridine, imidazo[1,2-a]pyrimidine, imidazo[1,2-c]pyrimidine,Attorney Docket No.52378-704.601 imidazo[1,5-a]pyrimidine, imidazo[1,5-c]pyrimidine, pyrrolo[2,3-b]pyridine, pyrrolo[2,3cjpyridine, pyrrolo[3,2-c]pyridine, pyrrolo[3,2-b]pyridine, pyrrolo[2,3- d]pyrimidine, pyrrolo[3,2-d]pyrimidine, pyrrolo[2,3-b]pyrazine, pyrazolo[1,5-a]pyridine, pyrrolo[1,2-b]pyridazine, pyrrolo[1,2-c]pyrimidine, pyrrolo[1,2-a]pyrimidine, pyrrolo[1,2- a]pyrazine, triazo[1,5-a]pyridine, pteridine, purine, carbazole, acridine, phenazine, phenothiazene, phenoxazine, 1,2-dihydropyrrolo[3,2,1-hi]indole, indolizine, pyrido[1,2- a]indole, 2(1H)-pyridinone, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H- pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Some exemplary heteroaryl groups include, but are not limited to, pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, 2- amino-4-oxo-3,4-dihydropteridin-6-yl, tetrahydroisoquinolinyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms of each ring may be substituted by a substituent.

[0103] The term “cyclyl” or “cycloalkyl” refers to saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons. Cxcyclyl and Cx-Cycylcyl are typically used where X and Y indicate the number of carbon atoms in the ring system. The cycloalkyl group additionally can be optionally substituted, e.g., with 1, 2, 3, or 4 substituents. C3-C10cyclyl includes cyclopropyl,Attorney Docket No.52378-704.601 cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl, cycloheptyl, cyclooctyl, bicyclo[2.2.2]octyl, adamantan-l-yl, decahydronaphthyl, oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 2-oxobicyclo[2.2.1]hept-l-yl, and the like.

[0104] Aryl and heteroaryls can be optionally substituted with one or more substituents at one or more positions, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or the like.

[0105] The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Cx heterocyclyl and Cx-Cy heterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent. Exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyl and the like.

[0106] The terms “bicyclic” and “tricyclic” refers to fused, bridged, or joined by a single bond polycyclic ring assemblies.

[0107] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[0108] “Administering” when used in conjunction with a therapeutic means to administer a therapeutic systemically or locally, as directly into or onto a target tissue, or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted. “Administering” a pharmaceutical composition may be accomplished by injection, topical administration, and oral administration or by other methods alone or in combination with other known techniques.

[0109] As used herein, the term “linker” means an organic moiety that connects two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR4, C(O), C(O)NH, C(O)O, NHC(O)O, OC(O)O, SO, SO2, SO2NH or a chainAttorney Docket No.52378-704.601 of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkyl aryl alkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, NR4, C(O), C(O)NH, C(O)O, NHC(O)O, OC(O)O, SO2NH, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R4is hydrogen, acyl, aliphatic or substituted aliphatic.

[0110] In some embodiments, the linker is a branched linker. The branchpoint of the branched linker may be at least trivalent, but can be a tetravalent, pentavalent or hexavalent atom, or a group presenting such multiple valencies. In some embodiments, the branchpoint is —N, —N(Q)-C, —O—C, —S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, — N(Q)C(O)—C, or —N(Q)C(O)O—C; wherein Q is independently for each occurrence H or optionally substituted alkyl. In some embodiments, the branchpoint is glycerol or derivative thereof.

[0111] A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood or serum of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0112] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. ExamplesAttorney Docket No.52378-704.601 of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; amidases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific) and proteases, and phosphatases.

[0113] A linker can include a cleavable linking group that is cleavable by a particular enzyme. A linking group cleavable by an enzyme can be an enzyme substrate that undergoes cleavage by the enzyme. Such a substrate is also referred to as cleavable enzyme substrate herein. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases. In some embodiments, the linker can include a cleavable linking group that is cleavable by Cathepsin G.

[0114] Exemplary cleavable linking groups include, but are not limited to, redox cleavable linking groups (e.g., —S—S— and —C(R)2—S—S—, wherein R is H or C1- C6alkyl and at least one R is C1-C6alkyl such as CH3or CH2CH3); phosphate-based cleavable linking groups (e.g., —O—P(O)(OR)—O—, —O—P(S)(OR)—O—, —O—P(S)(SR)—O—, —S—P(O)(OR)—O—, —O—P(O)(OR)—S—, —S—P(O)(OR)—S—, —O—P(S)(ORk)- S—, —S—P(S)(OR)—O—, —O—P(O)(R)—O—, —O—P(S)(R)—O—, —S—P(O)(R)— O—, —S—P(S)(R)—O—, —S—P(O)(R)—S—, —O—P(S)(R)—S—, —O—P(O)(OH)— O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O— P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, — O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, — S—P(O)(H)—S—, and —O—P(S)(H)—S—, wherein R is optionally substituted linear or branched C1-C10 alkyl); acid cleavable linking groups (e.g., hydrazones, esters, and esters of amino acids, —C═NN— and —OC(O)—); ester-based cleavable linking groups (e.g., — C(O)O—); peptide-based cleavable linking groups, (e.g., linking groups that are cleaved by enzymes such as peptidases and proteases in cells, e.g., —NHCHRAC(O)NHCHRBC(O)—, where RAand RBare the R groups of the two adjacent amino acids). A peptide based cleavable linking group comprises two or more amino acids. In some embodiments, the peptide-based cleavage linkage comprises the amino acid sequence that is the substrate for a peptidase or a protease found in cells.Attorney Docket No.52378-704.601

[0115] In some embodiments, an acid cleavable linking group is cleavable in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.5, 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid.

[0116] Linkers according to the present invention also include prodrug moieties and nanoparticles. For a non-limiting example, a prodrug moiety can be a linker that is susceptible to “cleavage” to produce active form of the drug. More information may be found in Bundgard (1985, Design of Prodrugs, pp.7-9, 21-24, Elsevier, Amsterdam) and Silverman (1992, the Organic Chemistry of Drug Design and Drug Action, pp.352-401, Academic Press, San Diego, Calif.), which are incorporated herein by reference in their entirety as though fully set forth.

[0117] As used herein, the term “substituted” refers to independent replacement of one or more (typically 1, 2, 3, 4, or 5) of the hydrogen atoms on the substituted moiety with substituents independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified. In general, a non-hydrogen substituent can be any substituent that can be bound to an atom of the given moiety that is specified to be substituted. Examples of substituents include, but are not limited to, acyl, acylamino, acyloxy, aldehyde, alicyclic, aliphatic, alkanesulfonamido, alkanesulfonyl, alkaryl, alkenyl, alkoxy, alkoxycarbonyl, alkyl, alkylamino, alkylcarbanoyl, alkylene, alkylidene, alkylthios, alkynyl, amide, amido, amino, amino, aminoalkyl, aralkyl, aralkylsulfonamido, arenesulfonamido, arenesulfonyl, aromatic, aryl, arylamino, arylcarbanoyl, aryloxy, azido, carbamoyl, carbonyl, carbonyls (including ketones, carboxy, carboxylates, CF3, cyano (CN), cycloalkyl, cycloalkylene, ester, ether, haloalkyl, halogen, halogen, heteroaryl, heterocyclyl, hydroxy, hydroxy, hydroxyalkyl, imino, iminoketone, ketone, mercapto, nitro, oxaalkyl, oxo, oxoalkyl, phosphoryl (including phosphonate and phosphinate), silyl groups, sulfonamido, sulfonyl (including sulfate, sulfamoyl and sulfonate), thiols, and ureido moieties, each of which may optionally also be substituted or unsubstituted. In some cases, two substituents, together with the carbon(s) to which they are attached to, can form a ring.

