WO2018119418A1

WO2018119418A1 - Methods relating to the treatment of chemoresistant and immuno-resistant cancer

Info

Publication number: WO2018119418A1
Application number: PCT/US2017/068251
Authority: WO
Inventors: Nikita Shah; Nathaniel HORWITZ
Original assignee: Nivien Therapeutics Company
Priority date: 2016-12-24
Filing date: 2017-12-22
Publication date: 2018-06-28

Abstract

Disclosed herein are methods of treating, reducing, delaying, preventing or inhibiting the proliferation, growth, and/or migration of cancer, such as those that are chemoresistant or immune-resistant, or both. Such methods comprise administering an inhibitor of, or disrupting, components that activate the Hippo- Yap pathway, such as through phosphorylation of YAP, including MST1/2, LATS1/2, NF2, MAP4Ks, TAOK1/3, in combination with therapeutic agents, such as chemotherapy, chemotherapeutic agent, anticancer agent, antineoplastic agent, cytotoxic agent, anti-angiogenesis agent, and immune checkpoint inhibitor, or combinations thereof.

Description

METHODS RELATING TO THE TREATMENT OF CHEMORESISTANT AND

IMMUNO-RESISTANT CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Serial No: 62/439,022, filed on

December 24, 2016, the contents of which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The invention described herein relates to the adjuvant treatment of cancer, such as those that are chemoresistant and immuno-resistant.

BACKGROUND

Despite the innovation of new drug modalities to treat cancer, chemotherapy remains the mainstay of oncology (Holohan et al., 2013 Nature Reviews Cancer, 73(10), 714-726). Drug resistance is partly mediated through cellular mechanisms such as drug efflux and metabolism, which lead to a decline in the cellular concentration of

chemotherapies (Szakacs et al., 2006 Nature reviews Drug discovery, 5(3), 219-234). The heterogeneity of molecular drivers of cancer is believed to account for differences in patient's response to chemotherapy (Chang et al., 2014 Nature Reviews Cancer 14, 291- 292). For example, some cancers which do not respond to chemotherapy may have highly transcribed genes coding for cytoplasmic enzymes that break down chemo-toxins, thereby preventing damage to the cell. This allows tumorigenic cells to overtake somatic cells in the presence of chemotherapy. The cancer stem cell hypothesis postulates that tumors may be sustained exclusively by a small fraction of cells, which are resistant to or survive treatment (Donnenburg & Donnenbrug, 2005 The Journal of Clinical

Pharmacology, 45(β), 872-877). Effective methods to overcome cell resistance are needed to improve patient' s response to chemotherapy and thereby increase likelihood of remission.

In this passage, we describe a counter-intuitive approach for modulating the Hippo- Yap pathway to treat cancer in combination with chemotherapy. The Hippo- Yap pathway was first identified in a forward genetic screen for tumor suppressors in Drosophila; a loss-of-function mutation resulted in cell proliferation and reduced cell death (Zhao et al., 2010 Genes & development, 24(9), 862-874). This pathway is conserved in mammalian species, where it is responsible for regulating genes for cell fate, by receiving and transducing signals from the plasma membrane to the nucleus (Harvey et al., 2013 Nature Reviews. ' Cancer. 13(4), 246-257). Aberrations in this pathway are correlated with tumor progression and metastasis in cancer patients (Pan, 2010 Developmental cell, 19(4), 491-505). Permanent disruption of the Hippo- Yap pathway, in mice, leads to tumorigenesis (Pan, 2010 Developmental ce l, 19(4), 491-505). These findings point to using Hippo- YAP activation to treat cancer, up-regulating tumor suppressor activity to halt cancer progression. This has been the subject of interest in numerous scientific papers, and related drug discovery efforts in oncology.

The basic Hippo- Yap pathway is comprised of core Hippo kinases, upstream regulators and downstream effectors. Stimuli such as cellular stressors or growth inhibitory signals activate the kinase cassette Mstl/2, which phosphorylates large tumor suppressor kinase 1/2 (Latsl/2), another kinase. When activated Latsl/2 phosphorylates the proteins Yap/Taz, sequestering them in the cytoplasm, Yap/Taz is ubiquitinated and ultimately degraded. In the absence of Mstl/2 mediated inhibition, Yap translocates to the nucleus, where it complexes with transcription factors such as TEAD to activate gene expression, most notably genes involved in cell proliferation. Other inputs regulate the core pathway; neurofibromin 2 (NF2) recruits Latsl/2 to the cytoplasm, leading to the degradation of Yap. Homologues of Mats (Mobla/b) mediates activity of the core pathway by enhancing activation of Latsl/2. The kinases Taokl/3 and Map4ks work in parallel to Mstl/2, also leading to the phosphorylation of Latsl/2, and therefore Yap/Taz.

Despite extensive studies investigating the function of the Hippo- Yap pathway, it has not yet been identified or concluded that inhibiting the Hippo- Yap pathway, thereby increasing the amount of nuclear Yap, is a beneficial strategy for the treatment of cancer. For example, activation of Hippo- Yap is commonly proposed via inhibition of Yap/Taz or GPCRs, thought to be oncogenes, to treat cancer, rather than inhibition of Hippo kinases or other upstream activating components (Johnson & Haider, 2014 Nature reviews Drug discovery, 13(1), 63-79). However, the scientific literature allows, while Hippo- Yap is central to controlling tissue and organ size, tumor formation only occurs with prolonged or permanent dysregulation of the Hippo- Yap pathway (Fan et al., 2016 Science Translaiional Medicine, 5(352), 352ral08-352ral 08). Furthermore, while there is one study suggesting inhibition of the Hippo- Yap pathway could increase sensitivity to chemotherapy, the overarching conclusion that inactivation of the Hippo- YAP pathway could serve a therapeutic purpose was not codified by the researchers (Jeong et al, 2014 Anticancer research. 2014;34(2):811-817). No recommendation was made for applying this method to the treatment of cancer (Jeong et al, 2014 Anticancer research. 2014;34(2):811-817). A subset of studies suggest that Hippo- Yap may play a tissue type-dependent oncogenic or tumorigenic role, but this is a broad generalization that has not been confirmed.

