Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/69—Boron compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
  • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the technology described herein relates to the modulation of asymmetric cell division and proliferation in cancer cells.
• Tumors can comprise both rapidly proliferating and slowly proliferating cells. Tumors comprising particularly rapidly proliferating cells clearly are capable of faster growth and progression. But these tumors also contain many slowly proliferating cancer cells that may complicate treatment by resisting cancer therapeutics which preferentially target fast proliferators. While clonal selection theory clearly explains how rapidly proliferating cancer cells evolve, it remains difficult to understand within this framework why even advanced tumors contain so many slowly proliferating cancer cells (P. C. Nowell, Science 194, 23 (Oct 1, 1976)).
• asymmetric proliferation In culture, cancer cells have been observed to occasionally divide in such a manner that one daughter cell will have a markedly slower proliferative rate than the other, a phenomenon referred to as "asymmetric proliferation.” The occurrence of asymmetric proliferation is generally assumed to simply reflect random variation among individual cancer cells in the many genetic and non-genetic factors that influence transit through the cell cycle (J. Massague, Nature 432, 298 (Nov 18, 2004)).
• Figures 1A-1P demonstrate that mTORC2-AKTl signaling regulates asymmetric cancer cell division.
• Figure 1A depicts a crystal structure of AKTl protein with mutated sites noted.
• Figures IB- IE and 1G-1P the bar graphs depict percentages of H3K9me2 low /MCM2 low /HESl Mgh asymmetric cells and GO-like cells.
• Figure IB depicts the results of AKT1/2 7" HCTl 16 cells replaced with AKTl and AKT2 cDNAs, HCTl 16 wild type (WT).
• Figure 1C depicts the results of mutation of AKT, e.g.
• FIG. 16 depicts the results of HCTl 16 and MCF7 cells, respectively treated with DMSO, control, TORIN1, AZD8055, ⁇ 128, Palomid 529, Rapamycin and RAD-001, for 72 h.
• Figures 1G-1K depict the results of RICTOR knockdown in HCTl 16 (Figure 1G), MCF7 (Figure 1H), MDA-MB-231 ( Figure II), PC9 ( Figure 1J) and A375 (Figure IK) cells with control, non- silencing(NS) hairpin(hp) and RICTOR knockdown hp 1,4.
• Figures 1L-1M depict the results of HCTl 16 and MCF7 cells, respectively treated with DMSO, control and kinase inhibitors AZD5363, GDC0068, for 72 hours.
• Figure IN depicts the results of AKT1/2 7" HCTl 16 cells replaced with AKT1-E17K cDNA, HCTl 16 WT.
• Figures 10- IP depicts the results of HCTl 16 and MCF7 cells, respectively treated with DMSO, control and AKT allosteric inhibitors AKT1/2, MK2206, for 72 h. Error bars indicate mean ⁇ SEM.
• Figure IF depicts the results of a Western blot for RICTOR in HCTl 16 cells.
• Figures 2A-2F demonstrate that a TTC3-proteasome pathway is necessary for GO-like cells.
• Figure 2A depicts the results of a Western blot for TTC3 in HCTl 16 cells.
• Figures 2B-2F depict bar graphs of percentages of H3K9me2 low /MCM2 low /HESl Mgh .
• Figures 2B-2C depict GO-like cells in HCTl 16 ( Figure 2B) and MCF7 ( Figure 2C) cells with control, NS hp, and TTC3 knockdown hp3-5.
• Figure 2D depicts the results of AKT1/2 7" HCTl 16 cells replaced with AKT1-K8R, AKT1- K14R and AKT1-K8R/K14R cDNAs, HCTl 16 WT.
• Figures 2E-2F depict the results of HCTl 16 ( Figure 2E) and MCF7 ( Figure 2F) cells treated with DMSO, control, MG-132 and Bortezomib for 24hours. Error bars indicate mean ⁇ SEM. Arrow indicates a GO-like TTC3 + cell.
• Figure 3 depicts a graph demonstrating that mTORC2 signaling induces slow
• Figures 4A-4N demonstrate that asymmetrically dividing cancer cells and slow proliferators promote tumorigenesis in vivo.
• Figures 4A-4E depict the results of control, NS (squares) and RICTOR knockdown shRNAs hp 1,4 (triangles and diamonds, respectively) of HCTl 16 (Figure 4A), MCF7 (Figure 4B), MDA-MB-231 ( Figure 4C), PC9 ( Figure 4D) and A375 ( Figure 4E) cells were injected in Nu/Nu mice and their tumor growth followed over a number of days.
• Figures 4F-4J, 4L, and 4N demonstrate that tumor forming potential of cells treated with DMSO, control (squares) or AKT 1/2 inhibitor for 72 h (diamonds) in ( Figures 4F-4J) HCTl 16 and ( Figures 4L and 4M) MCF7 cells, respectively.
• Serial dilutions of ( Figures 4F,4L) 5 l0 6 , (4G,4M) 5 l0 5 , (4H) 5x l0 4 , (41) 5x l0 3 , (4J) 5x l0 2 cells were used to inject mice and the tumor formation and growth was monitored over several days. Error bars indicate mean ⁇ SEM.
• Figures 4K and 4N depict images of mice injected with (4K) 5x l0 5 HCTl 16 cells, (4N) 5x l0 6 MCF7 cells, C (control, DMSO treated), I (induced, AKT1/2 inhibitor treated).
• Figure 5 depicts a working model for asymmetric cancer cell division.
• Figures 6A-6E demonstrate that RICTOR knockdown does not alter proliferation in vitro.
• Proliferation assay were performed over 5 days for control, NS shRNA (squares) and RICTOR knockdown shRNAs hp 1,4 (diamonds and triangles, respectively) in (6A) HCT1 16, (6B) MCF7, (6C) MDA-MB-231 , (6D) PC9 and (6E) A375 cell lines, under normal conditions. Error bars indicate mean ⁇ SEM.
• Figures 7A-7J demonstrate that i-integrin-FAK-mTORC2-AKTl signaling regulates the production of slow proliferators.
• Figure 7A depicts a schematic model of AKT1 protein.
• Figures 7B- 7H depict graphical representation of percentage of change of H3K9me21ow/MCM21ow/HES lhigh asymmetrically dividing and GO-like cells compared to control in HCT1 16 and MCF7 cell lines. Error bars indicate mean ⁇ SEM.
• Figures 7I-7J depict plots for percentage of sibling pairs with cell cycle time difference ⁇ t.
• Figure 8 demonstrates the interaction of FAK with RICTOR.
• RICTOR was immunoprecipitated with anti-FAK and immunoblotted with anti-mTOR, anti- RICTOR and anti-RAPTOR antibody.
• FAK was immunoprecipitated with anti- RICTOR and immunoblotted with anti-mTOR and anti-FAK, in Gl as well as M phase of the cell cycle.
• Figures 9A-9C demonstrate that slow proliferators promote tumorigenesis in vivo.
• Figure 9A depicts the experimental procedure and results for mice with subcutaneous tumors treated with TS2/16 antibody once a week for 5 weeks or untreated (control).
• Figure 9B depicts the experimental procedure and results when Inducible Non-Silencing shRNA (control) or RICTOR knockdown shRNAs hp 1 ,4 of 5 different cell lines were injected into mice.
• Figure 9C depicts the experimental procedure and results when serial dilutions of HCT1 16 and MCF7 cells incubated with DMSO (control) or ⁇ 1/2 ⁇ for 72hours were injected into mice. Tumor volume was followed weekly. Error bars indicate mean ⁇ SEM for five mice per group.
• Figure 10 depicts a working model for asymmetric cancer cell division.
• Figures 1 lA-1 1C demonstrate knockdown of proteins in HCT116 cells.
• Non-Silencing (NS) as control shRNA Figure 1 1C depicts a graphical representation of percentage of change of H3K9me21ow/MCM21ow/HES lhigh asymmetrically dividing and GO-like cells compared to control in RICTOR knockdown cell lines. Error bars indicate mean ⁇ SEM.
• Figures 12A-120 demonstrate that RICTOR knockdown does not alter proliferation in vitro.
• Figures 12A-120 depict graphs of the results of proliferation assay over 5 days for control, NS hp (squares) and RICTOR knockdown shRNAs hp 1,4 (triangles and circles, respectively) in (Figure
• Figures 13A- 13 J demonstrate that RICTOR knockdown does not alter proliferation in vitro.
• Figures 13A- 13E depict graphs of proliferation assay over 5 days for control, NS hp (squares) and RICTOR knockdown shRNAs hp 1,4 (triangles and circles, respectively) in (Figure 13 A) HCT1 16, ( Figure 13B) MCF7, ( Figure 13C) MDA-MB-231, ( Figure 13D) PC9 and (Figure 13E) A375 cell lines, under low glucose conditions.
• Figures 13F-13J depict the results of clonogenic assay over 2weeks after irradiation for control, NS hp and RICTOR knockdown shRNAs hp 1 ,4 in (Figure 13F) HCT1 16, ( Figure 13G) MCF7, ( Figure 13H) MDA-MB-231, ( Figure 131) PC9 and ( Figure 13 J) A375 cell lines. Error bars indicate mean ⁇ SEM.
• Figures 14A-14D demonstrate that RICTOR knockdown does not alter invasion in vitro.
• Figures 14A-14D depict the results of invasion assay over 24hours for control, NS hp (first bar) and RICTOR knockdown shRNAs hp 1,4 (second and third bars, respectively) in ( Figure 14A) HCT1 16, ( Figure 14B) MCF7, ( Figure 14C) PC9 and ( Figure 14D) A375 cell lines. Error bars indicate mean ⁇ SEM.