[0118] By “pharmaceutically acceptable”, it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0119] A “pharmaceutically acceptable salt”, as used herein, is intended to encompass any compound described herein that is utilized in the form of a salt thereof, especially where the salt confers on the compound improved pharmacokinetic properties as compared to the free form of compound or a different salt form of the compound. The pharmaceuticallyAttorney Docket No.52378-704.601 acceptable salt form can also initially confer desirable pharmacokinetic properties on the compound that it did not previously possess and may even positively affect the pharmacodynamics of the compound with respect to its therapeutic activity in the body. An example of a pharmacokinetic property that can be favorably affected is the manner in which the compound is transported across cell membranes, which in turn may directly and positively affect the absorption, distribution, biotransformation and excretion of the compound. While the route of administration of the pharmaceutical composition is important, and various anatomical, physiological and pathological factors can critically affect bioavailability, the solubility of the compound is usually dependent upon the character of the particular salt form thereof, which it utilized. One of skill in the art will appreciate that an aqueous solution of the compound will provide the most rapid absorption of the compound into the body of a subject being treated, while lipid solutions and suspensions, as well as solid dosage forms, will result in less rapid absorption of the compound.

[0120] The term “pharmaceutical composition” means a composition comprising at least one active ingredient, such as a steroid or a pharmaceutically acceptable salt thereof or a dual inhibitor of GSK3-β and HDAC or a pharmaceutically acceptable salt thereof, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

[0121] A “therapeutically effective amount” or “effective amount” as used herein refers to the amount of active compound or pharmaceutical agent that elicits a biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and / or symptomatology), and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and / or symptomatology).Attorney Docket No.52378-704.601

[0122] The terms “treat,” “treated,” “treatment,” or “treating” as used herein refers to both therapeutic treatment in some embodiments and prophylactic or preventative measures in other embodiments, wherein the object is to prevent or slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. A prophylactic benefit of treatment includes prevention of a condition, retarding the progress of a condition, stabilization of a condition, or decreasing the likelihood of occurrence of a condition. As used herein, “treat,” “treated,” “treatment,” or “treating” includes prophylaxis in some embodiments.

[0123] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. EXAMPLES

[0124] The following examples further illustrate the invention but should not be construed as in any way limiting its scope. In particular, the processing conditions are merely exemplary and can be readily varied by one of ordinary skill in the art.

[0125] All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the inventionAttorney Docket No.52378-704.601 unless otherwise claimed. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Example 1. Assessment of Compound 1 as mono- and combo-therapy in Human PDAC organoids

[0126] A library of 36 was characterized in molecular and functional terms to develop an experimentally tractable preclinical model system for further investigating particularly useful treatments with Compound 1, including, for example, when used as a dual inhibitor. For example, de novo transcriptomic signatures (TS) modeling drug response to Compound 1 (IC50) and Compound 1 plus irinotecan, respectively, was developed. Single Sample Gene Set Enrichment Analysis was performed to generate continuous TS scores, and further analyzed against a separate cohort of primary PDAC tissues characterized by RNASeq and multiplex immunofluorescence.

[0127] Compound 1 (an inhibitor of GSK3-β and histone deacetylase) when combined with the cytotoxics gemcitabine, irinotecan (IR), 5FU, oxaliplatin, and paclitaxel was synergistic when tested in human PDAC organoids (hPDOs). Basal-subtype hPDOs were more sensitive to Compound 1 and there was synergy with the Compound 1+IR combination in Classical-subtype hPDOs with increased apoptosis and autophagy. Continuous Transcriptomic signature (TS) scores derived by Single Sample Gene Set Enrichment Analysis from the hPDOs were evaluated in primary PDAC tissues. Compound 1-TSHIwere enriched for mRNA splicing and DNA repair processes, were associated with Basal-tissues but also with GATA6+ve-chemo-naïve samples and were higher following gemcitabine but not FOLFIRINOX. In contrast, Compound 1+IR-TSHIwere enriched for TP53 pathways, were associated with Classical-pretreatment samples and with GATA6+ve / KRT17+vehybrid cell types following FOLFIRINOX, but not gemcitabine treatment, and were unrelated to transcriptional subtype. Compound 1 as a single agent and in combination with irinotecan offers novel strategies for treating pancreatic cancer.

[0128] Methods

[0129] Patient Characteristics. Thirty PDAC tissues for human organoid (hPDO) generation were obtained from patients undergoing surgical resection plus three metastatic biopsy specimens from the University Clinic Heidelberg and three primary tumor biopsies from the Ludwig-Maximillian University (LMU), Munich. There were 29 hPDOs derived from patients that had not received any chemotherapy (chemo-naïve) and seven hPDOs generated from patients that had received prior chemotherapy. Companion RNASeq dataAttorney Docket No.52378-704.601 was obtained from 35 of these - PDO h32 did not have companion RNASeq data. For testing transcriptomic signatures (TS) derived from these hPDOs a separate cohort of 47 cryo- preserved PDAC tissues with both RNASeq and companion multiplex IF data was used. Samples from patients who received chemoradiation at any time were excluded from the present analysis. All samples were confirmed as PDAC tumors by specialist pancreatic cancer pathologists. Patient characteristics were extracted from the clinical database and anonymized. The study has been approved by the Ethics Committee of Heidelberg University for use of pancreatic cancer tissue and organoid generation (Project Nos. S-018 / 2020, S- 708 / 2019 and S-083 / 2021) and the Ludwig Maximilian University (LMU) of Munich. All patients provided informed consent for use of their tissue and clinical data in accordance with the Principles of Helsinki. The patient demographics are provided in Table 1. Table 1. Patient demographics and pathologic variablesAttorney Docket No.52378-704.601Values in parentheses are percentages; *values are median [IQR]; staging 8thAJCC edition.

[0130] Biological agents. Purchased reagents and antibodies are detailed in Table 2. The primary antibodies for IF and IHC were diluted in antibody diluents (with Background Reducing Components). The primary antibodies for WB were diluted in 0.1% TBS-T with 5% BSA. Table 2. Antibodies for IF / IHC and WB.Attorney Docket No.52378-704.601

[0131] Cell culture experiments and organoid generation and propagation. AsPC1, BxPC3, MiaPaCa2, and PANC1 cell lines were purchased from American Tissue Culture Collection (Manassas, VA) and grown in RPMI-1640 supplemented with 10% fetal bovine serum and 1% of antibiotic / antimycotics solution. The patient-derived Mayo-5289 cell line (PXC) (D Mukhopadhyay Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Jacksonville, FL 32224, USA) was cultured in Advanced DMEM / F-12 supplemented with 10% fetal bovine serum (FBS), 1X L-glutaMAX, 10 mM HEPES. Cell lines were maintained in 75 cm2flasks at 37°C and 5% CO2. The medium was changed twice a week, and cells were passaged when they achieved 80% confluence. hPDOs were derived and cultivated according to Tuveson (https: / / tuvesonlab.labsites.cshl.edu / protocolsreagents / ).

[0132] RNA and DNA sequencing. RNA and DNA was extracted from snap-frozen hPDO pellet samples using the AllPrep DNA / RNA / miRNA Universal Kit (Qiagen). Sanger sequencing. The organoid cell lines were initially checked for KRAS mutations by DNA Sanger sequencing. Primers sequences for amplification and sequencing of exons of the KRAS gene that contain the G12 / 13 codons were:

[0133] KRAS G12 / 13 Forward: 5′-CTGGTGGAGTATTTGATAGTG-3′.