Until now, molecular mechanisms governing Yap's transcriptional program involved in chemotherapy sensitization were not fully understood. It has been established that when Yap enters the nucleus it initiates the transcription of several proto-oncogenes leading to cell growth and division (Harvey, et al., 2013 Nature Reviews Cancer, 13(4), 246-257). A novel finding is that Yap also causes the down regulation of several multidrug efflux transporters and drug metabolizing enzymes, through its action in the nucleus, including ABCG2, MVP, ABCC3, ABCC5, as well as a drug metabolizing enzyme cytidine deaminase (CDA). Because drug resistance is partly mediated by low levels of intracellular drug, turning off cellular programs that would otherwise reduce drug concentrations may be one plausible mechanism by which tumor-drug sensitivity is restored by inactivation of the Hippo- Yap pathway and the subsequent increase of Yap in the nucleus. In fact, inhibition of drug efflux and drug inactivating enzymes has been a popular target for the development of anticancer drugs, with disappointing clinical results so far due to ineffective inhibition (Szakacs et al., 2006 Nature reviews Drug discovery, 5(3), 219-234: Gillet & Gottesman, 2010 Multi- drug resistance in cancer, 47-76: Holohan et al., 2013 Nature Reviews Cancer, 73(10), 714-726). One potential explanation is that candidates involved in the inhibition of a single drug efflux transporter may have little impact relative to the inactivation of Hippo- Yap, which leads to the simultaneous down- regulation of four transporters and a key drug metabolizing enzyme (Szakacs et al., 2006 Nature reviews Drug discovery, 5(3), 219-234: Gillet & Gottesman, 2010 Multi- drug resistance in cancer, 47-76: Holohan et al., 2013 Nature Reviews Cancer, 73(10), 714- 726). This understanding of the drug-resistance mechanism provides impetus to pursue a therapeutic approach of inactivating Hippo- Yap to treat patients suffering from

chemoresistant cancer.

Herein, we describe the first use of the inhibition of the Hippo- Yap pathway to treat cancer, in particular metastatic, advanced or unresectable cancer, or as an adjuvant therapy for resectable or local cancer. SUMMARY

In one embodiment of the present invention, a method is provided for the treatment of chemoresistant and immuno-resistant cancer, including pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, mesothelioma, stomach cancer, bowel cancer, and other cancers of the pancreas.

In another embodiment of the present invention, this method is described as a combination therapy to be administered in combination with a chemotherapy or

chemotherapeutic agent, typically one prescribed as the front-line treatment, including gemcitabine, 5-fluorouracil, cladribine, cytarabine, mercaptopurine, irinotecan, leucovorin, oxaliplatin, tioguanine, etoposide, teniposide, mitoxantrone, topotecan, ixabepilone, mitomycin, epirubicin, imatinib, and methotrexate, for the treatment of cancers, such as, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer,

mesothelioma, stomach cancer, bowel cancer, and other cancers of the pancreas.

In another embodiment of the present invention, this method is described as a combination therapy to be administered in combination with checkpoint inhibitors, including PD-1 inhibitors (Pembrolizumab, Nivolumab), PD-L1 inhibitor (Atezolizumab), and CTLA-4 targeting monoclonal antibody (Ipilimumab), for the treatment of cancers, such as, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, mesothelioma, stomach cancer, bowel cancer, and other cancers of the pancreas.

In another embodiment of the present invention, this chemotherapy is administered as an adjuvant, second-line, or third-line treatment for the treatment of cancers, such as, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, mesothelioma, stomach cancer, bowel cancer, and other cancers of the pancreas.

In another embodiment of the present invention, the methods are administered in combination with both the chemotherapy and another adjuvant therapy.

In another embodiment of the present invention, the method is administered in combination with both the checkpoint inhibitor and another adjuvant therapy.

One aspect of the invention relates to a method of treating cancer, comprising the step of: (a) administering an inhibitor targeting at least one component of the Hippo- YAP pathway in combination with; (b) administering a therapeutic agent; and wherein the at least one component is selected from the group consisting of NF2, LATS1, LATS2, MST1, MST2, MOB IA, MOBIB, MAP4K, TAOKl, TAOK3, and SAVl, or combinations thereof. Another aspect of the invention relates to a method of treating cancer, comprising the step of: (a) disrupting at least one component of the Hippo- YAP pathway in combination with; (b) administering a therapeutic agent; and wherein the at least one component is selected from the group consisting of F2, LATS1, LATS2, MST1, MST2, MOB1A, MOB IB, MAP4K, TAOK1, TAOK3, and SAV1, or combinations thereof.

In some embodiments of any of the aforemention methods, the cancer is immuno- resistant, chemoresistant, or both.

In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, liver cancer, stomach cancer, and lung cancer.

In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma.

In some embodiments, the liver cancer is hepatocellular adenocarcinoma.

In some embodiments, lung cancer is mesothelioma.

In some embodiments, the therapeutic agent is selected from the group consisting of chemotherapy, chemotherapeutic agent, anti-cancer agent, antineoplastic agent, cytotoxic agent, anti-angiogenesis agent, and immune checkpoint inhibitor, or combinations thereof.

In some embodiments, the therapeutic agent is selected from the group consisting of gemcitabine, 5-fluorouracil, methotrexate, cladribine, cytarabine, mercaptopurine, irinotecan, leucovorin, oxaliplatin, tioguanine, etoposide, teniposide, mitoxantrone, topotecan, ixabepilone, mitomycin, epirubicin, and imatinib.

In some embodiments of any of the aforemention methods, step (a) comprises a method of gene-editing, knock-down, or silencing.

In some embodiments, the inhibitor is selected from the group consisting of a a small molecule, an antibody, a nucleic acid encoding an antibody, an antigen binding fragment, a RNA interfering agent, a peptide, a peptidomimetic, a synthetic ligand, and an aptamer.

In some embodiments, step (a) is a method of gene-editing.

In some embodiments, the gene-editing is selected from CRISPR/Cas9, ZFN, or TALEN, or combinations thereof.

In some embodiments, step (a) is a method of silencing or knock-down.

In some embodiments, the silencing or knock-down is selected from the group consisting of siRNA, shRNA, or miRNA, or combinations thereof.

In some embodiments, the component is a gene, protein, polypeptide, DNA, RNA, or nucleotide component, or combinations thereof. In some embodiments, the at least one component is the gene NF2.

In some embodiments, the at least one component is the protein F2.

In some embodiments, the at least one component is the gene MST1 or MST2.

In some embodiments, the at least one component is the protein MST1 or MST2.

In some embodiments, the at least one component is the gene LATS1 or LATS2.

In some embodiments, the at least one component is the protein LATS1 or LATS2.

In some embodiments, the at least one component is the gene MOB 1 A or MOB 1 B .

In some embodiments, the at least one component is the protein MOB1A or MOB IB.

In some embodiments, the at least one component is the gene MAP4K.

In some embodiments, the at least one component is the protein MAP4K.

In some embodiments, the at least one component is the gene TAOK1 or TAOK3.

In some embodiments, the at least one component is the protein TAOK1 or TAOK3.

In some embodiments, the at least one component is the gene SAV1.

In some embodiments, the at least one component is the protein SAV1.