• Embodiments of the technology described herein relate to the inventor's discovery of a signaling pathway controlling asymmetric cell division and proliferation of cancer cells. Briefly, asymmetric proliferation is induced by the degradation of AKT1 protein. Modulating the rate of degradation of AKT1 protein can thus increase or decrease the rate of asymmetric proliferation and therefore the level of slow proliferator cancer cells within a population of cancer cells. In some embodiments, the degradation of AKT1 can be asymmetric. Methods relating to this modulation are described herein.
• “reduction”, “decrease”, or “inhibit” can mean a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or more or any decrease of at least 10% as compared to a reference level.
• the terms can represent a 100% decrease, i.e. a non-detectable level as compared to a reference level.
• a "decrease” is a statistically significant decrease in such level.
• the decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.
• the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
• the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
• a "increase” is a statistically significant increase in such level.
• a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
• Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
• the subject is a mammal, e.g., a primate, e.g., a human.
• the terms, "individual,” “patient” and “subject” are used interchangeably herein.
• the subject is a mammal.
• the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer.
• a subject can be male or female.
• a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for cancer or the one or more complications related to cancer.
• a subject can also be one who has not been previously diagnosed as having cancer or one or more complications related to cancer.
• a subject can be one who exhibits one or more risk factors for cancer or one or more complications related to cancer or a subject who does not exhibit risk factors.
• a "subject in need" of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
• a “cancer” or “tumor” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems.
• a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastatses. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
• a cancer cell can be a cell obtained from a tumor.
• Metastasis is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.
• MRI magnetic resonance imaging
• CT computed tomography
• cancers include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;
• lung cancer e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung
• lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's
• protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
• protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
• modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
• polypeptide are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
• protein and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
• exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
• nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
• the nucleic acid can be either single-stranded or double-stranded.
• a single-stranded nucleic acid can be one strand nucleic acid of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA.
• the nucleic acid can be DNA.
• nucleic acid can be RNA.
• Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.
• the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer.
• the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer therapy. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
• treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
• Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
• treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
• the term "pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
• a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
• pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site.
• Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
• statically significant or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
• compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.
• compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
• the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
• AKTl or "v-akt murine thymoma viral oncogene homolog 1” refers to a serine-threonine protein kinase activated by platelet-derived growth factor.
• the sequence of AKTl for a number of species is well known in the art, e.g. human AKTl (e.g. NCBI Ref Seq: NP 001014431 ; NCBI Gene ID : 207).
• mTORC2 refers to mTOR complex 2, a multi-protein complex comprising RICTOR, mTOR, G L, and MAPKAP 1 , and which phosphorylates Akt.
• the sequences of the components of mTORC2 are well known in the art, eg. human mTOR (e.g. NCBI Ref Seq: NP 004949; NCBI Gene ID: 2475), human G L (e.g. NCBI Ref Seq: NP 001 186102; NCBI Gene ID: 64223), and human MAPKAP 1 (e.g. NCBI Ref Seq: NP 001006618; NCBI Gene ID: 79109).
• RICTOR or "RPTOR independent companion of MTOR, complex 2” refers to a subunit of the mTORC2 complex.
• the sequence of RICTOR for a number of species is well known in the art, e.g. human RICTOR (e.g. NCBI Ref Seq: NP 689969; NCBI Gene ID: 253260).
• TTC3 or "tetratricopeptide repeat domain 3” refers to an E3 ligase that controls the ubiquitination of AKTl .
• the sequence of TTC3 for a number of species is well known in the art, e.g. human TTC3 (e.g. NCBI Ref Seq: NP 001001894; NCBI Gene ID: 7267).
• FAK focal adhesion kinase
• PTK2 in humans
• the sequence of FAK for a number of species is well known in the art, e.g. human FAK (e.g. NCBI Ref Seq: NP 005598; NCBI Gene ID: 5747).
• integrin refers to a class of transmembrane receptors that mediate the attachment of a cell to surrounding materials, e.g. extracellular matrix (ECM) or other cells, as well as transduce signals relating to the chemical and mechanical status of the surrounding materials and/or transduce signals from the cell to the surrounding materials. Integrins function as heterodimers, comprising an alpha chain and a beta chain. Mammalian genomes contain eighteen alpha subunits and eight beta subunits. In some embodiments of any of the aspects described herein, an integrin can be a ⁇ - integrin.
• ECM extracellular matrix
• ⁇ - integrin refers to a complete integrin heterodimer comprising a ⁇ beta chain and any of the eighteen possible alpha chains (e.g. l-al 1, aD, E, aL, M, aV, aX or a2B).
• the sequence of the ⁇ beta chain (i.e. ITGB 1) for a number of species is well known in the art, e.g., human ITGB1 (e.g. NCBI Gene ID: 3688; (mRNA: NCBI Ref Seq: NM 00221 1) (polypeptide NCBI Ref Seq:NP_002202).
• stem cell refers to a cell in an undifferentiated or partially differentiated state that has the property of self-renewal and has the developmental potential to naturally differentiate into a more differentiated cell type, without a specific implied meaning regarding developmental potential (i.e. , totipotent, pluripotent, multipotent, etc.).
• self-renewal is meant that a stem cell is capable of proliferation and giving rise to more such stem cells, while maintaining its developmental potential.
• stem cell refers to any subset of cells that have the developmental potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retain the capacity, under certain circumstances, to proliferate without substantially differentiating.
• somatic stem cell is used herein to refer to any stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Natural somatic stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Exemplary naturally occurring somatic stem cells include, but are not limited to, mesenchymal stem cells and hematopoietic stem cells. In some embodiments, the stem or progenitor cells can be embryonic stem cells.
• embryonic stem cells refers to stem cells derived from tissue formed after fertilization but before the end of gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10- 12 weeks gestation. Most frequently, embryonic stem cells are totipotent cells derived from the early embryo or blastocyst. Embryonic stem cells can be obtained directly from suitable tissue, including, but not limited to human tissue, or from established embryonic cell lines. In one embodiment, embryonic stem cells are obtained as described by Thomson et al. (U.S. Pat. Nos.
• Exemplary stem cells include embryonic stem cells, adult stem cells, pluripotent stem cells, neural stem cells, liver stem cells, muscle stem cells, muscle precursor stem cells, endothelial progenitor cells, bone marrow stem cells, chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells, hematopoietic stem cells, central nervous system stem cells, peripheral nervous system stem cells, and the like.
• Descriptions of stem cells, including method for isolating and culturing them, may be found in, among other places, Embryonic Stem Cells, Methods and Protocols, Turksen, ed., Humana Press, 2002; Weisman et al., Annu. Rev. Cell. Dev. Biol. 17:387 403; Pittinger et al., Science, 284: 143 47, 1999; Animal Cell Culture, Masters, ed., Oxford University Press, 2000;
• stromal cells including methods for isolating them, may be found in, among other places, Prockop, Science, 276:71 74, 1997; Theise et al., Hepatology, 31 :235 40, 2000; Current Protocols in Cell Biology, Bonifacino et al., eds., John Wiley & Sons, 2000 (including updates through March, 2002); and U.S. Pat. No. 4,963,489.
• progenitor cells refers to cells in an undifferentiated or partially differentiated state and that have the developmental potential to differentiate into at least one more differentiated phenotype, without a specific implied meaning regarding developmental potential (i.e. , totipotent, pluripotent, multipotent, etc.) and that does not have the property of self-renewal.
• progenitor cell refers to any subset of cells that have the developmental potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype.
• asymmetric proliferation refers to a process of cell division in which one daughter cell proliferates at the same rate as the parent cell while the other daughter cell proliferates at a statistically significantly slower rate.
• slow proliferators these slowly proliferating daughter cells are referred to herein as “slow proliferators.”
• slow proliferator or “GO-like cell”, which are used interchangeably herein, refer to a cancer cell which proliferates at a statistically significantly slower rate than the rate observed for at least 70% of cancer cells obtained from the same tumor.
• slow proliferators can be cancer cells which have statistically significantly decreased levels of expression of Aktl, H3K9me2, and MCM2 and statistically significantly increased levels of expression of TTC3 and Hes 1 as compared to the levels of expression found in at least 70% of cancer cells obtained from the same tumor.
• the level of expression of these markers can be the level of polypeptide expression product.
• a slow proliferator can revert to a normal, fast-proliferator phenotype, e.g. the slow proliferator phenotype can be reversible.
• a method of modulating the rate of asymmetric proliferation in a cell comprising: contacting the cell with a modulator of AKT1 degradation; wherein an increase in AKT1 degradation increases the rate of asymmetric proliferation in the cell; and wherein a decrease in AKT1 degradation decreases the rate of asymmetric proliferation in the cell.
• the rate of asymmetric proliferation in a population of cells can be modulated.
• AKT1 degradation refers to the ubiquitination and proteasome-mediated degradation of AKT1.
• the cell can be a cancer cell.
• the cell can be a stem and/or progenitor cell.
• the cell can be a cell engaged in wound repair, e.g. a cell located at a site of a wound and/or defect.
• the cell can be a cell undergoing asymmetric division, e.g. cells whose daughter cells comprise slow proliferators. Methods of identifying slow proliferator cells are described elsewhere herein.
• a modulator of AKT1 can be an agonist of ATK1 degradation or an inhibitor of AKT1 degradation.
• Modulators can be agents of any type and/or structure.
• the term "agent” refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject.