[0134] KRAS G12 / 13 Reverse: 5′-CTGTATCAAAGAATGGTCCTG-3′.

[0135] PCR products were purified using a QIAquick PCR purification kit, then sent and sequenced by Eurofins and sequence analysis was undertaken using Mutation Surveyor software (SoftGenetics, USA).

[0136] RNA Seq. Sequencing libraries were prepared using the Illumina TruSeq mRNA stranded Kit following the manufacturer’s instructions. Briefly, mRNA was purified from 500ng of total RNA using oligo(dT) beads. Then poly(A)+ RNA was fragmented to 150 bp and converted to cDNA. The cDNA fragments were then end-repaired, adenylated on the 3′ end, adapter ligated and amplified with 15 cycles of PCR. The final libraries were validatedAttorney Docket No.52378-704.601 using Qubit (Invitrogen) and Tapetstation (Agilent Technologies). 2x 100 bp paired-end sequencing was performed on the Illumina NovaSeq 6000 according to the manufacturer’s protocol. At least 54 Mio. reads per sample were generated.

[0137] Whole-Exome sequencing. Libraries were generated using the SureSelectXT Automation Reagent Kit and SureSelectXT Human All Exon v7 Capture Library (Agilent Technologies) following the manufacturer’s instructions. In brief, 200ng of gDNA was fragmented to ~150bp using a Covaris LE220 ultrasonicator (Covaris, Inc.). Subsequently, library preparation was performed on a Bravo automated liquid handler (Agilent Technologies) including end- repair, A-tailing, adaptor ligation and amplification. The concentration of amplified, adaptor- ligated DNA library was determined using the TapeStation (Agilent Technologies). In the subsequent steps 750ng of amplified, adaptor- ligated DNA library was used for the hybridization reaction with the SureSelectXT All Exon v7 bait set. The DNA-library / bait hybrids were captured using streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin T1 by Thermo Fisher Scientific). Index tags were added in the course of PCR-amplification of the captured libraries.

[0138] Sequencing data is available at: https: / / ega- archive.org / studies / EGAS00001007143

[0139] Pharmacological assay of cell lines and organoids. The survival of cell lines was measured by MTT assay. The organoids were dissociated before plating 1000 cells in 10 μL Matrigel per well in white 96-well plates (Greiner). Cytotoxic drugs were dissolved in DMSO (concentrations were normalized to 0.25% DMSO) and added 72 hours after plating. All drugs were tested in triplicate at concentrations ranging from 1.0 × 10−7to 1.0 × 10−3mol / L for 5-FU, irinotecan and oxaliplatin; from 1.0 × 10−10to 1.0 × 10−6mol / L for gemcitabine (Sigma), paclitaxel (Selleckchem), and SN38 (Sigma) and for from 1.0 × 10−7to 4.0 × 10−4mol / L for Compound 1. After 96 hours of treatment, cell viability was assessed using the CellTiter-Glo 3D cell viability assay (Promega). A four-parameter log-logistic function with an upper limit equal to the mean of the DMSO values was fitted to the drug response curve and IC50’s were calculated.

[0140] RNA Isolation and quantitative real-time RT-PCR. RNA from cell lines and hPDOs was extracted by using the Trizol-Chloroform method. Two-step quantitative PCR (qPCR) was performed using a SYBR Green PCR Master Mix kit (Thermo Fisher). Expression values of the targeted gene in a given sample was normalized to the corresponding expression of GAPDH as ΔCT. The 2-ΔΔCt method was used to calculate relative expression of the targeted genes after the treatment.Attorney Docket No.52378-704.601

[0141] Histology for organoids. Organoids were fixed in 4% paraformaldehyde solution and embedded in paraffin. Sections were subjected to H&E and immunofluorescence (IF) staining. Images of H&E and IF staining were acquired using imaging system Tissue-FAXS software (Tissue Gnostics, Austria). H&E images were acquired using a 20X objective lens using a bright field. IF images were acquired using a 20X objective lens with light-emitting diodes (LED) and with specific light filters. IF images of negative control sections were used to set the appropriate gating to exclude background immunofluorescence and non-specific binding signals. The expression level of each protein was calculated by the percentage of protein-positive stained cells in DAPI-positive cells.

[0142] Western blotting. Protein extracts from organoids were lysed in RIPA Lysis Buffer 50 with a protease inhibitor cocktail (Sigma) and phosphatase inhibitor (Sigma) and quantified using Pierce BCA protein assay kit (ThermoFisher). Following SDS-PAGE and transfer to PVDF membranes (Bio-Rad, 1704273), the membranes were blocked in Tris- buffered saline containing 5% BSA and 0.1% Tween 20 (TBS-T) for 1 hour before incubation with the primary antibody overnight at 4°C. After being washed three times in TBS-T and then incubated with species corresponding secondary antibodies (Anti-Mouse IgG, LI-COR, 1:10000; Anti-Rabbit IgG, LI-COR, 1:10000), the membrane was then visualized with an ODYSSEY CLx (LI-COR) image system.

[0143] Statistical analyses. GraphPad Prism was used to conduct statistical analyses utilizing the student t- test, one-way analysis of variance, and Fisher’s exact test (GraphPad Software, La Jolla, CA). A P value less than or equal to 0.05 was considered statistically significant. The ggstatsplot R package was used to generate boxplots and assess the significance of sample comparisons. WES and RNAseq analysis: paired exome sequencing data was aligned, and SNVs and indels were called using the DKFZ-ODCF workflows.

[0144] Alignment and QC workflows: https: / / github.com / DKFZ- ODCF / AlignmentAndQCWorkflows

[0145] SNV calling workflow: https: / / github.com / DKFZ-ODCF / SNVCallingWorkflow.

[0146] Indel calling workflow: https: / / github.com / DKFZ-ODCF / IndelCallingWorkflow.

[0147] SNV and Indel output was converted from VCF to MAF format using a custom R script and then .maf files were summarized and visualized using maftools R package CNV calling and CNV visualization was done using cnvkit tool. RNA-sequencing data were aligned and expression was quantified using the DKFZ-ODCF RNAseq workflow 28: https: / / github.com / DKFZ-ODCF / RNAseqWorkflow.Attorney Docket No.52378-704.601

[0148] For the downstream analysis log2(TPM+1) gene expression values were used. For classical / basal annotation the PurIST score was calculated ; “classical” label was assigned for organoids with PurIST score <= 0.05, and “basal” label for organoids with PurIST score > 0.05. For expression visualization complex heatmap R package was used.

[0149] Synergistic scores: For each cell line and hPDOs tested in vitro drug synergistic assay, the synergistic score of drugs combination was calculated by SynergyFinder, a web application that uses essential functions of the R-package.

[0150] Signature Generation: Drug response data obtained from PDOs was used to generate gene expression signatures representing response to Compound 1 or synergistic response to Compound 1 plus irinotecan. To generate signatures the dNetPipeline function in the dnet R package was initially used and P-values representing the significance of differential gene expression between high (greater than 66 percentile) and low (lower than 33 percentile) response / synergy values to identify a maximum scoring subgraph from the STRING human functional protein association network. Each gene signature is the set of nodes (genes) representing the maximum scoring subgraph with coefficients of the P-values representing gene weights.

[0151] Reactome pathway enrichment was assessed using the clusterProfiler R package.

[0152] Signature Scores: Gene signature scores representing the signed average of the set of genes making up each gene signature were calculated from normalized RNAseq data using the sig.score function as implemented in the genefu R package. Gene signature scores representing each patient sample were ordered by increasing value and signature genes visualized using the ComplexHeatmap R package.