In some embodiments, the at least one component is at least two, three, four, five, six, seven, eight, nine, or ten components, or combinations thereof.

In some embodiments of any of the aforemention methods, the method further comprises administering a checkpoint inhibitor.

In some embodiments, the checkpoint inhibitor targets PD-1, PD-L1, and CTLA-4.

In some embodiments, the checkpoint inhibitor of PD-1 is selected from

Pembrolizumab or Nivolumab.

In some embodiments, the checkpoint inhibitor of PD-L1 is Atezolizumab.

In some embodiments, the checkpoint inhibitor of CTLA-4 is Ipilimumab.

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein: FIG. 1 depicts a line graph showing that Hippo- YAP inactivation increases patient survival. The red line (far most left line) represents the number of patients surviving with an activated Hippo- YAP pathway when treated for Pancreatic Ductal Adenocarcinoma with gemcitabine monotherapy, based on data from the FDA Label for Gemzar. The blue line (far most right line) represents the expected survival of patients treated with both gemcitabine and the future Hippo- YAP inactivator Nivien (in development), based on two retrospective studies of PDAC patients with a naturally-occurring mutation that inactivates the Hippo- YAP pathway.

FIG. 2 is a cartoon depiction of the Hippo- YAP Pathway, showing when the pathway is ON and when the pathway is OFF (Moroishi et al. (2015) Nat Rev Cancer 15(2):73-79). Inactivation of the Hippo- YAP pathway leads to dephosphorylated YAP localizing to the nucleus, where it promotes the transcription of downstream factors that down regulate drug efflux transporters and reduce the metabolism of chemotherapeutic agents. NF2, LATSl/2, MST1/2, MOB 1 A/IB, Sav family WW domain-containing protein 1 (SAV1), and other components of the Hippo- YAP pathway are promising targets for inhibition in the adjuvant treatment of pancreatic ductal adenocarcinoma and other chemo-resistant cancers. The traditional conception of the Hippo- YAP pathway is that activation prevents cancer; we propose methods based on the opposite conclusion.

FIG. 3 is a line graph Gemzar (gemcitabine) and 5-FU (5-fluorouracil) in the treatment of pancreatic cancer (FDA Label for Gemzar by Eli Lilly). Gemzar was approved for marketing by Eli Lilly as the front-line chemotherapy for pancreatic cancer in 1996, based in part on this data from the Gemzar FDA Label. The fraction of patients surviving when treated with Gemzar was shown to be greater than the rate of those treated with the previous front-line chemotherapy, another anti-metabolite, called 5-fluorouracil. Neither therapy is particularly effective alone or in combination with each other or other common chemotherapies like methotrexate.

FIG. 4 depicts the chemical composition of gemcitabine (FDA Label for Gemzar by Eli Lilly). Gemcitabine is 2'-deoxy-2',2'-difluorocytidine monohydrochloride (β-isomer). Formula: C9H11F2N3O4.

FIG. 5 are line graphs depicting survival rates from two retrospective studies (Quan et al. (2015) Cancer Res 75(22):4778-4789). Patients with high vs. low NF2 expression, and therefore High vs. low Hippo- YAP pathway activation with regards to YAP phosphorylation, nuclear localization, and activation of downstream agents.

FIG. 6 is a cartoon depiction of the Hippo- YAP pathway. Inactivation of the Hippo- YAP pathway leads to dephosphorylated YAP localizing to the nucleus, where it promotes the transcription of downstream factors that downregulate drug efflux transporters and reduce the metabolism of chemotherapeutic agents. F2, LATS1/2, MST1/2, MOB1A/1B, SAV1, and other components of the Hippo- YAP pathway are promising targets for inhibition in the adjuvant treatment of pancreatic ductal

adenocarcinoma and other chemo-resistant cancers. The traditional conception of the Hippo- YAP pathway is that activation prevents cancer; we propose methods based on the opposite conclusion.

Further features and advantages will become apparent from the following Detailed Description and Claims.

DETAILED DESCRIPTION AND EXEMPLIFICATIONS

The current invention is based, in part, on the discovery that suppression of activity of the Hippo- Yap pathway in combination with a variety of therapeutic agents, including chemotherapy, chemotherapeutic agents, anti-cancer agents, antineoplastic agents, cytotoxic agents, anti-angiogenesis agents, and immune checkpoint inhibitors, is effective in promoting the regression of various types of cancers in mouse and human 3D spheroid cultures.

The present invention is, in part, based on the discovery that manipulation of the activity of the Hippo- Yap pathway using a therapeutically applicable modality such as shRNA (or other anti-sense or RNAi technologies) can be used in combination with anticancer agents such as chemotherapy and checkpointing inhibitors to cause the regression of various types of cancers. The results achieved by treating tumors with Hippo- Yap inhibitors in combination with therapeutic agents, such as anti-cancer agents, is superior to the results achieved by the anti-cancer agents alone, in vitro and in vivo. Accordingly, the present invention is a method of treating cancer using a combination treatment involving chemotherapy or checkpoint inhibitors, with Hippo- Yap inhibitors, where combination therapy is shown to increase efficacy of treatment.

Another aspect of the present invention relates to methods of treating or alleviating any symptoms of cancer, or what is considered precancerous conditions, in a patient by administering to the subject a therapeutically effective amount of Hippo- Yap pathway inhibitors, in addition to one or more therapeutic agents.

In a certain embodiment, the cancer is a carcinoma. In a certain embodiment, the cancer is a pancreatic ductal adenocarcinoma. In a certain embodiment, the cancer is a non-small cell lung carcinoma. In a certain embodiment, the cancer is a breast carcinoma. In a certain embodiment, the cancer is a mesothelium carcinoma. In a certain embodiment, the cancer is an ovary carcinoma. In a certain embodiment, the cancer is a colon carcinoma. In a certain embodiment, the cancer is a head and neck carcinoma. In a certain embodiment, the cancer is a sarcoma carcinoma. In a certain embodiment, the cancer is a cholangiocarcinoma carcinoma.

Additional cancers include, but not limited to, cancers of the: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, lipoma and teratoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal system, for example, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), gastric, pancreas (ductal

adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract, for example, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and/or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,

fibroadenoma, adenomatoid tumors, lipoma); liver, for example, hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma); bone, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system, for example, neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma],

glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); reproductive system, for example, gynecological, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre- tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal

rhabdomyosarcoma), fallopian tubes (carcinoma) and other sites associated with female genital organs; placenta, penis, prostate, testis, and other sites associated with male genital organs; hematologic system, for example, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cavity, for example, lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx; skin, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids; adrenal glands: neuroblastoma; and other tissues including connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa, breast, head or/and neck, anal region, thyroid, parathyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites

In certain embodiments, the carcinoma is non-mutant or Hippo- Yap wild-type carcinoma. In certain embodiments, the carcinoma is mutant, but not deactivating Hippo- Yap carcinoma. In certain embodiments, the mutant Hippo- Yap comprises one or more mutations in its upstream Hippo kinases or central pathway mediators.