• An agent can be selected from a group comprising: polynucleotides;
• polypeptides are polypeptides; small molecules; antibodies; or functional fragments thereof.
• a polynucleotide can be RNA or DNA, and can be single or double stranded, and can be selected from a group comprising: nucleic acids and nucleic acid analogues that encode a polypeptide.
• a polypeptide can be, but is not limited to, a naturally-occurring polypeptide, a mutated polypeptide or a fragment thereof that retains the function of interest.
• agents include, but are not limited to a nucleic acid (DNA or RNA), small molecule, aptamer, protein, peptide, antibody, polypeptide comprising an epitope-binding fragment of an antibody, antibody fragment, peptide -nucleic acid (PNA), locked nucleic acid (LNA), small organic or inorganic molecules; saccharide; oligosaccharides;
• an agent can be applied to the media, where it contacts the cell and induces its effects.
• an agent can be intracellular as a result of introduction of a nucleic acid sequence encoding the agent into the cell and its transcription resulting in the production of the nucleic acid and/or protein environmental stimuli within the cell.
• the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities.
• the agent is a small molecule having a chemical moiety.
• chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof.
• Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
• the modulator of AKTl degradation can be an agonist and/or promoter of AKTl degradation.
• An agonist of AKTl degradation can be any agent that increases the level and/or rate of ATK1 degradation, either through direct or indirect action.
• the term "agonist" refers to an agent which increases the expression and/or activity of the target by at least 10% or more, e.g. by 10% or more, 50% or more, 100% or more, 200% or more, 500% or more, or 1000 % or more.
• Non- limiting examples of agonists of AKTl degradation include allosteric inhibitors of AKTl and clustered homology domain inhibitors of AKTl .
• an agonist of AKTl degradation can be a dual-specific (e.g. in inhibits AKTl and AKT2) inhibitor or an AKT1- specific inhibitor.
• Allosteric inhibitors and clustered homology domain inhibitors of AKTl are known in the art and include, by way of non-limiting example, AKT1/2; ARQ 092; and MK2206. Allosteric inhibitors of AKTl are also described, e.g. in U.S. Patent No. 8, 183,249; Cherrin et al.
• an agonist of AKTl degradation is not a catalytic inhibitor of AKTl .
• contacting a cancer cell with an agonist of ATK1 degradation leads to the production, or the increased production, of slow proliferator cancer cells.
• AKTl degradation is negatively regulated by FAK activity.
• an agonist of ATK1 degradation can include, by way of non- limiting example, an inhibitor of FAK expression and/or activity.
• a modulator of AKTl degradation can be an inhibitor of AKTl degradation.
• An inhibitor of AKTl degradation can be any agent that decreases the level and/or rate of AKTl degradation, whether by direct or indirect action.
• the term "inhibitor” refers to an agent which reduces the expression and/or activity of the target by at least 10%, e.g. by 10% or more, 20% or more, 30% or more, 50% or more, 75% or more, 90% or more, 95% or more, 98% or more, or 99% or more.
• AKT1 degradation is positively regulated by mTORC2, RICTOR, and TTC3. Accordingly, inhibiting these proteins and/or expression of these proteins can inhibit AKT1 degradation.
• inhibitors of AKT1 degradation include inhibitors of mTORC2 signaling, inhibitors of mTORC2, inhibitors of mTORC2 expression, inhibitors of RICTOR, inhibitors of RICTOR expression, inhibitors of TTC3, and inhibitors of TTC3 expression.
• AKT1 degradation is negatively regulated by ⁇ -integrin activity. Accordingly, activating or increasing ⁇ -integrin expression or activity can inhibit AKT1 degradation.
• inhibitors of AKT1 degradation include activators of ⁇ -integrin activity and activators of ⁇ -integrin expression.
• irregular concentrations of collagen in the extracellular environment can create polar activation of ⁇ -integrin by the collagen, which can increase A T1 degradation. Accordingly, providing a substrate or growth medium for a cell such that the individual cell is exposed to a homogeneous concentration of collagen can inhibit AKT1 degradation.
• Inhibitors of the expression of a given gene can be an inhibitory nucleic acid.
• gene silencing or RNAi can be used.
• contacting a cell with the inhibitor results in a decrease in the target mRNA level in a cell of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the miRNA or RNA interference molecule.
• the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.
• the inhibitor can comprise an expression vector or viral vector comprising the RNAi molecule.
• RNAi refers to any type of interfering RNA, including but are not limited to RNAi, siRNA, shRNA, endogenous microRNA and artificial microRNA. For instance, it includes sequences previously identified as siRNA, regardless of the mechanism of down-stream processing of the RNA (i.e. although siRNAs are believed to have a specific method of in vivo processing resulting in the cleavage of mRNA, such sequences can be incorporated into the vectors in the context of the flanking sequences described herein).
• RNAi and "RNA interfering" with respect to an agent of the technology described herein, are used interchangeably herein.
• RNA refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is present or expressed in the same cell as the target gene.
• the double stranded RNA siRNA can be formed by the complementary strands.
• a siRNA refers to a nucleic acid that can form a double stranded siRNA.
• the sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof.
• the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).
• shRNA small hairpin RNA
• stem loop is a type of siRNA.
• shRNAs are composed of a short, e.g. about 19 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand.
• the sense strand can precede the nucleotide loop structure and the antisense strand can follow.
• RNAi may be delivered with the help of nanoparticles as described for example in Schiffelers and Storm, Expert Opin Drug Deliv. 2006 May;3(3):445-54 or liposomes (e.g. Hughes et al., Methods Mol Biol. 2010;605:445-59).
• Inhibitors of mTORC2 are known in the art and include, by way of non-limiting example, TORIN1, AZD8055, ⁇ 128, and Palomid-529. Further examples of mTORC2 inhibitors include OSI-027; MK8669; TOP216; TORISEL; CERTICAN; ABI-009; KU-0063794; AZD2014; NVP- BGT226; PF-04691502; PP242; XL765; EXEL-2044; EXEL-3885; EXEL-4431 ; EXEL-7518 and those described, e.g. in US Patent Publication 2012/0165334; 201 1/0224223; 2012/01 14739;
• an inhibitor of mTORC2 can be an inhibitor of mTORCl and mTORC2. In some embodiments, an inhibitor of mTORC2 can be specific for inhibition of mTORC2.
• Inhibitors of RICTOR are known in the art and include, by way of non- limiting example, NVP-BEZ235.
• Inhibitors of TTC3 are known in the art and include, by way of non- limiting example, MG-132 and bortezomib.
• Inhibitors of FAK are known in the art and include, by way of non- limiting example, PF- 562271 and NVP-TAE226.
• Inhibitors of ⁇ -integrin activity are known in the art and include, by way of non- limiting example, the monoclonal antibodies A2B2 and P4C10.
• therapies which target fast proliferator cells can be ineffective in decreasing populations of slow proliferators (see, e.g. Dey-Guha et al. PNAS 2011 108: 12845- 12850; which is incorporated by reference herein in its entirety).
• described herein is a method of treating cancer in a subject in need thereof, the method comprising: administering an inhibitor of AKT1 degradation to the subject (i.e. decreasing the number of slow proliferators in the subject).
• the method can further comprise administering a cancer therapy that targets fast proliferator cancer cells.
• the inhibitor of AKT1 degradation can be administered before the administration of a cancer therapy that targets fast proliferator cancer cells.
• the inhibitor of AKT1 degradation can be administered at least 1 day before the administration of a cancer therapy that targets fast proliferator cancer cells, e.g. 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or further before. In some embodiments, the inhibitor of AKT1 degradation can be administered at least 3 days before administration of a cancer therapy that targets fast proliferator cancer cells.
• Cancer therapies that target fast proliferator cells are well known in the art and include, by way of non-limiting example, therapies that degrade or disrupt nucleic acids, e.g. doxorubicin, alkylating agents, nitrogen mustard alkylating agents, agents that intercalate DNA;
• cyclophosphamide or therapies that inhibit cell division, e.g. mitotic inhibitors, paclitaxel.
• an inhibitor of AKT1 degradation can be administered to reduce and/or reverse the growth of a cancer. In some embodiments, an inhibitor of AKT1 degradation can be administered to reduce the rate of the growth of a cancer. In some embodiments, an inhibitor of AKT1 degradation can be administered to prevent the growth of a cancer. In some embodiments, an inhibitor of AKT1 degradation can be administered to prevent relapse and/or development of a cancer.
• agents that can be fast proliferator targeting agents include, but are not limited to chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies (e.g., Herceptin®), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (Tarceva®)), platelet derived growth factor inhibitors (e.g., GleevecTM (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-be
• cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
• the term is intended to include radioactive isotopes (e.g. At 211 , 1 131 , 1 125 , Y 90 ,
• chemotherapeutic agents such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
• chemotherapeutic agent refers to any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancer as well as diseases characterized by hyperplastic growth.
• Chemotherapeutic agents as used herein encompass both chemical and biological agents. These agents function to inhibit a cellular activity upon which the cancer cell depends for continued survival.
• chemotherapeutic agents include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most if not all of these agents are directly toxic to cancer cells and do not require immune stimulation.
• a chemotherapeutic agent is an agent of use in treating neoplasms such as solid tumors.
• a chemotherapeutic agent is a radioactive molecule.
• chemotherapeutic agent of use e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et ah, Chemotherapy, Ch.
• the modulators of AKT1 degradation described herein can be used in conjunction with additional chemotherapeutic agents.
• radiation therapy is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one-time administration and typical dosages range from 10 to 200 units (Grays) per day.
• the subject can be one who has been identified has having slow proliferator cells.