[0153] Results

[0154] Genetic and transcriptomic profiling of organoids. Organoids were obtained from 36 patients with histologically confirmed PDAC in 31 cases from primary tumor following resection and in five cases from biopsies of the primary in two and three from liver metastases. (Table 1; FIGS.8A-8E). There were 29 hPDOs derived from patients that had not received any chemotherapy (chemo-naïve) and seven hPDOs generated from patients that had received prior chemotherapy (post-chemotherapy, h03, h20, h43, h44, h48, h51, h57). KRAS codon-12 / 13 mutations were found in 31 of 36 (86%) hPDOs (Sanger sequencing in 34, whole exome sequencing (WES) in 29). FIG.8B shows results of whole exome sequencing in 29 organoids showing the tumor mutational burden (TMB), and specific gene alterations. Whole-exome sequencing in 29 organoids (7 post-chemotherapy and 22 chemo- naïve) revealed that the main driver mutated genes were KRAS (83%), TP53 (66%),Attorney Docket No.52378-704.601 CDKN2A (41%), and SMAD4 (34%) (FIG.8B). FIG.8C shows a chromosomal analysis heat map showing the log2 of the copy number (CN) ratio (when this value is 0 it means no CN gain or loss, a single CN gain = 0.585, and a single CN loss = -1.0).The mutational burden (single-nucleotide variants and insertion-deletion) was similar between the chemo-naive and post-chemotherapy groups, although copy number variation, and copy number gain, was more apparent in the post-chemotherapy group indicating accumulated chromosomal instability (FIGS.8C-8D). No significant difference in gene losses between chemo-naive versus post-chemotherapy organoids but a significant increase in gene gains in the post- treatment organoids (FIG.8D). A heat map of RNASeq of all of the organoids showing the PurIST classification based on the continuous score and the expression levels of the eight most common Basal-like genes (including KRT5), and the eight most common Classical-like genes (including GATA6) is shown in FIG.8E. Transcriptome profiling identified nine PurIST Basal-like (h08, h69, h43, h63, h74, h03, h40, h36, and h33) and 27 Classical-like organoids (FIG.8E).

[0155] Phenotypic characterization of organoids. Representative chemo-naïve hPDOs, eight Classical-like and one Basa-like (h03) were shown to express mRNA levels of HDACs 1-10 as well as GSK-3β (FIG.9A). mRNA levels are shown for ten individual HDACs 1-10, and GSK-3β relative to GAPDH in chemo-naïve organoids. h03 was Basal-like and the remaining organoids were Classical-like. Organoid h19 shown to be very chemo-resistant (Figure 2) had GSK-3β mRNA level 2-5 folds higher than in the other organoids tested. In addition, the mRNA levels of HDACs 5, 6, 7, 8, and 9 were the highest in the resistant organoid h19 compared to the other organoids. The increase in the level of HDAC9 was 5 to 50 folds compared to the other organoids. HDACs 1 to 4 and 10 were also highly expressed in the resistant h19 organoid. These data indicate a strong association between the levels of GSK-3β and HDACs, especially HDAC9, and the resistance to chemotherapy. In some cases, Compound 1 induced a significant decrease in the mRNA level of HDAC9 (FIG.9B). mRNA levels for HDAC9 at baseline (h’n’) and following treatment with Compound 1 (h’n’MT). HDAC9 was the only HDAC to demonstrate mRNA HDAC following Compound 1 treatment.

[0156] Correspondingly with the reduced expression of HDAC9 there was an increase in the acetylated-H3K9 protein levels after Compound 1 treatment (FIG.10). Previously mRNA levels of the stem cell markers Sox2, Nanog and CD133 were shown to be reduced in the MiaPaCa2, and BxPC3 cell lines by Compound 1. Here mRNA and protein levels were reduced in the cancer stemness markers Sox2 and CD44 in the organoids followingAttorney Docket No.52378-704.601 Compound 1 treatment (FIG.10). Compound 1 treatment caused strong H3K9 acetylation in all eight human derived PDAC organoids, previously only shown in experimental systems.

[0157] Molecular subtype responses to Compound 1. First, the effects of Compound 1 were compared on the ASPIC1 and PXC Classical-like and the BxPC3, MiaPaCa2 and PANC1 Basal-like cells. To confirm activity of Compound 1 protein levels of serine-9 phosphorylation of GSK-3β (p-GSK3β) and acetylation of H3K9 (ace-H3K9) were measured in PDAC cell lines and hPDOs by western blotting after 72 hours of treatment with the indicated concentrations of Compound 1. Blots were re-probed for GAPDH to confirm equal loading (n=3; *p<0.05). The protein and mRNA expression levels of serine-9 phosphorylation of GSK-3β (p-GSK3β) and acetylation of H3K9 (ace-H3K9) in BxPC3 and PANC1 cell lines were significantly upregulated after Compound 1 (Figure 1A-1C). Increased cellular protein expression of p-GSK-3β and ace-H3K9 in BxPC3 cells of Compound 1 was demonstrated by immunofluorescence and reproduced using tideglusib (TDG) and SAHA the respective inhibitors of GSK-3β and HDAC (classes I and II) (Figure 1E-1F). Intracellular activity was confirmed of Compound 1 protein levels of p-GSK3β and ace-H3K9 by immunofluorescence localization in the PDAC cell lines using Compound 1 or the positive controls tideglusib (TDG) and SAHA (the respective inhibitors of GSK-3β and HDAC) (scale bar=20 µm). Cells were stained for p-GSK-3β (green), ace-H3K9 (red), and DAPI (blue) (FIGS.1E-1F). The Basal-like subtype cell lines (e.g., BxPC3, MiaPaCa2, PANC1) were relatively more sensitive to Compound 1 than the Classical-like cell lines (e.g., AsPC1, PXC) (Figure 1G). The Basal-like cell lines were also more sensitive to GSK-3β inhibition by tideglusib but not to HDAC acetylation by SAHA (Figure 1H-1J). FIGS.1H- 1I shows a similar MTT assay response in Classical-like and Basal-like cell lines to mono- inhibitor treatment for 72 hours was found for the GSK-3β inhibitor tideglusib whereas all five cell lines were highly sensitive to the HDAC inhibitor SAHA (n=3). The specific IC50 values in response to 72h treatment with Compound 1, tideglusib and SAHA in PDAC cell lines are shown (n=3) in FIG.1J. Second, it was shown that in organoids there was also increased protein levels of p-GSK3β inhibitory phosphorylation and ace-H3K9 after Compound 1 (Figure 1D). As in the cell lines the Basal-like hPDOs were more sensitive to Compound 1 than Classical-like hPDOs but there was no difference between the chemo-naive and post-treatment groups (mean ± SEM) (Figure 1K). The increased sensitivity of Basal- like cells may be linked to intrinsically higher GSK-3β protein expression (Figures 1L-1N). Protein GSK-3β expression of PDAC cell lines re-probed for GAPDH shows increased expression in the Basal like cell-lines compared to the Classical-like cell lines (n=3). (*pAttorney Docket No.52378-704.601 <0.05, **p <0.01, 2-sided unpaired t test) (FIG.1L). Representative immunofluorescent co- localization images stained for GSK-3β (green) and DAPI (blue) also showing greater expression of GSK-3β in Basal-like (FIG.1N) compared to Classical-like organoids (FIG. 1M) (scale bar=100 µm). (*p <0.05, **p <0.01, 2-sided unpaired t test).