In certain embodiments of the invention, the Hippo- Yap inhibitor is a small molecule. In certain embodiments of the invention the Hippo- Yap inhibitor is a miRNA. In certain embodiments of the invention the Hippo- Yap inhibitor is shRNA. In certain embodiments of the invention the Hippo- Yap inhibitor is gene therapy. In certain embodiments of the invention the Hippo- Yap inhibitor is siRNA.

Another aspect of the invention is a method for inhibiting the conversion of non- phosphorylated Yap to phosphorylated Yap, in a subject. Another aspect of the invention is a method for inhibiting the retention of Yap in the cytoplasm, stopping Yap from entering the nucleus in a subject. Another aspect of the invention is a method for inhibiting the ubiquitination and degradation of Yap in a subject. The inhibition can involve inhibiting the the conversion of non-phosphorylated LATSl/2 to phosphorylated LATSl/2, conversion of non-phosphorylated MST1/2 to phosphorylated MST1/2, the transport of LATSl/2 to the cytoplasm assisted by NF2, the enhanced interaction between MST1/2 and LATSl/2 by Savl, or any other mechanism which leads to the phosphorylation, cytoplasmic retention or degradation of Yap.

As used herein "MST1" refers to "Mammalian STE20-like protein kinase 1" that is a stress-activated, pro-apoptotic kinase which, following caspase-cleavage, enters the nucleus and induces chromatin condensation followed by internucleosomal DNA fragmentation. Nucleic acid and polypeptide sequences for MST1 are known, for example, but not limited to, human NM_006282.4, UniProtKB Q13043.2, U18297.1, AAA83254.1, U60207.1, AAB 17262.1, AK315238.1, BAG37665.1, Z93016.2, AL109839. i l, CH471077.2, EAW75882.1, BC029511.1, AAH29511.1, BC058916.1, AAH58916.1, BC093768.1, AAH93768.1, NP_006273.1, and XP_005260590.1.

As used herein "MST2" refers to "Mammalian STE20-like protein kinase 2" that is a stress-activated, pro-apoptotic kinase which, following caspase-cleavage, enters the nucleus and induces chromatin condensation followed by internucleosomal DNA fragmentation. Nucleic acid and polypeptide sequences for MST2 are known, for example, but not limted to, human UniProtKB XM 017013756.1, NM 006281, Q13188.2, U26424.1, AAC50386.1, U60206.1, AAB 17261.1, AK131363.1, BAG54769.1, AK291837.1, BAF84526.1,

AC016877. i l, AP002087.2, AP003355.2, AP003467.2, AP003551.2, CH471060.1,

EAW91781.1, BC010640.2, AAH10640.1, Z25422.1, CAA80909.1, 138212,

NP_001243241.1, and NP_006272.2.

The methods of the present invention may comprise disrupting at least one component of the Hippo- YAP pathway through gene editing, silencing, or knock-down methods or techniques known in the art. Such gene editing methods may comprise

CRISPR/Cas9, Zinc Finger Technology (ZFN), or Transcription activator-like effector nucleases (TALEN), or combinations thereof. Other methods to disrupt at least one component of the Hippo- YAP pathway may comprise silencing or knock-down methods, such as anti-sense and RNAi technologies known in the art. Such silencing or knock-down methods including use of siRNA, shRNA, or miRNA, or combinations thereof. In some embodiments, the inhibitor, or methods of disrupting at least one component of the Hippo YAP pathway, specifically targets MST1, MST2, or both (MST1/2). In certain emdoments, the inhibitor or gene editing, silencing, or knock-down methods target the MST1 or MST2 nucleic acid or polypeptide sequences, or both, as set forth above.

The inhibition is a level of inhibition of the target comparable to a non-inhibited control. In one embodiment, inhibition is at least ten percent relative to a non-inhibited control. This is to say the amount of protein available to participate in enzymatic activity is less than or equal to ninety percent of the equivalent amount of protein or rate of enzymatic activity in the absence of the inhibitor. In other embodiments, inhibition may be at least 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90 or 95 percent of inhibition of a non- inhibited control.

The therapeutic agents (e.g., chemotherapy, chemotherapeutic agent, anti-cancer agent, antineoplastic agent, cytotoxic agent, anti-angiogenesis agent, and immune checkpoint inhibitor) listed below are illustrative not limiting. The present invention includes one or more therapeutic agents from those below. The present invention can include more than one therapeutic agent, two, three, four or five agents, such that the composition of the present invention may act according to its intended function. In another embodiment, the second, third, fourth or fifth therapeutic agent is a chemotherapeutic agent, known as an antineoplastic agent or anti-proliferative agent, chosen from a group including 5-fluorouracil, gemcitabine, cladribine, clofarabine, mercaptopurine, topotecan, imatinib, epirubicin, mitomycin, methotrexate, ixabepilone, tioguanine, etoposide, teniposide, mitoxantrone, cytarabine, cladribine, clofarabine.

In some embodiments, the anti-cancer agent used in the methods described herein is an anti-angiogenesis agent (e.g., an agent that stops tumors from developing new blood vessels). Examples of anti-angiogenesis agents include for example VEGF inhibitors, VEGFR inhibitors, TIE-2 inhibitors, PDGFR inhibitors, angiopoetin inhibitors, PKC.beta. inhibitors, COX-2 (cyclooxygenase II) inhibitors, integrins (alpha-v/beta-3), MMP-2

(matrix-metalloproteinase 2) inhibitors, and MMP-9 (matrix-metalloproteinase 9) inhibitors. Preferred anti-angiogenesis agents include sunitinib (Sutent®), bevacizumab (Avastin®), axitinib (AG 13736), SU 14813 (Pfizer), and AG 13958 (Pfizer).