• the subject has been determined to have a subpopulation of cancer cells expressing increased levels of one or more genes selected from the group consisting of: Hesland TTC3; and/or decreased levels of one or more genes selected from the group consisting of: AKT1 ; H3K9me2; and MCM2; wherein an increased level is a level statistically significantly higher than the level of expression found in at least 70% of cancer cells obtained from the same tumor and a decreased level is a level statistically significantly lower than the level of expression found in at least 70% of cancer cells obtained from the same tumor.
• the subject can have been determined to have cancer cells expressing increased levels of TTC3; and, optionally, increased levels of Hesl and/or decreased levels of one or more genes selected from the group consisting of: AKT1 ; H3K9me2; and MCM2.
• the subject can have been determined to have cancer cells expressing increased levels of Hesland TTC3 and decreased levels AKT1 ; H3K9me2; and MCM2.
• an increased level can be at least 2x higher than the level of expression found in at least 70% of cancer cells obtained from the same tumor, e.g. at least 2x, at least 3x, at least 4x, at least 5x, at least lOx, or higher.
• an increased level can be at least 50% or less than the level of expression found in at least 70% of cancer cells obtained from the same tumor, e.g. 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or less.
• the expression level of a gene can be the level of mRNA or polypeptide expression product. In some embodiments, the level of expression can be determined, e.g. by in situ
• the expression level of an mRNA expression product can be determined, e.g. by RT-PCR, quantitative RT-PCR, RNA-seq, Northern blot, or microarray based expression analysis.
• the level of expression of a gene can be the level of polypeptide expression product. Methods for measuring polypeptide expression products are known in the art and include, by way of non-limiting example ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, immunohistochemistry, and immunofluorescence using detection reagents such as an antibody or protein binding agent.
• the expression level can be determined by ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, immunohistochemistry, and immunofluorescence using detection reagents such as an antibody or protein binding agent.
• the expression level can be determined by ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, immunohistochemistry, and immunofluorescence using detection rea
• IHC immunohistochemistry
• ICC immunocytochemistry
• Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells.
• the antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience, e.g. a change in color, upon encountering the targeted molecules or upon treatment with a chemical agent.
• signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker signal or marker activity (e.g. an enzyme activity), follows the application of a primary target-specific antibody.
• kits for determining if a subject has slow proliferator cells can comprise a detection agent specific for an expression product of TTC3.
• the kit can comprise a detection agent specific for an expression product of at least one of the genes selected from the group consisting of: TTC3; Hesl ; AKT1 ;
• a detection agent can be any agent which can specifically detect the presence of the target (e.g. bind specifically to the target) according to an assay described herein, e.g. a detection reagent can be a nucleic acid probe or primer specific for the target or an agent which specifically binds to a target polypeptide.
• the detection reagent can comprise a detectable signal or be capable of generating a detectable signal.
• the detection agent can be an antibody reagent.
• the detection agent can be a monoclonal antibody and/or comprise CDRs of a monoclonal antibody.
• Non-limiting examples of antibody reagents specific for the described slow proliferator markers are described in the Examples herein.
• the kit can further comprise reagents necessary for performing the assay, e.g. buffers and/or reagents for generating and/or detecting a detectable signal.
• the kit can further comprise instructions.
• an antibody reagent refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen.
• An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody.
• an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody.
• an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
• an antibody in another example, includes two heavy (H) chain variable regions and two light (L) chain variable regions.
• antibody reagent encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies.
• An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof).
• Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
• VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” ("CDR"), interspersed with regions that are more conserved, termed “framework regions” ("FR").
• CDR complementarity determining regions
• FR framework regions
• the extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties).
• Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• antigen-binding fragment or "antigen-binding domain”, which are used interchangeable herein are used herein to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest.
• binding fragments encompassed within the term "antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546; which is incorporated by reference herein in its entirety), which consists of
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv).
• scFv single chain Fv
• Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those of skill in the art.
• “monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody” or “monoclonal antibody composition,” which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.
• specific binding refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
• specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.
• label refers to a composition capable of producing a detectable signal indicative of the presence of an antibody reagent (e.g. a bound antibody reagent). Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
• the rate of growth of a cancer can be reduced by increasing the percentage of the cells which are slow proliferators.
• a method of treating cancer in a subject in need thereof comprising: administering an agonist of AKTl degradation to the subject.
• Administration of an agonist of ATK1 degradation can increase the number of slow proliferators present in a tumor, causing the overall growth rate of the tumor to decrease.
• the subject can be a subject selected from the group consisting of: a subject with early stage cancer; a subject who is in remission or is likely to be in remission; a subject at risk of developing cancer and/or a subject at risk of having a cancer and/or tumor grow to the extent that it is clinically dangerous.
• the cancer to be treated can be any type of cancer in any location.
• the cancer can be breast cancer, lung cancer, prostate cancer, colorectal cancer, lung cancer, and/or melanoma.
• the cancer can comprise a metastasis
• a method of producing slow proliferator cancer cells comprising: (i) contacting a cancer cell with an agonist of AKTl degradation; (ii) maintaining the cancer cells treated in step (i).
• the cells can be maintained in vivo or in vitro.
• Conditions suitable for maintaining cells in culture are well known in the art and can vary depending on the precise identity of the cells.
• slow proliferators can be maintained under the same cell culture conditions as the cancer cells from which they originated. Examples of suitable cell culture conditions are described in the Examples herein.
• the method can further comprise the step of enriching the cells of step (ii) for slow proliferators by selecting for cells having increased levels of expression of TTC3 and optionally, increased levels of expression of Hes 1 or decreased levels of expression of AKT1 ; H3K9me2; MCM2; MK167; CDC6; GMN ; AURKA; PLKl ; H3S10ph; H3K4me2; H3K9me2; H3K27me3; H4K12ac; or H4K16ac.
• the method can further comprise the step of enriching the cells of step (ii) for slow proliferators by selecting for cells having increased levels of expression of TTC3 and Hesl and decreased levels of expression of AKT1 ; H3K9me2; MCM2; MK167; CDC6; GMNN; AURKA; PLKl ; H3S10ph;
• Enriching can encompass selecting for slow proliferators (e.g. treating with an agent specific for fast proliferators) or sorting slow proliferators from other cells, e.g. by FACS sorting.
• an anti-slow proliferator agent is any agent which can either 1) cause slow proliferators to convert to a fast proliferator phenotype (e.g.
• an anti-tumor effect can comprise a reduction in the growth of a tumor, a reduction in signs or symptoms of cancer, a reduction in mortality, cytotoxic activity, cytotoxicity specific for slow proliferators, reduction in relapse after remission, a reduction in invasiveness, and/or a reduction of metastasis.
• a reduction in the growth of a tumor e.g. by cell viability assays, or by measuring the size of tumors over time.
• test agent refers to a compound or agent and/or compositions thereof that are to be screened to determine whether they possess anti- tumor and/or anti- slow proliferator activity, as identified herein.
• a test agent can be a nucleic acid (DNA or RNA), small molecule, aptamer, protein, peptide, antibody, polypeptide comprising an epitope-binding fragment of an antibody, antibody fragment, peptide-nucleic acid (PNA), locked nucleic acid (LNA), small organic or inorganic molecules;
• saccharide oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi, or mammalian cells or tissues; naturally occurring or synthetic compositions; peptides; aptamers; and antibodies, or fragments thereof.
• biological macromolecules e.g., peptides, proteins, and peptide analogs and derivatives
• peptidomimetics nucleic acids
• nucleic acid analogs and derivatives extracts made from biological materials such as bacteria, plants, fungi, or mammalian cells or tissues; naturally occurring or synthetic compositions; peptides; aptamers; and antibodies, or fragments thereof.
• the methods of screening described herein can be performed in vitro or in vivo.
• the cancer cell contacted with the agonist of AKTl degradation is located and/or maintained in vivo, e.g. in an animal model of cancer.
• the cancer cell contacted with the agonist of AKTl degradation is located and/or maintained in vitro, e.g. in cell culture.
• the method can further comprise selecting for slow proliferator cells after step (i), e.g. sorting cells by FACS using the slow proliferator markers described herein (e.g. TTC3).
• contacting a population of cancer cells with a agonist of AKTl degradation can increase the number and/or proportion of slow proliferators in the population by a statistically significant amount. In some embodiments, contacting a population of cancer cells with a agonist of AKTl degradation can increase the number and/or proportion of slow proliferators in the population by at least 2x, e.g. 2x or more, 3x or more, 4x or more, 5x or more, 6x or more, 7x or more, 8x or more, 9x or more, lOx or more, 20x or more, 50x or more, or lOOx or more.
• 2x e.g. 2x or more, 3x or more, 4x or more, 5x or more, 6x or more, 7x or more, 8x or more, 9x or more, lOx or more, 20x or more, 50x or more, or lOOx or more.
• the methods of screening described herein can also be adapted to screen for agents which cause slow proliferators to retain a slow proliferator phenotype (i.e. cause a lower rate of reversion to a fast proliferator phenotype as compared to untreated cells) or agents which cause slow proliferators to enter a dormant or quiescent state.
• agents which cause slow proliferators to retain a slow proliferator phenotype i.e. cause a lower rate of reversion to a fast proliferator phenotype as compared to untreated cells
• agents which cause slow proliferators to enter a dormant or quiescent state e.g. by measuring proliferation rates and/or metabolic rates.
• a method comprising; (i) obtaining a tumor biopsy from a subject; (ii) determining the expression level of TTC3 in cells obtained from the subject; (iii) identifying the presence of slow proliferators in the tumor when cells with increased levels of expression of TTC3 are detected.