[0158] Organoid sensitivity to Compound 1 and individual cytotoxics. The therapeutic response to 5-FU, oxaliplatin, and irinotecan, gemcitabine and paclitaxel cytotoxic reagents was assessed in all 36 hPDOs. Drug response of organoids from different subtypes and treatment groups showed heterogeneity both for the same drug and the different drugs (Figure 2). Basal hPDOs were more sensitive to Compound 1 and gemcitabine. Different IC50 distribution for the 36 hPDOs treated with Compound 1 and five standard cytotoxics are shown in FIG.2A. The white to yellow scale represents the relative sensitive and resistant responses as a continuum. Each box shows the actual IC50 value (n=3). Individual drug organoid sensitivities, comparing Basal versus Classical-like hPDOs and organoids derived from chemo-naive versus post-chemotherapy PDAC tissues (FIGS.2B- 2G). Compound 1 (FIG.2B) (p=0.001) and gemcitabine (FIG.2C) (p=0.046) were each more potent in the Basal-like organoids.

[0159] Compound 1 induces autophagy mediated apoptosis. Compound 1-treated organoids showed morphological apoptosis-like characteristic cell blebbing and shrinkage, nuclear fragmentation, condensation and fragmentation of genetic materials (FIG.11A). Apoptosis-like morphology was observed in hPDOs following treatment with Compound 1 (20 μM) for 72 hours, representative images for h02 are shown (FIG.11A). Western blot analysis of the corresponding cell death proteins in BxPC3 and PANC1 cells after 24-72 hours of treatment with Compound 1 (0,5,20 μM) versus the control group (n=3; *p=0.05) are shown in FIGS.11B-11E. In BxPC3 and PANC1 cell lines Compound 1 demonstrated features of apoptosis with cleavage of poly-ADP-ribose polymerase (c-PARP), and autophagy with lipidation of microtubule-associated protein 1 light chain 3 to generate the electrophoretically mobile form II (LC3-II), but not necroptosis as shown by the marker levels of phosphorated Mixed lineage kinase domain-like protein (p-MLKL) (FIGS.11B- 11E), and supported by immunofluorescent levels of consisted of staining of c-PARP, cleaved-Caspase3, and p-MLKL with corresponding death inducers as positive control (FIGS.11F-11H). Multiplexed IF images of representative BxPC3 cells after Compound 1 treatment (0, 20 μM) and positive controls (irinotecan 10 μM; TNF-α 10 μM) stained with c- PARP (green), cleaved-Caspase 3 (red), p-MLKL (yellow) and DAPI (blue) areshown in FIGS.11F-11H. Scale bars indicated in the figures are 20 µm.Attorney Docket No.52378-704.601

[0160] As autophagy is a dynamic multistep process and elevated LC3-II levels are linked to autophagosome production or turnover, Compound 1-induced autophagic flux was evaluated next using a tandem mCherry-GFP-LC3 reporter fluorescence experiment (FIG. 11I). BxPC3 cells were transfected with the mCherry-GFP-LC3 plasmid. After 24 hours, cells were treated with Compound 1 (0,5,20 μM) for another 72 hours. Chloroquine (CQ) (20 μM) which inhibits autophagy by impairing autophagosome fusion with lysosomes used as a control. Fluorescent microscopy revealed mCherry-GFP-LC3 as a diffuse cytoplasmic pool for the untreated group. The homogeneous fusion of the red and green fluorescence was exhibited. The treated group showed that Compound 1 exposure dose-dependently led to significantly increased numbers of mCherry-tagged LC3 protein puncta. In contrast, the number of GFP puncta did not increase due to the acid environment resulting from autophagosome fusion and lysosome. This observation suggests that Compound 1 stimulates the formation of the autophagosomes and activates the autophagic flux in BxPC3 cells. In contrast, additional yellow labeled puncta (overlay between mCherry and GFP puncta) were identified in the negative control group due to the fusion block of the autophagosome and lysosome by chloroquine. BxPC3 and PANC1 cells were treated with Compound 1 (0,5,20 μM) for 72 hours in the presence or absence of 24 hours of CQ pre-treatment. The expression levels of LC3-II and p62 are shown (FIG.11J). *P <0.05. In addition, after blocking the fusion of autophagosome and lysosome pretreated with chloroquine, Compound 1 treatment still caused upregulated LC3-II expression in a dose-dependent fashion (FIG.11J). Next, a rescue assay was performed on BxPC3 and PANC1 cell lines by pretreatment with necroptosis inhibitors (necrostatin-1), an apoptosis inhibitor (ZVAD-FMK) plus autophagy inhibitors chloroquine and 3-methyladenine before receiving Compound 1 treatment. BxPC3 and PANC1 cells were treated with Compound 1 in the absence or presence of cell death inhibitors necrostatin-1 (targets RIP1), ZVAD-FMK (pan-caspase inhibitor), CQ, and 3- Methyladenine (3-MA, inhibits blocks autophagosome formation via class III PI3K complex inhibition) for 24 hours. Cell viability was assayed by MTT (n=3, *p < 0.05) (FIG.11K). Scale bars indicated in the figures are 20 µm. Only the early-stage autophagy inhibitor 3- methyladenine restored cell viability (FIG.11K).

[0161] Synergistic effects of Compound 1 and cytotoxics. The dose-dependent matrix viability assay in PDAC cell lines was initially used to determine drug sensitivity at various dose combinations to assess any potential synergy effect between Compound 1 and the cytotoxic drugs. Five established cell lines received Compound 1 in combination with either gemcitabine, irinotecan, 5-FU, oxaliplatin, or paclitaxel in different doses (Figure 3). TheAttorney Docket No.52378-704.601 synergistic score is calculated by the Zero interaction potency (ZIP) model. Values over 10 indicating strong synergy are depicted in dark red, and values over three but less than ten (indicating low synergy are depicted in light red. Scores between -3 and 3 were considered as dual drug additives. Values less than 10 indicating strong antagonistic are depicted in dark blue, and values less than -3 but more than -10 indicating low synergy are depicted in light blue. The Basal-like AsPC1, and PXC cells, and the Classical-like BxPC3, MIA PaCa2, and PANC1 cells, were treated for 72 hours with up to 19.2 μM cytotoxic drug (n=3) (FIG.3A). FIG.3B shows synergistic scores of Compound 1 combined with other drugs (n=3). Values over 10 indicating strong synergy are depicted in dark red, and values over three but less than ten (indicating low synergy are depicted in light red. Scores between -3 and 3 were considered as dual drug additives. Values less than 10 indicating strong antagonistic are depicted in dark blue, and values less than -3 but more than -10 indicating low synergy are depicted in light blue. Classical-like organoids were more synergistic than Basal-like hPDOs (p=0.001), and organoids derived from chemo-naive tissues were also more synergistic than organoids derived from tissues following chemotherapy (p=0.017) (FIG.3C). Compound 1+IR had significantly higher scores in the Classical-like hPDOs (p=0.0095) (FIG.3E). Red indicates a high synergistic effect, white a neutral effect is white, and dark blue a high antagonistic effect. Overall Compound 1 exhibited a relatively strong but variable synergistic effect when combined with gemcitabine, irinotecan and paclitaxel in all five of the PDAC cell lines. In some cases, a variable synergistic effect of Compound 1 when combined individually with all five tested cytotoxics was also seen across the 36 hPDOs. The synergistic effect was greater in the Classical than the Basal-like organoids and similarly in the chemo-naïve than in the post-treatment derived organoids. Synergy was observed in 22 of the 36 hPDOs treated with gemcitabine, in 24 with irinotecan, 18 with 5-FU, 22 with oxaliplatin, and in 15 treated with paclitaxel. In some cases, the synergistic effect of Compound 1 with irinotecan, was also most evident in the Classical-like organoids. FIGS. 12A-12B show western blot analysis of different cell death proteins (c-PARP, p-MLKL and LC3-I / II) in BxPC3 cells after 72 hours of treatment with Compound 1 and cytotoxics as shown, re-probed for GAPDH to confirm equal loading (n=3). Compound 1+irinotecan induced higher levels of c-PARP (p=0.012) and LC3-I / II (p=0.014). FIGS.12C-12D show representative IF localization images c-PARP (red), LC3-II (green), and DAPI (blue) after 72 hour treatment of Compound 1, irinotecan and gemcitabine or combined (scale bar =100 µm). Compound 1 greatly increased apoptosis and autophagy when combined with irinotecan (FIGS.12A-12D).Attorney Docket No.52378-704.601