Additional anti-angiogenesis agents include vatalanib (CGP 79787), Sorafenib (Nexavar®), pegaptanib octasodium (Macugen®), vandetanib (Zactima®), PF-0337210 (Pfizer), SU 14843 (Pfizer), AZD 2171 (AstraZeneca), ranibizumab (Lucentis®),

Neovastat® (AE 941), tetrathiomolybdata (Coprexa®), AMG 706 (Amgen), VEGF Trap (AVE 0005), CEP 7055 (Sanofi-Aventis), XL 880 (Exelixis), telatinib (BAY 57-9352), and CP-868,596 (Pfizer). Other anti-angiogenesis agents include enzastaurin (LY 317615), midostaurin (CGP 41251), perifosine (KRX 0401), teprenone (Selbex®), UCN 01 (Kyowa Hakko), celecoxib (Celebrex®), parecoxib (Dynastat®), deracoxib (SC 59046), lumiracoxib (Preige®), valdecoxib (Bextra®), rofecoxib (Vioxx®), iguratimod (Careram®), IP 751 (Invedus), SC-58125 (Pharmacia), etoricoxib (Arcoxia®), exisulind (Aptosyn®), salsalate (Amigesic®), diflunisal (Dolobid®), ibuprofen (Motrin®), ketoprofen (Orudis®)

nabumetone (Relafen®), piroxicam (Feldene®), naproxen (Aleve®, Naprosyn®) diclofenac (Voltaren®), indomethacin (Indocin®), sulindac (Clinoril®), tolmetin (Tolectin®), etodolac (Lodine®), ketorolac (Toradol®), oxaprozin (Daypro®), ABT 510 (Abbott), apratastat (TMI 005), AZD 8955 (AstraZeneca), incyclinide (Metastat®), PCK 3145 (Procyon), acitretin (Neotigason®), plitidepsin (Aplidine®), cilengtide (EMD 121974), combretastatin A4 (CA4P), fenretinide (4 HPR), halofuginone (Tempostatin®), Panzem® (2-methoxyestradiol), PF-03446962 (Pfizer), rebimastat (BMS 275291), catumaxomab (Removab®), lenalidomide

(Revlimid®) squalamine (EVIZON®), thalidomide (Thalomid®), Ukrain® (NSC 631570),

Vitaxin® (MEDI 522), and zoledronic acid (Zometa®). In some embodiments, the anti-cancer agent is a signal transduction inhibitor (e.g., inhibiting the means by which regulatory molecules that govern the fundamental processes of cell growth, differentiation, and survival communicated within the cell). Signal transduction inhibitors include small molecules, antibodies, and antisense molecules. Signal transduction inhibitors include for example kinase inhibitors (e.g., tyrosine kinase inhibitors or serine/threonine kinase inhibitors) and cell cycle inhibitors. More specifically signal transduction inhibitors include, for example, ALK inhibitors, ROS1 inhibitors, TrkA inhibitors, TrkB inhibitors, TrkC inhibitors, farnesyl protein transferase inhibitors, EGF inhibitor, ErbB-1 (EGFR), ErbB-2, pan erb, IGFIR inhibitors, MEK, c-Kit inhibitors, FLT-3 inhibitors, K-Ras inhibitors, PI3 kinase inhibitors, JAK inhibitors, STAT inhibitors, Raf kinase inhibitors, Akt inhibitors, mTOR inhibitor, P70S6 kinase inhibitors, inhibitors of the WNT pathway and so called multi-targeted kinase inhibitors, gefitinib (Iressa®), cetuximab (Erbitux®), erlotinib (Tarceva®), trastuzumab (Herceptin®), sunitinib (Sutent®) imatinib (Gleevec®), PD325901 (Pfizer), BMS 214662 (Bristol-Myers Squibb), lonafarnib

(Sarasar®), pelitrexol (AG 2037), matuzumab (EMD 7200), nimotuzumab (TheraClM h- R3®), panitumumab (Vectibix®), Vandetanib (Zactima®), pazopanib (SB 786034), ALT 1 10 (Alteris Therapeutics), BIBW 2992 (Boehringer Ingelheim), Cervene® (TP 38), PF- 2341066 (Pfizer), PF-299804 (Pfizer), canertinib (CI 1033), pertuzumab (Omnitarg®), Lapatinib (Tycerb®), pelitinib (EKB 569), miltefosine (Miltefosin®), BMS 599626 (Bristol- Myers Squibb), Lapuleucel-T (Neuvenge®), NeuVax® (E75 cancer vaccine), Osidem®

(IDM 1), mubritinib (TAK-165), CP-724,714 (Pfizer), panitumumab (Vectibix®), lapatinib (Tycerb®), PF-299804 (Pfizer), pelitinib (EKB 569), (Omnitarg®), ARRY 142886 (Array Biopharm), everolimus (Certican®), zotarolimus (Endeavor®), temsirolimus (Torisel®), AP 23573 (ARIAD), VX 680 (Vertex), XL 647 (Exelixis), sorafenib (Nexavar®), LE-AON (Georgetown University), and GI-4000 (Globelmmune), ABT 751 (Abbott), alvocidib (flavopiridol), BMS 387032 (Bristol Myers), EM 1421 (Erimos), indisulam (E 7070), seliciclib (CYC 200), BIO 1 12 (One Bio), BMS 387032 (Bristol-Myers Squibb), PD

0332991 (Pfizer), AG 024322 (Pfizer), LOXO-101 (Loxo Oncology), crizotinib, and ceritinib.

In some embodiments, antineoplastic agents include, but are not limited, to hormonal modulators such as hormonal, anti -hormonal, androgen agonist, androgen antagonist and anti-estrogen therapeutic agents, histone deacetylase (HDAC) inhibitors, gene silencing agents or gene activating agents, ribonucleases, proteosomics, Topoisomerase I inhibitors, Camptothecin derivatives, Topoisomerase II inhibitors, alkylating agents, antimetabolites, poly(ADP-ribose) polymerase- 1 (PARP-1) inhibitor, microtubulin inhibitors, antibiotics, plant derived spindle inhibitors, platinum-coordinated compounds, gene therapeutic agents, antisense oligonucleotides, vascular targeting agents (VTAs), and statins.

Examples of antineoplastic agents used in the methods disclosed herein, include, but are not limited to, glucocorticoids, such as dexamethasone, prednisone, prednisolone, methylprednisolone, hydrocortisone, and progestins such as medroxyprogesterone, megestrol acetate (Megace), mifepristone (RU-486), Selective Estrogen Receptor Modulators (SERMs; such as tamoxifen, raloxifene, lasofoxifene, afimoxifene, arzoxifene, bazedoxifene, fispemifene, ormeloxifene, ospemifene, tesmilifene, toremifene, trilostane and CHF 4227 (Cheisi)), Selective Estrogen-Receptor Downregulators (SERD's; such as fulvestrant), exemestane (Aromasin), anastrozole (Arimidex), atamestane, fadrozole, letrozole (Femara), gonadotropin-releasing hormone (GnRH; also commonly referred to as luteinizing hormone- releasing hormone [LHRH]) agonists such as buserelin (Suprefact), goserelin (Zoladex), leuprorelin (Lupron), and triptorelin (Trelstar), abarelix (Plenaxis), bicalutamide (Casodex), cyproterone, flutamide (Eulexin), megestrol, nilutamide (Nilandron), and osaterone, dutasteride, epristeride, finasteride, Serenoa repens, PHL 00801, abarelix, goserelin, leuprorelin, triptorelin, bicalutamide, tamoxifen, exemestane, anastrozole, fadrozole, formestane, letrozole, and combinations thereof.