• the expression level of TTC3 and optionally Hesl, AKTl ; H3K9me2; MCM2; MK167; CDC6; GMN ; AURKA; PLK1 ; H3S10ph; H3K4me2; H3K9me2; H3K27me3; H4K12ac; or H4K16ac can be determined; wherein the presence of slow proliferators in the tumor is indicated when cells with increased levels of expression of TTC3 and optionally increased levels of expression of Hesl or decreased levels of expression of AKTl ;
• the method can further comprise administering an inhibitor of AKTl degradation to the subject.
• the method can further comprise treating the cancer with an inhibitor of AKTl degradation according to any of the embodiments described herein.
• the determination of the expression level of the expression products foregoing genes can be performed as described in, e.g. Dey- Guha et al.
• a method of screening for a biomarker of anti-slow proliferator cells comprising: (i) contacting a cancer cell with an agonist of AKT1 degradation; (ii) measuring the expression of one or more genes in the cell of (i) and comparing that to the level of expression to a reference level (e.g. the level in the cell prior to step (i) or to a cell not treated according to step (i)), wherein a gene having expression after step (i) which varies by a statistically significant amount is identified as a biomarker of slow proliferator status.
• a reference level e.g. the level in the cell prior to step (i) or to a cell not treated according to step (i)
• a gene having expression after step (i) which varies by a statistically significant amount is identified as a biomarker of slow proliferator status.
• the methods described herein relate to treating a subject having or diagnosed as having cancer with a modulator of AKT1 degradation.
• Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and/or
• cancer complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, growth of a tumor, impaired function of the organ or tissue harboring cancer cells, etc.
• Tests that may aid in a diagnosis of, e.g. cancer include, but are not limited to, tissue biopsies and histological examination.
• a family history of cancer or exposure to risk factors for cancer can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.
• compositions and methods described herein can be administered to a subject having or diagnosed as having cancer.
• the methods described herein comprise administering an effective amount of compositions described herein, e.g. modulators of ATK1 degradation to a subject in order to alleviate a symptom of a cancer.
• modulators of ATK1 degradation e.g. modulators of ATK1 degradation to a subject in order to alleviate a symptom of a cancer.
• "alleviating a symptom of a cancer” is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
• a variety of means for administering the compositions described herein to subjects are known to those of skill in the art.
• Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
• the term "effective amount” as used herein refers to the amount of a modulator of ATK1 degradation needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
• therapeutically effective amount therefore refers to an amount of a modulator of ATK1 degradation that is sufficient to effect a particular anti-tumor effect when administered to a typical subject.
• An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate "effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
• Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dosage can vary depending upon the dosage form employed and the route of administration utilized.
• the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
• Compositions and methods that exhibit large therapeutic indices are preferred.
• a therapeutically effective dose can be estimated initially from cell culture assays.
• a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a modulator of AKT1 degradation, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model.
• IC50 i.e., the concentration of a modulator of AKT1 degradation, which achieves a half-maximal inhibition of symptoms
• Levels in plasma can be measured, for example, by high performance liquid chromatography.
• the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor growth, among others.
• the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
• the technology described herein relates to a pharmaceutical composition
• a pharmaceutical composition comprising a modulator of ATK1 degradation as described herein, and optionally a pharmaceutically acceptable carrier.
• Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
• materials which can serve as pharmaceutically - acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as e
• wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
• the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
• the carrier inhibits the degradation of the active agent, e.g. a modulator of ATKl degradation as described herein.
• the pharmaceutical composition comprising a modulator of ATKl degradation as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient.
• parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
• controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, administration DUROS ® -type dosage forms, and dose-dumping.
• Suitable vehicles that can be used to provide parenteral dosage forms of a modulator of ATKl degradation as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
• Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a modulator of ATKl degradation as disclosed herein can also be incorporated into the parenteral dosage forms
• compositions comprising a modulator of ATKl degradation can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil- in-water emulsion, or a water-in-oil emulsion.
• Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005); which is incorporated by reference herein in its entirety.
• Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
• controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
• controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
• the agent can be administered in a sustained release formulation.
• Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts.
• the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
• Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
• Controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
• a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5, 120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365, 185 Bl ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example,
• hydroxypropylmethyl cellulose other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
• OROS Alza Corporation, Mountain View, Calif. USA
• the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy.
• a second agent and/or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carbop latin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI- 103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide,
• pancratistatin a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
• neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, ADPJAMYCIN® doxorubicin (including morpholino-doxorubicin,
• dromostanolone propionate epitiostanol, mepitiostane, testolactone
• anti-adrenals such as aminoglutethimide, mitotane, trilostane
• folic acid replenisher such as frolinic acid
• aceglatone
• aldophosphamide glycoside aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;
• pirarubicin losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton,
• chloranbucil GEMZAR® gemcitabine
• 6-thioguanine 6-thioguanine
• mercaptopurine methotrexate
• platinum analogs such as cisplatin, oxalip latin and carboplatin
• vinblastine platinum
• platinum etoposide (VP- 16);
• ifosfamide mitoxantrone; vincristine; NAVELBINETM. vinorelbine; novantrone; teniposide;
• edatrexate edatrexate
• daunomycin aminopterin
• xeloda xeloda
• ibandronate irinotecan (Camptosar, CPT-1 1)
• irinotecan including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine;
• combretastatin combretastatin
• leucovorin LV
• oxaliplatin including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TykerbTM); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
• an effective dose of a composition comprising a modulator of ATKl degradation as described herein can be administered to a patient once.
• an effective dose of a composition comprising a modulator of ATKl degradation can be administered to a patient repeatedly.
• subjects can be administered a therapeutic amount of a composition comprising a modulator of ATKl degradation such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
• a composition comprising a modulator of ATKl degradation can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
• the administration can be repeated, for example, on a regular basis, such as hourly for 3 hours, 6 hours, 12 hours or longer or such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer.
• the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.
• Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. cancer by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
• the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
• the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active agent.
• the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
• administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months.
• dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.
• the dosage ranges for the administration of a modulator of ATK1 degradation according to the methods described herein depend upon, for example, the form of a modulator of ATK1 degradation, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for cancer growth or the extent to which, for example, tumor size are desired to be induced.
• the dosage should not be so large as to cause adverse side effects.
• the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art.
• the dosage can also be adjusted by the individual physician in the event of any complication.
• a modulator of ATK1 degradation in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician.
• a treatment is considered "effective treatment," as the term is used herein, if any one or all of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
• Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. tumor size. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g.
• An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
• Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. reduction in tumor growth rate). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. tumor size.
• a method of modulating the rate of asymmetric proliferation in a cell comprising:
• AKT1 degradation decreases the rate of asymmetric proliferation in the cell.
• a cancer cell a stem cell; a progenitor cell; and a cell engaged in wound repair.
• AKTl degradation selected from the group consisting of:
• AKTl an allosteric inhibitor of AKTl ; AKT1/2; MK2206; an inhibitor of ⁇ -integrin expression; an inhibitor of ⁇ -integrin activity; A2B2: P4C10; an inhibitor of focal adhesion kinase (FAK) expression; an inhibitor of FAK activity; PF-562271 ; and NVP-TAE226.
• AKTl an allosteric inhibitor of AKTl ; AKT1/2; MK2206; an inhibitor of ⁇ -integrin expression; an inhibitor of ⁇ -integrin activity; A2B2: P4C10; an inhibitor of focal adhesion kinase (FAK) expression; an inhibitor of FAK activity; PF-562271 ; and NVP-TAE226.
• FAK focal adhesion kinase
• inhibitor of AKTl degradation selected from the group consisting of:
• mTORC2 mTOR complex 2
• TORIN1 ; AZD8055; ⁇ 128; Palomid-529; inhibitors of mTORC2 expression; inhibitors of RPTOR independent companion of MTOR, complex 2 (RICTOR); inhibitors of RICTOR expression; an inhibitor of tetratricopeptide repeat domain 3 (TTC3); MG- 132; bortezomib; activators of ⁇ -integrin activity; activators of ⁇ - integrin expression.
• the modulator of AKTl degradation is a substrate or growth medium which provides a homogeneous concentration of collagen to an individual cell;
• AKTl degradation is inhibited by the symmetric activation of ⁇ -integrin by the homogeneous concentrations of collagen.
• a method of treating cancer in a subject in need thereof comprising:
• inhibitor of AKTl degradation is selected from the group consisting of:
• inhibitors of mTOR complex 2 mTORC2 signaling; inhibitors of mTORC2; TORIN1 ; AZD8055; ⁇ 128; Palomid-529; inhibitors of mTORC2 expression; inhibitors of RPTOR independent companion of MTOR, complex 2 (RICTOR); inhibitors of RICTOR expression; an inhibitor of tetratricopeptide repeat domain 3 (TTC3); MG-132; bortezomib; activators of ⁇ -integrin activity; activators of ⁇ - integrin expression.
• mTORC2 mTOR complex 2
• melanoma lung cancer; colorectal cancer; and breast cancer.
• the method of paragraph 10 wherein the inhibitor of AKTl degradation is administered before the administration of a cancer therapy that targets fast proliferator cancer cells.
• the method of paragraph 1 wherein the inhibitor of AKTl degradation is administered at least 1 day before the administration of a cancer therapy that targets fast proliferator cancer cells.
• an increased level is a level statistically significantly higher than the level of expression found in at least 70% of cancer cells obtained from the same tumor and a decreased level is a level statistically significantly lower than the level of expression found in at least 70% of cancer cells obtained from the same tumor.