[0162] Transcriptomic Signatures. Drug response data obtained from hPDOs (IC50s) were used to generate continuous gene expression signatures (TS) – by Single Sample Gene Set Enrichment Analysis – representing response to Compound 1 or synergistic response to Compound 1+IR. These TSs were evaluated in 47 primary PDAC tissues that had both RNASeq analysis and multiplex immunofluorescence (IF) including GATA6, CYP3A5 and KRT17. The clinical data are summarized in Table 1. Compound 1-TSHIscores were significantly associated with hPDOs exhibiting a Basal-like phenotype and were enriched for mRNA splicing and DNA repair molecular processes (Figure 4). FIG.4A displays heatmap showing the relative expression of signature genes between Compound 1 (Cmpd1) High and Low IC50 values. High and Low Cmpd1 IC50 groups represent the upper 33% quantile of IC50 and lower 33% quantile of Compound 1 IC50 values, respectively. FIG.4B displays network of signature genes representing the maximum scoring subgraph obtained from the set of genes differentially expressed between high and low Cmpd1 IC50 groups. Nodes (circles) represent genes and lines represent functional protein associations as curated by STRING. The degree of network centrality is shown by the size of each node. Node size denotes a higher degree of interconnectedness. FIG.4C displays reactome pathway enrichment analysis showing significantly enriched pathways associated with the set of Cmpd1 signature genes. P-values are adjusted by Bonferroni-Hochberg correction. Compound 1-TSHIscores were higher in Basal-like transcriptional states in resected chemo-naïve patient samples (Figure 5). FIG.5A Top panel, Bar charts showing Compound 1 (Cmpd1) score and percent tumor enrichment of GATA6 / CYP3A / KRT17 cell populations as determined by multiplexed IF. A LOESS regression line has been added to each tumor cell enrichment bar plot. Bottom panel, Heatmap showing the relative mRNA expression of Cmpd1 signature genes. Heatmap annotation shows sample characteristics. Patient samples in top and bottom panels are identical (n=47) and similarly ordered according to Cmpd1 sensitivity. FIG.5B displays boxplots showing Cmpd1 signature scores stratified according to treatment as indicated. FIG.5C displays boxplots showing Cmpd1 signature scores stratified according to the Moffitt subtype in chemo-naive patient samples. FIG.5D displays boxplots showing Cmpd1 signature scores stratified according to treatment type in post-CTX patient samples. FIG.5E displays boxplots showing Cmpd1 signature scores stratified according to the Moffitt subtype in post-CTX patient samples. Mann-Whitney rank sum test (two-sided) P-values are shown on the plots. Boxplots show the median (line), the interquartile range (IQR) between the 25th and 75th percentiles (box) and 1.5× the IQR ± the upper and lower quartiles. P-values were not adjusted for multiple testing.Attorney Docket No.52378-704.601

[0163] Compound 1-TSHIscores were higher following neoadjuvant chemotherapy with gemcitabine but not with FOLFIRINOX. In chemo-naïve samples whilst Compound 1-TSHIscores were associated with a Basal-like phenotype they were but also associated with GATA6+vetissues. Compound 1-TSHIscores were not significantly associated with transcriptional states in post-chemotherapy patient samples. In contrast, Compound 1+IR- TSHIscores were enriched for TP53 regulatory processes and or functions and were significantly higher in post-chemotherapy patient samples (Figure 6). FIG.6A displays a heatmap showing the relative expression of signature genes between Compound 1 (Cmpd1) plus irinotecan High and Low synergy scores. High and Low synergy scores represent the upper 33% quantile and lower 33% quantile of synergy scores, respectively. FIG.6B displays network of signature genes representing the maximum scoring subgraph obtained from the set of genes differentially expressed between samples exhibiting high and low Cmpd1 plus irinotecan synergy scores. Nodes (circles) represent genes and lines represent functional protein associations as curated by STRING. The degree of network centrality is shown by the size of each node. Node size denotes the degree of interconnectedness. FIG.6C displays reactome pathway enrichment analysis showing significantly enriched pathways associated with the set of Cmpd1 plus irinotecan signature genes. P-values are adjusted by Bonferroni- Hochberg correction.

[0164] Compound 1+IR-TSHIscores were significantly associated with Classical-like transcriptional states in resected chemo-naïve patient samples, consistent with the findings in hPDOs (Figure 7). FIG.7A Top panel, Bar charts showing Compound 1 (Cmpd1) plus irinotecan synergy scores and the percent tumor enrichment of GATA6 / CYP3A / KRT17 cell populations as determined by multiplexed IF. A LOESS regression line has been added to each tumor cell enrichment bar plot. Bottom panel, Heatmap showing the relative mRNA expression of Cmpd1 plus irinotecan signature genes. Heatmap annotation shows sample characteristics. Patient samples in top and bottom panels are identical (n=47) and similarly ordered according to Cmpd1 plus irinotecan synergy score. FIG.7B displays boxplots showing Cmpd1 plus irinotecan synergy scores stratified according to treatment as indicated. FIG.7C displays boxplots showing Cmpd1 plus irinotecan synergy scores stratified according to the Moffitt subtype in chemo-naive patient samples. FIG.7D displays boxplots showing Cmpd1 plus irinotecan synergy scores stratified according to treatment type in post- CTX patient samples. FIG.7E displays boxplots showing Cmpd1 plus irinotecan synergy scores stratified according to the Moffitt subtype in post-CTX patient samples. Mann- Whitney rank sum test (two-sided) P-values are shown on the plots. Boxplots show theAttorney Docket No.52378-704.601 median (line), the interquartile range (IQR) between the 25th and 75th percentiles (box) and 1.5× the IQR ± the upper and lower quartiles. P-values were not adjusted for multiple testing.

[0165] Compound 1+IR-TSHIscores were significantly higher in post-chemotherapy patient samples after FOLFIRINOX but not after gemcitabine and were unrelated to transcriptional subtype. Compound 1+IR-TSHIscores were associated with GATA6+ve / KRT17+vehybrid cell types in predominantly post-chemotherapy patient samples.