Other examples of antineoplastic agents include, but are not limited to, suberolanilide hydroxamic acid (SAHA, Merck Inc./Aton Pharmaceuticals), depsipeptide (FR901228 or FK228), G2M-777, MS-275, pi valoyloxy methyl butyrate and PXD-101; Onconase

(ranpirnase), PS-341 (MLN-341), Velcade (bortezomib), 9-aminocamptothecin, belotecan, BN-80915 (Roche), camptothecin, diflomotecan, edotecarin, exatecan (Daiichi), gimatecan, 10-hydroxy camptothecin, irinotecan HC1 (Camptosar), lurtotecan, Orathecin (rubitecan, Supergen), SN-38, topotecan, camptothecin, 10-hydroxycamptothecin, 9- aminocamptothecin, irinotecan, SN-38, edotecarin, topotecan, aclarubicin, adriamycin, amonafide, amrubicin, annamycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, etoposide, idarubicin, galarubicin, hydroxy carbamide, nemorubicin, novantrone

(mitoxantrone), pirarubicin, pixantrone, procarbazine, rebeccamycin, sobuzoxane, tafluposide, valrubicin, Zinecard (dexrazoxane), nitrogen mustard N-oxide, cyclophosphamide, AMD-473, altretamine, AP-5280, apaziquone, brostallicin, bendamustine, busulfan, carboquone, carmustine, chlorambucil, dacarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine, mafosfamide,

mechlorethamine, melphalan, mitobronitol, mitolactol, mitomycin C, mitoxatrone, nimustine, ranimustine, temozolomide, thiotepa, and platinum-coordinated alkylating compounds such as cisplatin, Paraplatin (carboplatin), eptaplatin, lobaplatin, nedaplatin, Eloxatin (oxaliplatin, Sanofi), streptozocin, satrplatin, and combinations thereof.

In some embodiments, the inhibitors are used in the methods described herein are dihydrofolate reductase inhibitors (such as methotrexate and NeuTrexin (trimetresate glucuronate)), purine antagonists (such as 6-mercaptopurine riboside, mercaptopurine, 6- thioguanine, cladribine, clofarabine (Clolar), fludarabine, nelarabine, and raltitrexed), pyrimidine antagonists (such as 5-fluorouracil (5-FU), Alimta (premetrexed disodium, LY231514, MTA), capecitabine (Xeloda®), cytosine arabinoside, Gemzar® (gemcitabine, Eli Lilly), Tegafur (UFT Orzel or Uforal and including TS-1 combination of tegafur, gimestat and otostat), doxifluridine, carmofur, cytarabine (including ocfosfate, phosphate stearate, sustained release and liposomal forms), enocitabine, 5-azacitidine (Vidaza), decitabine, and ethynylcytidine) and other antimetabolites such as eflornithine, hydroxyurea, leucovorin, nolatrexed (Thymitaq), triapine, trimetrexate, N-(5-[N-(3,4-dihydro-2-methyl-4- oxoquinazolin-6-ylmethyl)-N-methylamino]~ 2-thenyl)-L-glutamic acid, AG-014699 (Pfizer Inc.), ABT-472 (Abbott Laboratories), INO-1001 (Inotek Pharmaceuticals), KU-0687

(KuDOS Pharmaceuticals), and GPI 18180 (Guilford Pharm Inc), or combinations thereof.

Other examples of antineoplastic cytotoxic agents used in the methods described herein include, but are not limited to, Abraxane (Abraxis Bioscience, Inc.), Batabulin (Amgen), EPO 906 (Novartis), Vinflunine (Bristol-Myers Squibb Company), actinomycin D, bleomycin, mitomycin C, neocarzinostatin (Zinostatin), vinblastine, vincristine, vindesine, vinorelbine (Navelbine), docetaxel (Taxotere), Ortataxel, paclitaxel (including Taxoprexin a DHA/paciltaxel conjugate), cisplatin, carboplatin, Nedaplatin, oxaliplatin (Eloxatin), Satraplatin, Camptosar, capecitabine (Xeloda), oxaliplatin (Eloxatin), Taxotere alitretinoin, Canfosfamide (Telcyta®), DMXAA (Antisoma), ibandronic acid, L-asparaginase, pegaspargase (Oncaspar®), Efaproxiral (Efaproxyn®~radiation therapy)), bexarotene

(Targretin®), Tesmilifene (DPPE—enhances efficacy of cytotoxics)), Theratope® (Biomira),

Tretinoin (Vesanoid®), tirapazamine (Trizaone®), motexafin gadolinium (Xcytrin®) Cotara® (mAb), and NBI-3001 (Protox Therapeutics), polyglutamate-paclitaxel (Xyotax®) and combinations thereof.

Further examples of antineoplastic agents used in the methods described herein include, but are not limited to, as Advexin (ING 201), TNFerade (GeneVec, a compound which express TNF alpha in response to radiotherapy), RB94 (Baylor College of Medicine), Genasense (Oblimersen, Genta), Combretastatin A4P (CA4P), Oxi-4503, AVE-8062, ZD- 6126, TZT-1027, Atorvastatin (Lipitor, Pfizer Inc.), Provastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.), Simvastatin (Zocor, Merck Inc.), Fluvastatin (Lescol, Novartis), Cerivastatin (Baycol, Bayer), Rosuvastatin (Crestor, AstraZeneca), Lovostatin, Niacin (Advicor, Kos Pharmaceuticals), Caduet, Lipitor, torcetrapib, and combinations thereof.