• AKTl AKT1 ; H3K9me2; MCM2; MK167; CDC6; GMN ; AURKA; PLK1 ; H3S 10ph; H3K4me2; H3K9me2; H3K27me3; H4K12ac; and H4K16ac.
• a method of treating cancer in a subject in need thereof comprising:
• agonist of AKTl degradation is selected from the group consisting of:
• AKTl an allosteric inhibitor of AKTl ; AKT1/2; MK2206; an inhibitor of ⁇ -integrin expression; an inhibitor of ⁇ -integrin activity; A2B2: P4C10; an inhibitor of focal adhesion kinase (FAK) expression; an inhibitor of FAK activity; PF-562271 ; and NVP-TAE226.
• AKTl an allosteric inhibitor of AKTl ; AKT1/2; MK2206; an inhibitor of ⁇ -integrin expression; an inhibitor of ⁇ -integrin activity; A2B2: P4C10; an inhibitor of focal adhesion kinase (FAK) expression; an inhibitor of FAK activity; PF-562271 ; and NVP-TAE226.
• FAK focal adhesion kinase
• a subject with early stage cancer a subject who is in remission or is likely to be in remission; and a subject at risk of developing cancer.
• a method of screening for an anti-slow proliferator agent comprising:
• step (ii) contacting the cell obtained from step (i) with a test agent
• test agent (iv) identifying a test agent as an anti-slow proliferator agent when a statistically significant anti-tumor effect is observed.
• a method comprising;
• H3K27me3; H4K12ac; or H4K16ac are detected.
• a method of producing slow proliferator cancer cells comprising:
• step (ii) maintaining the cancer cells treated in step (i).
• the method further comprises the step of enriching the cells of step (ii) for slow proliferators by selecting for cells having increased levels of expression of TTC3 and optionally, increased levels of expression of Hes 1 or decreased levels of expression of AKT1 ; H3K9me2; MCM2; MK167; CDC6; GMNN; AURKA; PLK1 ;
• H3S10ph H3K4me2; H3K9me2; H3K27me3; H4K12ac; or H4K16ac.
• step (ii) for slow proliferators by selecting for cells having increased levels of expression of TTC3 and Hesl and decreased levels of expression of AKT1 ; H3K9me2;
• H3K27me3 H4K12ac; and H4K16ac.
• kit of paragraph 31 wherein the kit comprises a detection agent specific for an expression product of TTC3.
• kit comprises a detection agent specific for an expression product of at least one of the genes selected from the group consisting of: TTC3; Hesl ; AKTl ; H3K9me2; MCM2; MK167; CDC6; GMN ; AURKA; PLK1 ; H3S10ph; H3K4me2; H3K9me2; H3K27me3; H4K12ac; and H4K16ac.
• an inhibitor of AKTl degradation to treat cancer, the use comprising administering an inhibitor of AKTl degradation to a subject in need of treatment for cancer.
• mTORC2 mTOR complex 2
• TORIN1 ; AZD8055; ⁇ 128; Palomid-529; inhibitors of mTORC2 expression; inhibitors of RPTOR independent companion of MTOR, complex 2 (RICTOR); inhibitors of RICTOR expression; an inhibitor of tetratricopeptide repeat domain 3 (TTC3); MG-132; bortezomib; activators of ⁇ -integrin activity; activators of ⁇ - integrin expression.
• melanoma lung cancer; colorectal cancer; and breast cancer.
• paragraph 39 wherein the inhibitor of AKTl degradation is administered at least 1 day before the administration of a cancer therapy that targets fast proliferator cancer cells.
• paragraph 39 wherein the inhibitor of AKTl degradation is administered at least 3 days before the administration of a cancer therapy that targets fast proliferator cancer cells.
• any of paragraphs 35-41 wherein the subject has been determined to have a subpopulation of cancer cells expressing increased levels of one or more genes selected from the group consisting of:
• AKTl AKT1 ; H3K9me2; MCM2; MK167; CDC6; GMNN; AURKA; PLK1 ; H3S 10ph; H3K4me2; H3K9me2; H3K27me3; H4K12ac; and H4K16ac.
• an increased level is a level statistically significantly higher than the level of expression found in at least 70% of cancer cells obtained from the same tumor and a decreased level is a level statistically significantly lower than the level of expression found in at least 70% of cancer cells obtained from the same tumor.
• AKT1 AKT1 ; H3K9me2; MCM2; MK167; CDC6; GMN ; AURKA; PLK1 ; H3S 10ph; H3K4me2; H3K9me2; H3K27me3; H4K12ac; and H4K16ac.
• AKTl an allosteric inhibitor of AKTl ; AKT1/2; MK2206; an inhibitor of ⁇ -integrin expression; an inhibitor of ⁇ -integrin activity; A2B2: P4C10; an inhibitor of focal adhesion kinase (FAK) expression; an inhibitor of FAK activity; PF-562271 ; and NVP-TAE226.
• AKTl an allosteric inhibitor of AKTl ; AKT1/2; MK2206; an inhibitor of ⁇ -integrin expression; an inhibitor of ⁇ -integrin activity; A2B2: P4C10; an inhibitor of focal adhesion kinase (FAK) expression; an inhibitor of FAK activity; PF-562271 ; and NVP-TAE226.
• FAK focal adhesion kinase
• a subject with early stage cancer a subject who is in remission or is likely to be in remission; and a subject at risk of developing cancer.
• Cancer cells in culture usually divide to produce two daughters that will divide again in relative synchrony, but occasionally these cells will divide to produce one daughter cell with a markedly slower proliferative rate than the other. Since established cell lines have been grown for many years under experimental conditions that ought to favor purifying selection for a rapidly and uniformly dividing population, this asynchronicity in cell culture is quite puzzling and remains poorly understood. It is generally assumed to simply reflect random variation among individual cancer cells in the many genetic and non-genetic factors that influence transit through the cell cycle (8).
• cancer cells divide asymmetrically at low frequency (i.e. ⁇ 5% of cell divisions) in established lines. These asymmetrically dividing cancer cells produce one rapidly proliferating AKT hlgh daughter cell and another AKT low daughter that down-regulates multiple proliferation proteins and is very slowly cycling (e.g. MKI67 low , MCM2 low , CDC6 low , GMN low ) (7). AKT low cells also suppress multiple nuclear histone marks associated with both transcriptional activation and repression, mimicking an epigenomic profile that has been observed in quiescent cell populations (e.g.
• H3S 10ph low , H3K4me2 low , H3K9me2 low , H3K27me3 low Furthermore, AKT low cells up-regulate HES 1 , a transcription factor that marks cells that have exited the cell cycle into a GO state. Since AKT low cells do eventually divide, reverting to an AKT hlgh proliferative phenotype over time, the term "GO-like" is used herein to emphasize the temporary and reversible nature of this cell state.
• HCT1 16 colorectal cancer cells with adeno-associated virus (AAV)-mediated disruption of the AKT1 and AKT2 gene loci i.e. AKTl/2 7" cells
• AAV adeno-associated virus
• AKTl/2 7" cells do not have AKT1 or AKT2, nor do they express AKT3, but they are able to survive and proliferate in the complete absence of AKT signaling, presumably through compensatory changes that arose during their initial selection.
• Confocal microscopy was used to score the AKTl/2 " ' " cell line for rare, asymmetrically dividing and GO-like cancer cells that express the previously validated MCM2 low / H3K9me2 low / HES l high marker profile.
• TTC3 is a RING-type E3 protein- ligase known to ubiquitinate AKT1 at the lysine-8 and lysine- 14 residues to trigger its destruction by the proteasome (15).
• GO-like cells express high levels of TTC3 protein compared to proliferating cells, suggesting that this E3 ligase might play a special role in the production of these slowly cycling cells (data not shown).
• inducible shRNA knockdown of TTC3 with three different short hairpin RNAs dramatically suppressed the frequency of GO-like cells in both HCT1 16 and MCF7 ( Figures 2A-2C).
• Live-cell imaging experiments were performed to further define the role that mTORC2 signaling plays in regulating asymmetric cancer cell division.
• Serial images of HCT1 16 cells dividing over seven days in culture were obtained, either with or without RICTOR knockdown. These images were analyzed to identify individual dividing cells, creating lineage traces of these cells and their progeny to identify sibling pairs, and differences in mitotic times between sister cells arising from the same precursor were plotted.
• ninety percent of dividing cells produced two siblings that divided again within ten hours of each other ( Figure 3). However, approximately ten percent of cells divided more asymmetrically to produce daughters with larger differences in time to mitosis that were greater than ten hours.
• RICTOR (-) cancer cells with reduced frequency of asymmetric division were markedly less tumorigenic compared to RICTOR (+) cells across the cell line panel, resulting in tumors with that were 50 to 80% smaller in size ( Figures 4A-4E).
• HCTl 16 and MCF7 cells were pre- treated for 72 hours with low doses of an allosteric AKT inhibitor (i.e. AKT1/2), which had been found to induce a large fraction of asymmetrically dividing and GO-like cancer cells (see Figures 10, IP). Variable numbers of these pre -treated cells were implanted into nude mice and grown without further manipulation in vivo.
• AKT1/2 allosteric AKT inhibitor
• asymmetric cancer cell division may actually represent the execution of a novel cell cycle decision that involves mTORC2 signaling at a very specific point in late mitosis.
• the newborn cancer cell with suppressed AKT1 signaling assumes special characteristics (including a slowed cell cycle and GO-like features) that enable it to withstand harsh negative selective pressures during tumor formation, upon transplantation, or on exposure to cytotoxic insult.