[0166] DISCUSSION

[0167] In some cases, effective systemic therapy for PDAC is based on cytotoxic regimens but the ceiling in terms of survival has been reached with triplet therapies (NALIRIFOX and FOLFIRINOX). Targeted therapies based on specific genetic alterations such as BRCA1 / 2 mutations, microsatellite instability (MSI-Hi) or deficient mismatch repair, and NTRK1 / 2 / 3 fusions benefit no more than 5% of all patients. This study characterized the effectiveness of a first-in-class dual inhibitor named Compound 1 in treating pancreatic cancer. Strong inhibition of HDAC9, and increased GSK3-β serine-9 phosphorylation and H3K9 acetylation was confirmed in human derived PDAC organoids previously shown only in established cell lines and KPC mice. In some cases, cell death was observed in all of the organoids in response to Compound 1 with autophagy mediated apoptosis being the principal mechanism, which was increased by the synergistic combination of Compound 1 with irinotecan. Both GSK3-β and HDAC inhibitors have been shown to target autophagy. Herein is demonstrated that susceptibility to Compound 1 or Compound 1 plus irinotecan is associated with an increased enrichment of pathways associated with DNA damage repair, transcriptional regulation by TP53 and RNA splicing. TP53 mutations which occur in greater than 70% on PDAC can lead to impaired regulation of autophagy, increased replications stress, aberrant ribosome biogenesis and genomic instability. Compound 1 treatment of human organoids also strongly downregulated mRNA and protein levels of the cancer stem cell markers CD44 and SOX2 associated with EMT and chemotherapy resistance. A group of Basal-like subtype cell lines and hPDOs expressing relatively higher GSK-3β protein levels were more sensitive to Compound 1 than the Classical-subtype. This observation suggests that the PDAC molecular subtype as shown in cell-lines and hPDOs may influence their response to Compound 1. It has become apparent that PDAC tumors undergo plasticity over time and in response to certain types of chemotherapy notably FOLFIRINOX shifting to more Basal-like subtypes. In which case the use of chemotherapy in Basal-like tumors could be particularly amenable to Compound 1 treatment. The increased PDAC cell killing byAttorney Docket No.52378-704.601 GSK-3β inhibition and gemcitabine in cell lines has been ascribed to regulation of the TopBP1 / ATR / Chk1 DNA damage response pathway. In the present study using hPDOs synergistic cytotoxicity of Compound 1 with standard cytotoxics used to treat pancreatic cancer was shown. The combination of Compound 1 and irinotecan displayed significant synergistic anti-tumor effect in Classical-subtypes, and greatly increased apoptosis and autophagy. Irinotecan is a prodrug that is converted into the active metabolite SN-38 and resistance appears to be tumor-cell intrinsic metabolism by uridine diphosphate glucuronosyltransferase 1A1 and cytochrome P450 mediation. In some cases, the synergistic action of Compound 1 with irinotecan is that GSK-3β inhibitors have been shown to activate the WNT / β-catenin pathway that regulate the expression of CYP2E.

[0168] In some embodiments, it was found that that Compound 1-TSHIscores were significantly associated with hPDOs exhibiting a Basal-like phenotype and were enriched for mRNA splicing and DNA repair molecular processes, linking GSK3-β inhibition with Basal- like transcriptional states. In some cases, the association of Compound 1-TSHIand Compound 1+IR-TSHIscores with different molecular profiles may open up strategies for treating pancreatic cancer, as different cell regulatory mechanisms appear to be susceptible to single agent Compound 1 (mRNA splicing and DNA repair molecular processes) or to Compound 1+IR (regulation of TP53 molecular processes). In some cases, Compound 1+IR- TSHIscores were significantly higher in post-chemotherapy patient samples after FOLFIRINOX but not after gemcitabine and were associated with GATA6+ve / KRT17+vehybrid cell types that have recently been identified as persistent cells following with resistance to irinotecan therapy (and hence FOLFIRINOX). Precision clinical trials using Compound 1 alone or in combination with irinotecan are now being developed incorporating these signatures.

[0169] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No.52378-704.601 CLAIMS What is claimed is:

1. A method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises an increased amount or expression of a GATA gene, a keratin 17 (KRT17) gene, a TP56 gene, a CYP3A gene, or a combination thereof.

2. The method of claim 1, wherein said cancer has previously been identified as comprising the increased amount or expression of the GATA gene, the keratin 17 (KRT17) gene, the TP56 gene, a CYP3A gene, or a combination thereof.

3. The method of claim 1 or 2, wherein said GATA gene is a GATA6 gene.

4. The method of any one of claims 1-3, wherein said cancer comprises the increased amount or expression of the GATA gene and the KRT17 gene.

5. The method of any one of claims 1-4, wherein said cancer comprises the increased amount or expression of the GATA gene.

6. The method of any one of claims 1-5, wherein said cancer comprises the increased amount or expression of the KRT17 gene.

7. The method of any one of claims 1-6, wherein said cancer comprises the increased amount or expression of the TP56 gene.

8. The method of any one of claims 1-7, wherein said cancer comprises the increased amount or expression of the CYP3A gene.

9. The method of claim 8, wherein the CYP3A gene is a CYP3A5 gene.

10. The method of claim 1, wherein said cancer comprises the increased amount or expression of said GATA gene, said KRT17 gene, and said TP56 gene.

11. The method of any one of claims 1-10, wherein said subject has not previously received an anti-cancer agent.

12. The method of any one of claims 1-10, wherein said subject has previously received an anti-cancer agent.

13. The method of claim 12, wherein said anti-cancer agent comprises gemcitabine.

14. A method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor,Attorney Docket No.52378-704.601 wherein said cancer comprises a decreased amount or function of a GATA protein, an increased amount or function of a KRT17 protein, a decreased amount or function of p53, or a combination thereof.

15. The method of claim 14, wherein said cancer has previously been identified as comprising the decreased amount or function of the GATA protein, the increased amount or function of the KRT17 protein, the decreased amount or function of p53, or a combination thereof.

16. The method of claim 14 or 15, wherein said cancer comprises the decreased amount or function of the GATA protein.

17. The method of any one of claims- 14-16, wherein said cancer comprises the increased amount or function of the KRT17 protein.

18. The method of any one of claims 14-17, wherein said cancer comprises the decreased amount or function of the p53 protein.

19. The method of The method of claim 14, wherein said cancer comprises the decreased amount or function of the GATA protein and an increased amount or function of the KRT17 protein.

20. A method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said subject has previously received an anti-cancer agent.

21. The method of claim 20, wherein said anti-cancer agent is not said GSK3-β inhibitor and said HDAC inhibitor.

22. The method of claim 20, wherein said anti-cancer agent comprises an autophagy inhibitor.

23. The method of claim 22, wherein said autophagy inhibitor comprises a phosphatidylinositol 3-kinases (PI3K) inhibitor.

24. The method of claim 23, wherein said PI3K inhibitor is a PI3Kү inhibitor.

25. The method of claim 20, wherein said anti-cancer agent comprises gemcitabine, irinotecan, fluorouracil (5FU), oxaliplatin, paclitaxel, folinic acid, or a combination thereof.

26. The method of claim 20, wherein said anti-cancer agent comprises folinic acid, 5FU, irinotecan, and oxaliplatin (FOLFIRINOX).Attorney Docket No.52378-704.601 27. The method of claim 26, wherein said cancer expresses an increased amount or expression of GATA and KRT17.

28. The method of claim 26 or 27, wherein said cancer comprises classical-type cancer cells.

29. The method of any one of claims 20-26, wherein said subject has previously received an anti-cancer agent for treatment of the cancer.

30. The method of any one of claims 20-26, wherein said subject has previously received an anti-cancer agent for treatment of a previous cancer.

31. A method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said subject has not previously received an anti-cancer agent.

32. The method of claim 31, wherein said subject has not previously received an anti- cancer agent for treatment of the cancer.

33. The method of claim 31, wherein said subject has not previously received an anti- cancer agent for treatment of a previous cancer.

34. The method of claim 31, wherein said cancer comprises classical-like cancer cells.

35. A method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises classical-like cancer cells.

36. The method of claim 35, wherein said cancer was previously identified as comprising said classical-like cells.

37. The method of claim 35 or 36, wherein said classical-like cells are identified using a gene classifier.

38. A method of treating a cancer in a subject in need thereof, said method comprising administering a pharmaceutical composition comprising a glycogen synthase kinase-3 beta (GSK3-β) inhibitor and a histone deacetylase (HDAC) inhibitor, wherein said cancer comprises basal-like cancer cells.