Additional, anti-cancer agents include, but not limited to, trastuzumab, tamoxifen, docetaxel, paclitaxel, capecitabine, gemcitabine, vinorelbine, exemestane, letrozole, anastrozole, FOLFOX (a combination of 5-fluorouracil (5-FU) or capecitabine (Xeloda)), leucovorin and oxaliplatin (Eloxatin). Further examples of particular anti-cancer agents include those typically used in chemotherapy for metastatic disease, such as FOLFOX or FOLFOX in combination with bevacizumab (Avastin); and FOLFIRI, a combination of 5- FU or capecitabine, leucovorin and irinotecan (Camptosar). Further examples include 17- DMAG, ABX-EFR, AMG-706, AMT-2003, ANX-510 (CoFactor), aplidine (plitidepsin, Aplidin), Aroplatin, axitinib (AG-13736), AZD-0530, AZD-2171, bacillus Calmette-Guerin (BCG), bevacizumab (Avastin), BIO-117, BIO-145, BMS-184476, BMS-275183, BMS- 528664, bortezomib (Velcade), C-1311 (Symadex), cantuzumab mertansine, capecitabine (Xeloda), cetuximab (Erbitux), clofarabine (Clofarex), CMD-193, combretastatin, Cotara, CT-2106, CV-247, decitabine (Dacogen), E-7070, E-7820, edotecarin, EMD-273066, enzastaurin (LY-317615) epothilone B (EPO-906), erlotinib (Tarceva), flavopyridol, GC AN- 101, gefitinib (Iressa), huA33, huC242-DM4, imatinib (Gleevec), indisulam, ING-1, irinotecan (CPT-11, Camptosar) ISIS 2503, ixabepilone, lapatinib (Tykerb), mapatumumab (HGS-ETR1), MBT-0206, MEDI-522 (Abregrin), Mitomycin, MK-0457 (VX-680), MLN- 8054, NB-1011, NGR-TNF, NV-1020, oblimersen (Genasense, G3139), OncoVex, ONYX 015 (CI- 1042), oxaliplatin (Eloxatin), panitumumab (ABX-EGF, Vectibix), pelitinib (EKB-

569), pemetrexed (Alimta), PD-325901, PF-0337210, PF-2341066, RAD-001 (Everolimus),

RAV-12, Resveratrol, Rexin-G, S-l (TS-1), seliciclib, SN-38 liposome, Sodium stibogluconate (SSG), sorafenib (Nexavar), SU-14813, sunitinib (Sutent), temsirolimus (CCI 779), tetrathiomolybdate, thalomide, TLK-286 (Telcyta), topotecan (Hycamtin), trabectedin (Yondelis), vatalanib (PTK-787), vorinostat (SAHA, Zolinza), WX-UK1, and ZYC300, wherein the amounts of the active agent together with the amounts of the combination anticancer agents are effective in treating colorectal cancer.

In some embodiments, the inhibitors that may target immune checkpoint targets include, but are not limited to, 2B4 (CD244), A2aR, B7H3 (CD276), B7H4 (VTCN1), B7H6, B7RP1, BTLA (CD272), butyrophilins, CD103, CD 122, CD137 (4-1BB), CD 137L, CD 160, CD2, CD200R, CD226, CD26, CD27, CD28, CD30, CD39, CD40, CD48, CD70, CD73, CD80 (B7.1), CD86 (B7.2), CEACAM1, CGEN-15049, CTLA-4, DR3, GALS, GITR, GITRL, HVEM, ICOS, ICOSL (B7H2), IDOl, ID02, ILT-2 (LILRB 1), ILT-4 (LILRB2), KIR, KLRGl, LAG3, LAIRl (CD305), LIGHT (TNFSF 14), MARCO, KG2A, KG2D, OX-40, OX-40L, PD-1, PDL-1 (B7-H1, CD 274), PDL-2 (B7-DC, CD 273), PS, SIRPalpha (CD47), SLAM, TGFR, TIGIT, TFM1, TIM3 (HAVCR2), TIM4, or VISTA. In some embodiments, an immune checkpoint inhibitor is conjointly administered. Some immune checkpoint inhibitors are Enoblituzumab (e.g., MGA271), Ipilimumab (e.g., BMS- 734016, MDX-010), Tremelimumab (e.g., CP-675, CP-675,206), Lirilumab (e.g., BMS- 986015, IPH2102), BMS986016, Pembrolizumab (e.g., MK-3475, SCH 900475),

Nivolumab (e.g., BMS-936558, MDX-1 106, ONO-4538), Pidilizumab (e.g., CT-01 1, MDV9300), Atezolizumab (e.g., MPDL3280A, RG7446, R05541267), BMS-936559 (e.g., MDX-1 105), and Bavituximab.

The present invention includes methods for use as a combination therapy, in which composition of the combination therapy includes a Hippo- Yap activity inhibitor, and at least one therapeutic agents, listed above, administered to a subject for the treatment of disease such as cancer. The combination therapy may be administered to cancer cells to stop, reduce or reverse the proliferation of cancer cells or to cause cancer cell death. In one aspect, the inhibitor of Hippo- Yap is a pharmaceutically acceptable salt, administered prior to the administration of the one or more other therapeutic agents. In one aspect, the inhibitor of Hippo- Yap is a pharmaceutically acceptable salt administered subsequent to the administration of one or more therapeutic agents such that one or more therapeutic agents may be administered as a single or two or more compositions, administered sequentially, simultaneously or in alternation. In one aspect, the inhibitor of Hippo- Yap is a pharmaceutically acceptable salt administered prior to the administration of one or more therapeutic agents such that one or more therapeutic agents may be administered as a single or two or more compositions, administered sequentially, simultaneously or in alternation. Two or more therapeutic agents may be administered in a time frame of minutes, hours, days or weeks of one another.

In certain embodiments, the combination therapy is intended to involve the administration of one or more therapeutic agents in a sequential manner where each therapeutic agent may be administered at a different time or at least two therapeutics are administered simultaneously. Simultaneous administration may be accomplished by administration of a single capsule, or in multiple, single capsules at a fixed ratio of each therapeutic agent. Sequential or simultaneous administration can be done for each therapeutic agent by, but not limited to, intravenous, oral, intramuscular, subcutaneously, intra-, peri-, or intertumorally, or direct routes. Therapeutic agents may be administered sequentially or simultaneously by one or more routes of administration.

Such combination therapy may comprise conjoint administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either

concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.

In certain aspects of the invention, the combination therapy, including the inhibitor of an activating component of Hippo Yap, and the chemotherapy or checkpoint inhibitor, that is the present invention results in a synergistic effect for the treatment of cancer. The synergistic effect may be defined as an effect where the efficacy of the combination treatment is greater than the sum of each individual treatment given alone. The synergistic effect may also be one that is not achievable by the administration of a single therapeutic agent. The synergistic effect may be applicable to, but is not limited to, an effect on cancer through inhibiting, reducing, or delaying tumor growth, killing cancer cells or extending patient survival.

In certain aspects of the invention, the combination therapy, including the inhibitor of an activating component of Hippo Yap and the chemotherapy or checkpoint inhibitor, also may be administered in further combination with non-drug therapies, such as surgery or radiation therapy, or other biologically active ingredients. For example, the non-drug treatment may be administered at a suitable time, requiring that the administration of the combination therapy consisting of the inhibitor of an activating component of Hippo Yap and the chemotherapy or checkpoint inhibitor may still be allowed to have a synergistic effect.