• This model will naturally evoke comparisons to prior work describing putative cancer stem cell populations that might be interesting to pursue using the theoretical, experimental, and mechanistic framework described herein (17-22).
• HCTl 16 colon, MCF7 breast, MDA-MB-231 breast, A375 melanoma, and PC9 lung cancer cells were purchased from the American Type Culture Collection (ATCC).
• HCTl 16 AKTl/2 7" cells were purchased from Horizon Discovery (Cambridge, UK).
• MCF7 and MDA-MB-231 cells were maintained in DMEM, 10% FCS, 40mMglutamine, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin.
• HCTl 16 and HCTl 16 AKT1-/AKT2- cells were maintained in McCoy's 5a medium supplemented with 10% FCS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin.
• PC9 cells were maintained in RPMI, 25% glucose, 1% sodium pyruvate, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin.
• A375 cells were maintained in DMEM supplemented with high glucose HEPES buffer, 10% FCS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin. All the cells were grown in a humidified atmosphere at 37°C and 5% C02.
• shRNA constructs Human TRIPZ lentiviral inducible shRNAmirs for Rictor (Clone ID: V2THS 120392, V2THS 120389, V2THS 38014, V2THS 225915 ), non- silencing, and empty vector were purchased from Open Biosystems and virus was generated using our standard protocol. Infection was performed 24 h later in MCF7, HCTl 16, A375, PC9 and MDA-MB-231 cell lines with the lentiviral particles followed by selection with 2 ⁇ puromycin. Following selection, cells were allowed to grow to confluency. The shR As were induced using 2 ⁇ g/ml doxycycline for 72 h. The TTC3 virus was purchased from Sigma- Aldrich and infected in HCT1 16 and MCF7 cells and the standard protocol for selection was followed.
• AKT1 mutant cell lines AKTl(WT) cDNA was purified using PCR after cutting PDD AKTl(WT) with restriction enzymes BamHI and Xhol. Following purification, the product was ligated into pMSCVpuro-C-tag-mCherry cut with Bglll and Sail. All the AKT1 mutants were generated using the QuikChange site directed mutagenesis kit (Agilent technologies) and the product was ligated into pMSCVpuro- C-tag-mCherry. The resulting vector pMSCV-puro-AKTl- mCherry was sub-cloned into DH5a competent cells (Invitrogen). Sequencing verification of the fusion product was performed by the MGH DNA Core Facility with primers pMSCV 5'- CCCTTGAACCTCCTCGTTCGACC-3'(SEQ ID NO: 1) and pMSCV 3'-
• Virus carrying the desired fusion gene was produced by transfecting 293-T cells with target vector pMSCV-puro- AKTl-mCherry and packaging vector pCL-Ampho using the Mirus TranIT-293 transfection reagent and established protocols. Virus was collected 24 h following transfection. Before infection, cells were plated in a six-well plate in DMEM, 10% FCS. Infection was performed 24 h later by adding 0.5 mL DMEM, 10% FCS, 0.5mL pooled virus, and ⁇ ⁇ ⁇ l,000x polybrene per well.
• a media change was performed the following day and cells were allowed to grow to confluency before splitting into a 10-cm dish and selection with 2 ⁇ puromycin. Following selection, cells were allowed to grow to confluency before clones were selected using single-cell sorting (Becton Dickinson FACSAria II). Single cells were filtered by gating on the brightest 5% of cells in the PETexas red channel and sorted into individual wells of a 96-well plate. Clones were harvested between 14 and 21 d.
• All secondary antibodies were Alexa Fluor conjugates (488, 555, 568, 633, and 647) (Invitrogen). Immunofluorescence imaging (on a Nikon Eclipse Ti A1R-A1 confocal microscope) and live-cell imaging (on the Nikon Biostation CT platform) were performed as previously described (/).
• Virus carrying the pMSCV-CMV- NLSmCerulean construct was produced by transfecting 293-T cells plated at 500,000 cells per well in a six-well plate. Twenty four hours later, these cells were transfected with 1 ⁇ g target vector pMSCV- CMVNLS- mCerulean, ⁇ g packaging vector pCL-Ampho, and 3 ⁇ ⁇ FuGENE HD mixed with ⁇ reduced serum solution (Opti- MEM; Invitrogen). Virus was collected 24 h following transfection. Before infection with virus, HCT1 16 cells were plated at 50,000 cells per well in a six-well plate in DMEM, 10% FCS.
• Infection was performed 24 h later by adding 0.5 mL DMEM, 10% FCS, 0.5 mL pooled virus, and ⁇ ⁇ ⁇ l,000x polybrene per well. A media change was performed the following day, and cells were allowed to grow to confluency before splitting into a 10-cm plate. MCF7/ NLS- mCerulean cells were selected using fluorescence-activated cell sorting (Becton Dickinson FACSAria II) and gating on the brightest 5% cells in the Pacific blue channel.
• HCT- 1 16 cells tagged with NLS-mCerulean and also a doxycycline-inducible non- silencing or Rictor knockdown shRNA (hp4) construct in glass-bottom 12-well plates (MatTek Product # P12G-1.0-10-F) treated with type IV collagen.
• Multi-point serial imaging was performed using an inverted microscope fitted with a tissue culture incubator (Nikon Ti-Eclipse) every 20 minutes at 20x magnification (CFI Plan Apo 20x) for 164 hours. Both phase and fluorescent images were captured. Cells were excited with an LED (Nikon C-HGFI Intensilight HG Ilium) and passed through a filter series (Nikon, C-FL CFP and RFP HC HISN Zero Shift Filter Set). All cell division events were tracked manually using the CFP images by recording the following
• characteristics for each cell ID based on initial frame of appearance and x/y coordinate, first frame, last frame, origin ID, progenitor IDs, and x/y coordinates for first and last frame, and end method (division, lost in tracking, lost to wash out, or lost to cell death). Analysis was performed using R v2.8.0 (The R Foundation for Statistical Computing, 2008) by analyzing all division events.
• mice 5 l0 5 cells (MCF7, HCT1 16, A375, PC9, MDA-MB-231 cell lines) carrying either doxycycline-inducible non-silencing or RICTOR- targeting shRNAs (120392, 225915) were injected subcutaneously into the flanks of 5-6 week old, female nude mice. The mice were given doxycycline in water at 20 mg/ml for hairpin induction.
• Example 3 A Mechanism for Slowly Proliferating Cancer Cells that Promote Tumor Growth
• Tumor growth is driven by rapidly dividing cancer cells that arise through mutation and natural selection. Clonal selection does not fully explain, however, why established tumors also contain slowly proliferating cancer cells.
• a dividing cancer cell experiences an asymmetric decrease in ⁇ -integrin signaling, it activates mTORC2 kinase signaling which induces degradation of AKT1 kinase through a TTC3 / proteasome mechanism, to produce a slowly proliferating AKT How daughter cell.
• a dividing cancer cell generally produces two daughter cells that divide again in relative synchrony within hours of each other in cell culture. Occasionally, however, a cancer cell divides to produce progeny that are asynchronous, with one daughter cell having a markedly slower cell division time, on the order of days, compared to the other.
• this asynchronicity relates to cancer cells asymmetrically suppressing AKT protein kinase levels by about ninety percent during mitosis just before cytokinesis. This asymmetry produces one AKThigh daughter cell that rapidly enters the next cell cycle and another AKTlow cell that remains dormant for a more prolonged time before dividing again.
• AKTlow cells reduce their production of reactive oxygen species (i.e., ROSlow), down-regulate proliferation proteins (e.g., MKI671ow, MCM21ow), suppress multiple nuclear histone marks similar to quiescent cell populations (e.g., H3K9me21ow), and up-regulate the HE SI transcription factor that may mark exit from the cell cycle into GO (i.e., HE S I high) (1).
• ROSlow reactive oxygen species
• MKI671ow MCM21ow
• H3K9me21ow suppress multiple nuclear histone marks similar to quiescent cell populations
• H3K9me21ow up-regulate the HE SI transcription factor that may mark exit from the cell cycle into GO (i.e., HE S I high) (1).
• GO-like is used to describe this temporary and reversible cell state.
• AKTlow cancer cells are found within actual human breast tumors where they preferentially survive therapy with combination chemotherapy, suggesting that these cells may constitute an important but unappreciated reservoir of treatment resistance in patients with breast cancer (1). Since AKTlow cells share a number of conceptual features with putative cancer stem cell populations (e.g., asymmetric division, slow cycling, ROSlow, treatment resistance), it was reasoned that understanding in molecular detail how AKTlow slow proliferators arise might provide fundamental insight into the dynamics of tumor growth (1,2).
• AKTlow cancer cells only partially suppress total AKT protein levels. To do so, HCT116 colorectal cancer cells with adeno-associated virus (AAV)-mediated disruption of the AKTl and AKT2 gene loci (i.e., AKTl/2-/- cells) were obtained (3).
• AKTl/2-/- cells do not express either AKTl or AKT2, nor do they express AKT3, and thus are able to survive and proliferate in the complete absence of AKT signaling, presumably through compensatory changes that arose during their initial selection.
• AKTl domains that might be required for its partial suppression during asymmetric division.
• a series of AKTl cDNA constructs with mutations in critical amino acids known to be important for various aspects of AKTl signaling were created (Fig. 7A). Each mutant AKTl construct was overexpressed in AKTl/2-/- cells and these engineered cells scored for both asymmetrically dividing and GO-like cancer cells.