39. The method of claim 38, wherein said cancer was previously identified as comprising said basal-like cells.Attorney Docket No.52378-704.601 40. The method of claim 38 or 39, wherein said basal-like cells are identified using a gene classifier.

41. The method of any one of claims 1-38, further comprising administering an additional anti-cancer agent.

42. The method of claim 38, wherein said additional anti-cancer agent comprises a chemotherapeutic agent.

43. The method of claim 42, wherein said chemotherapeutic agent comprises a topoisomerase inhibitor.

44. The method of claim 43, wherein said chemotherapeutic agent comprises a topoisomerase I inhibitor.

45. The method of claim 43, wherein said chemotherapeutic agent comprises a camptothecin derivative.

46. The method of any one of claims 43-45 , wherein said chemotherapeutic agent comprises irinotecan or a derivative thereof.

47. The method of claim 45, wherein the camptothecin derivative comprises irinotecan, SN38, topotecan, belotecan, silatecan, karenitecin, or a combination thereof.

48. The method of claim 46, wherein said chemotherapeutic agent comprises irinotecan or a derivative thereof.

49. The method of claim 48, wherein said chemotherapeutic agent comprises irinotecan.

50. The method of claim 46, wherein said chemotherapeutic agent comprises SN38 and derivatives thereof.

51. The method of claim 50, wherein said chemotherapeutic agent comprises SN38.

52. The method of claim 42, wherein said chemotherapeutic agent comprises an antimetabolite.

53. The method of claim 52, wherein said chemotherapeutic agent comprises gemcitabine.

54. The method of claim 52, wherein said chemotherapeutic agent comprises 5FU.

55. The method of claim 42, wherein said chemotherapeutic agent comprises an alkylating agent.

56. The method of claim 42, wherein said chemotherapeutic agent comprises a platinum- containing agent.

57. The method of claim 56, wherein said chemotherapeutic agent comprises oxaliplatin.

58. The method of claim 42, wherein said chemotherapeutic agent comprises a taxane.

59. The method of claim 58, wherein said chemotherapeutic agent comprises paclitaxel.Attorney Docket No.52378-704.601 60. The method of claim 42, wherein said chemotherapeutic agent comprises gemcitabine, irinotecan, 5FU, oxaliplatin, paclitaxel, folinic acid, or a combination thereof.

61. The method of claim 42, wherein chemotherapeutic agent comprises gemcitabine, irinotecan, paclitaxel, or a combination thereof.

62. The method of claim 42, wherein chemotherapeutic agent comprises 5FU, irinotecan, and oxaliplatin.

63. The method of claim 62, further comprising administering folinic acid.

64. The method of any one of claim 1-38, wherein said GSK3-β inhibitor and said HDAC inhibitor comprises a dual inhibitor of GSK3-β and HDAC.

65. The method of any one of claims 1-64, wherein said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (IV):wherein: L1 and L2 are in dependently a linker; R1is an aromatic moiety, alkyl, acyl, cyclyl or heterocyclyl, each of which can be optionally substituted; R2is hydrogen, lower alkyl, cyclyl, heterocyclyl , aryl, or heteroaryl, each of which can be optionally substituted; R3is absent or an aromatic moiety, which can be optionally substituted ; p is 0, 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10; and wherein –L1R1is linked to one nitrogen of the thiadiazolidine ring and– (CH2)p-R3- L2-C(O)NH OR2is linked to the other nitrogen of the thiadiazolidine ringAttorney Docket No.52378-704.601 66. The method of any one of claims 1-64, wherein said dual inhibitor of GSK3-β and HDAC comprises a compound represented by the structure of.

67. The method of any one of claim 1-66, wherein said cancer comprises a sarcoma.

68. The method of claim 67, wherein said pancreatic cancer comprises an epithelioid sarcoma.

69. The method of any one of claim 1-66, wherein said cancer comprises a carcinoma.

70. The method of claim 69, wherein said carcinoma comprises an adenocarcinoma.

71. The method of any one of claim 1-70, wherein said cancer comprises pancreatic cancer.

72. The method of claim 67, wherein said pancreatic cancer comprises pancreatic ductal adenocarcinoma.

73. A kit for treating a cancer, comprising: a. a test for determining whether a cancer comprises an increased amount or expression of a GATA gene, a keratin 17 (KRT17) gene, a TP56 gene, a CYP3A gene, or a combination thereof; and b. a GSK3-β inhibitor and a HDAC inhibitor.

74. The kit of claim 73, wherein said GSK3-β inhibitor and said HDAC inhibitor comprises a dual inhibitor of GSK3-β and HDAC.

75. The kit of any one of claims 73-74, wherein said dual inhibitor of GSK3-β and HDAC comprises a compound of Formula (IV):wherein: L1and L2are in dependently a linker; R1is an aromatic moiety, alkyl, acyl, cyclyl or heterocyclyl, each of which can be optionally substituted;Attorney Docket No.52378-704.601 R2is hydrogen, lower alkyl, cyclyl, heterocyclyl , aryl, or heteroaryl, each of which can be optionally substituted; R3is absent or an aromatic moiety, which can be optionally substituted ; p is 0, 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10; and wherein –L1R1is linked to one nitrogen of the thiadiazolidine ring and– (CH2)p- R3-L 2-C(O)NH OR2is linked to the other nitrogen of the thiadiazolidine ring.

76. The kit of claim 73 or 74, wherein said dual inhibitor of GSK3-β and HDAC comprises a compound represented by the structure77. The kit of any one of claims 73-76, further comprising an additional anti-cancer agent.

78. The kit of claim 77, wherein said additional anti-cancer agent comprises a chemotherapeutic agent.

79. The kit of claim 78, wherein said chemotherapeutic agent comprises a topoisomerase I inhibitor.

80. The kit of claim 79, wherein said chemotherapeutic agent comprises irinotecan.

81. The kit of claim 78, wherein said chemotherapeutic agent comprises an antimetabolite.

82. The kit of claim 81, wherein said chemotherapeutic agent comprises gemcitabine.

83. The kit of claim 81, wherein said chemotherapeutic agent comprises 5FU.

84. The kit of claim 78, wherein said chemotherapeutic agent comprises an alkylating agent.

85. The kit of claim 78, wherein said chemotherapeutic agent comprises a platinum- containing agent.

86. The kit of claim 85, wherein said chemotherapeutic agent comprises oxaliplatin.

87. The kit of claim 78, wherein said chemotherapeutic agent comprises a taxane.

88. The kit of claim 87, wherein said chemotherapeutic agent comprises paclitaxel.

89. The kit of claim 78, wherein said chemotherapeutic agent comprises gemcitabine, irinotecan, 5FU, oxaliplatin, paclitaxel, folinic acid, or a combination thereof.Attorney Docket No.52378-704.601 90. The kit of claim 78, wherein said chemotherapeutic agent comprises gemcitabine, irinotecan, paclitaxel, or a combination thereof.

91. The kit of claim 78, wherein said chemotherapeutic agent comprises 5FU, irinotecan, oxaliplatin, and folinic acid (FOLFIRINOX).

92. The kit of any one of claims 73-91, wherein said cancer comprises a sarcoma.

93. The kit of claim 92, wherein said pancreatic cancer comprises an epithelioid sarcoma.

94. The kit of any one of claim 73-93, wherein said cancer comprises a carcinoma.

95. The kit of claim 94, wherein said carcinoma comprises an adenocarcinoma.

96. The kit of any one of claim 73-95, wherein said cancer comprises pancreatic cancer.

97. The kit of claim 96, wherein said pancreatic cancer comprises pancreatic ductal adenocarcinoma.

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