In one embodiment of the invention, the pharmaceutical composition of the combination therapy is delivered as a unit based dose. The dosage can range from

O.Olmg/kg per day to 5000mg/kg per day. The quantity of the active ingredient should be delivered in an effective amount, which can be varied depending on the treatment. For example, variation in dose may be done depending on age, weight, route of delivery or condition. The therapeutically effective amount, or dosage range, will be estimated in cell culture assays or animal models, including rats, mice, rabbits, dogs or pigs. The therapeutic efficacy and toxicity are measured using standard pharmaceutical procedures to determine the ED50, the therapeutically effective dose in 50% of the population, and the LD50, the lethal dose in 50% of the population. The difference between the ratio of the LD50 to the ED50 is the therapeutic index. A large enough dose will be provided to maintain the desired effect, while avoiding side effects caused by toxicity. The exact dose will take into account the severity of the disease and age, weight, gender, diet and health of the subject. Dosage may be further adjusted based on patient's response.

In certain embodiments, the invention is targeted to subjects needing treatment for resistant cancer, which is a cancer that does not respond to conventional drugs including chemotherapy and checkpoint inhibitors. The cancer may be resistant when treatment is administered or become resistant during treatment. In certain embodiments, the subject may have cancer following previous treatment, or may have tailed other effective treatment for cancer.

The methods and uses described herin may include detecting patients without mutations suppressing activity of the Hippo- Yap pathway. A sample may be taken of the tumor of a patient with cancer, typically by the physician according to routine practice, before the combination therapy of this invention is given. The lack of deactivating mutations may indicate the subject will be responsive to combination therapy. In certain embodiments of the present invention it is a personalized medicine for the treatment of a subject with cancer or displaying the symptoms of cancer, after genetic screenings which may be used to predict the responsiveness of the subject to a therapeutically effective administration of the combination therapy. Responsiveness refers to the subject's objective therapeutic response when administered the drug, including tumor cell shrinkage, apoptosis or inhibition; it is used interchangeably with sensitivity. A responsive patient will have greater probability relative to a non treated patient population, of showing a therapeutic response.

REFERENCES

All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detail by way of illustration for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

1. A method of treating cancer, comprising the step of:

(a) administering an inhibitor targeting at least one component of the Hippo- YAP pathway in combination with;

(b) administering a therapeutic agent; and

wherein the at least one component is selected from the group consisting of F2, LATSl, LATS2, MSTl, MST2, MOBIA, MOBIB, MAP4K, TAOKl, TAOK3, and SAVl, or combinations thereof.

2. A method of treating cancer, comprising the step of:

(a) disrupting at least one component of the Hippo- YAP pathway in combination with;

(b) administering a therapeutic agent; and

3. The method of any one of claim 1 or 2, wherein the cancer is immuno-resistant, chemoresistant, or both.

4. The method of any one of claims 1-3, wherein the cancer is selected from the group consisting of pancreatic cancer, liver cancer, stomach cancer, and lung cancer.

5. The method of claim 4, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

6. The method of claim 4, wherein the liver cancer is hepatocellular adenocarcinoma.

7. The method of claim 4, wherein the lung cancer is mesothelioma.

8. The method of any one of claims 1-7, wherein the therapeutic agent is selected from the group consisting of chemotherapy, chemotherapeutic agent, anti-cancer agent, antineoplastic agent, cytotoxic agent, anti-angiogenesis agent, and immune checkpoint inhibitor, or combinations thereof.

9. The method of any one of claims 1-8, wherein the therapeutic agent is selected from the group consisting of gemcitabine, 5-fluorouracil, methotrexate, cladribine, cytarabine, mercaptopurine, irinotecan, leucovorin, oxaliplatin, tioguanine, etoposide, teniposide, mitoxantrone, topotecan, ixabepilone, mitomycin, epirubicin, and imatinib.

10. The method of claim 2, wherein step (a) comprises a method of gene-editing, knockdown, or silencing.

11. The method of claim 1, wherein the inhibitor is selected from the group consisting of a a small molecule, an antibody, a nucleic acid encoding an antibody, an antigen binding fragment, a RNA interfering agent, a peptide, a peptidomimetic, a synthetic ligand, and an aptamer.

12. The method of claim 2, wherein step (a) is a method of gene-editing.

13. The method of claim 12, wherein the gene-editing is selected from CRISPR/Cas9, ZFN, or TALEN, or combinations thereof.

14. The method of claim 2, wherein step (a) is a method of silencing or knock-down.

15. The method of claim 14, wherein the silencing or knock-down is selected from the group consisting of siRNA, shRNA, or miRNA, or combinations thereof.

16. The method of any one of claims 1-15, wherein the component is a gene, protein, polypeptide, DNA, RNA, or nucleotide component, or combinations thereof.

17. The method of any one of claims 1-16, wherein the at least one component is the gene NF2.

18. The method of any one of claims 1-16, wherein the at least onecomponent is the protein NF2.

19. The method of any one of claims 1-16, wherein the at least one component is the gene MSTl or MST2.

20. The method of any one of claims 1-16, wherein the at least one component is the protein MST1 or MST2.

21. The method of any one of claims 1-16, wherein the at least one component is the gene LATSl or LATS2.

22. The method of any one of claims 1-16, wherein the at least one component is the protein LATS1 or LATS2.

23. The method of any one of claims 1-16, wherein the at least one component is the gene MOB 1 A or MOB 1 B .

24. The method of any one of claims 1-16, wherein the at least one component is the protein MOB1A or MOB IB.

25. The method of any one of claims 1-16, wherein the at least one component is the gene MAP4K.

26. The method of any one of claims 1-16, wherein the at least one component is the protein MAP4K.

27. The method of any one of claims 1-16, wherein the at least one component is the gene TAOK1 or TAOK3.

28. The method of any one of claims 1-16, wherein the at least one component is the protein TAOK1 or TAOK3.

29. The method of any one of claims 1-16, wherein the at least one component is the gene SAV1.

30. The method of any one of claims 1-16, wherein the at least one component is the protein S AVI .

31. The method of any one of claims 1-30, wherein the at least one component is at least two, three, four, five, six, seven, eight, nine, or ten components, or combinations thereof.

32. The method of any one of claims 1-31, further comprising administering a checkpoint inhibitor.

33. The method of claim 32, wherein the checkpoint inhibitor targets PD-1, PD-L1, and CTLA-4.

34. The method of claim 33, wherein the checkpoint inhibitor of PD-1 is selected from Pembrolizumab or Nivolumab.

35. The method of claim 33, wherein the checkpoint inhibitor of PD-L1 is Atezolizumab.

36. The method of claim 33, wherein the checkpoint inhibitor of CTLA-4 is Ipilimumab.