• AKT1-K179M (a mutation in the kinase pocket that renders AKTl catalytically inactive) failed to restore production of asymmetrically dividing and GO-like cells in the AKTl/2-/- line, while AKT1- D292A (another kinase dead mutant) did so only weakly compared to wild-type AKTl (Fig. 7B) (4).
• AKTl enzymatic activity is necessary for asymmetric cancer cell division.
• AKTl protein is suppressed to produce slow proliferators.
• treating cancer cells with allosteric AKT inhibitors at low doses dramatically increases the frequency of both asymmetrically dividing and GO-like cells (i.e., AKT1/2, MK2206) (Fig. 7C) (1).
• These allosteric inhibitors are known to bind to the AKT1 pleckstrin homology domain, inducing conformational change and displacement of the protein from the cell membrane, promoting its ubiquitination and proteasome -mediated degradation (5). Therefore, it was hypothesized that asymmetric division might actually depend on the targeted degradation of the AKT1 protein.
• TTC3 is a RING-type E3 protein- ligase known to ubiquitinate AKT1 at its lysine-8 and lysine- 14 residues leading to its destruction by the proteasome (6).
• GO-like cells express high levels of TTC3 protein compared to proliferating cells, consistent with a potential role for this E3 ligase in producing AKTllow cells (data not shown).
• inducible shRNA knockdown of TTC3 with three different short hairpin RNAs suppressed the frequency of GO-like cells in both HCTl 16 and MCF7 without affecting overall cell proliferation ( Figures 7D and 2A).
• AKT1-K8R, AKT1-K14R, and AKT1-K8R / K14R double mutant proteins failed to rescue the formation of GO-like cells in the AKT1/2-/- line (Fig. 7D).
• two different small molecules that inhibit proteasome function reduced the frequency of GO-like cells in both HCTl 16 and MCF7 when used at doses that do not affect overall cell proliferation (i.e., MG-132, Bortezomib) (Fig. 7D).
• MG-132, Bortezomib i.e., MG-132, Bortezomib
• AKT1 kinase Two different upstream signaling pathways are known to activate AKT1 kinase: PDPK1 kinase phosphorylates AKT1 at the T308 residue, while the mTORC2 kinase complex phosphorylates the AKT1-S473 and AKT1-T450 sites (7,8). It was therefore asked whether any of these canonical AKT1 residues were necessary for asymmetric cancer cell division. Similar to wild-type AKT1, overexpression of the AKT1-T308A mutant (which cannot be phosphorylated by PDPK1) in AKT1/2-/- cells completely restored the production of asymmetrically dividing and GO-like cells (Fig. 7E).
• AKT1-S473A, AKT1- T450A, and an AKT1-T308A / AKT1-S473A double mutant did not produce phenotypic rescue (Fig. 7E).
• inducible shRNA knockdown of RICTOR an obligate member of the mTORC2 signaling complex
• two different short hairpin RNAs suppressed the production of both asymmetrically dividing and slowly proliferating GO-like cells in a panel of five different human cancer cell lines, including those with a functional dependency on mutant PI3K (i.e., HCT116 (PIK3CAmutant), MCF7 (PIK3CAmutant), MDA-MB-231 breast, PC9 lung, and A375 melanoma) ( Figures 7E, IF, and 11C).
• RICTOR (-) cells did not differ from RICTOR (+) cells with respect to overall proliferation, response to stress (i.e., low serum, low glucose, or hypoxic conditions), or invasion in vitro.
• stress i.e., low serum, low glucose, or hypoxic conditions
• mTORC2 specifically induces asymmetric division and the production of slow proliferators, independent of PI3K or mTORCl activity and without altering other important cancer cell functions ( Figures 12A-120, 12A-13J and 14A-14D).
• Live-cell imaging experiments were performed to confirm mTORC2 regulation of asymmetric cancer cell division.
• Serial images of HCT116 and MCF7 cells dividing over seven days in culture either with or without RICTOR knockdown were obtained. These images were analyzed to identify individual dividing cells, lineage traces of these cells and their progeny created in order to identify sibling pairs, and differences plotted in mitotic times between sister cells arising from the same precursor.
• Eighty to ninety percent of dividing cells produced two siblings that divided again within five hours of each other ( Figure 71, 7J). However, approximately ten to twenty percent of cells divided more asymmetrically to produce daughters with larger differences in time to mitosis.
• IP immunoprecipitation
• Integrins are a family of heterodimeric transmembrane receptors that transduce signals from the extracellular matrix by activating a number of well-described signaling intermediaries within the cell, including FAK, to regulate cell cycle, shape, and motility in cancer and normal cells (9). It was reasoned that decreased integrin signaling might cause a loss of FAK activity resulting in mTORC2 activation during asymmetric division. In fact, inducible shRNA knockdown of ⁇ -integrin (i.e., ITGB1, CD29) with two different short hairpins increased the fraction of asymmetrically dividing and GO-like cells in both HCT116 and MCF7 ( Figures 7G and 1 IB).
• ⁇ -integrin i.e., ITGB1, CD29
• TS2/16 treatment resulted in markedly slower tumor growth compared to control across this spectrum of solid tumor models, which included melanoma, lung, colorectal, and breast cancers (Figure 9A).
• This finding was notable given that integrin signaling is generally thought to promote cancer cell proliferation, survival, and invasion (12). Since TS2/16 specifically activates human ⁇ - integrin, moreover, these anti-tumor effects most likely resulted from the direct targeting human cancer cells rather than mouse stroma in these xenografts.
• RNA interference which also reduces asymmetric division and slow proliferators without altering general cancer cell viability
• AKT How cancer cell temporarily arrests its cell cycle, and superficially expresses a quiescent marker profile (i.e., MCM21ow, H3K9me21ow, HESlhigh), but can begin cycling again if its ⁇ -integrin sensor is ligated optimally (1).
• Asymmetrically dividing cancer cells are not a fixed subpopulation, but rather appear to arise randomly depending on interaction with extracellular matrix proteins like collagen. Furthermore, slowly proliferating AKT How cancer cells do not differentiate as far as we know.
• colorectal cancer cells clarifies their roles in tumor growth regulation. Proceedings of the National Academy of Sciences of the United States of America 107, 2598 (Feb 9, 2010).
• HCTl 16 colon, MCF7 breast, MDA-MB-231 breast, A375 melanoma, and PC9 lung cancer cells were purchased from the American Type Culture Collection (ATCC).
• HCTl 16 AKTl/2-/- cells we purchased from Horizon Discovery
• MCF7 and MDA-MB-231 cells were maintained in DMEM, 10% FCS, 40mM glutamine, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin.
• HCTl 16 and HCTl 16 AKTl/2-/- cells were maintained in McCoy's 5a medium supplemented with 10% FCS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin.
• PC9 cells were maintained in RPMI, 25% glucose, 1%) sodium pyruvate, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin.
• A375 cells were maintained in DMEM supplemented with high glucose HEPES buffer, 10% FCS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin. All the cells were grown in a humidified atmosphere at 37°C and 5% C02.
• shRNA constructs Human TRIPZ lentiviral inducible shRNAmirs for RICTOR (Clone ID: V2THS 120392, V2THS 120389, V2THS 38014, V2THS 225915 ), FAK (Clone ID: V2THS 57326, V2THS 325805), ⁇ -integrin (Clone ID: V2THS 133469, V2THS 390997), non- silencing, and empty vector were purchased from Open Biosystems and virus was generated using a standard protocol. Infection was performed 24 h later in MCF7, HCT1 16, A375, PC9 and MDA-MB-231 cell lines with the lentiviral particles followed by selection with 2 ⁇ puromycin.
• the shRNAs were induced using 2 ⁇ g/ml doxycycline for 72 h.
• the TTC3 virus was purchased from Sigma-Aldrich and infected in HCT1 16 and MCF7 cells and the standard protocol for selection was followed.
• AKTl(WT) cDNA was purified using PCR after cutting PDD AKTl(WT) with restriction enzymes BamHI and Xhol. Following purification, the product was ligated into pMSCVpuro-C-tag-mCherry cut with Bglll and Sail. All the AKTlmutants were generated using the QuikChange site directed mutagenesis kit (Agilent technologies) and the product was ligated into pMSCVpuro- C-tag-mCherry. The resulting vector pMSCV-puro-AKTl-mCherry was sub-cloned into DH5a competent cells (Invitrogen).
• Virus carrying the desired fusion gene was produced by trans fecting 293 -T cells with target vector pMSCV-puro- AKTl-mCherry and packaging vector pCL-Ampho using the Minis TranlT- 293 transfection reagent and established protocols. Virus was collected 24 h following transfection. Before infection, cells were plated in a six-well plate in DMEM, 10% FCS.
• Infection was performed 24 h later by adding 0.5 mL DMEM, 10% FCS, 0.5mL pooled virus, and ⁇ ⁇ ⁇ 1 ,000 ⁇ polybrene per well.
• a media change was performed the following day and cells were allowed to grow to confluency before splitting into a 10-cm dish and selection with 2 ⁇ puromycin. Following selection, cells were allowed to grow to confluency before clones were selected using single-cell sorting (Becton Dickinson FACSAria IITM). Single cells were filtered by gating on the brightest 5% of cells in the PETexas red channel and sorted into individual wells of a 96-well plate. Clones were harvested between 14 and 21 days.
• Virus carrying the pMSCV-CMV- NLSmCerulean construct was produced by transfecting 293-T cells plated at 500,000 cells per well in a six-well plate. Twenty four hours later, these cells were transfected with ⁇ g target vector pMSCV-CMVNLS- mCerulean, ⁇ g packaging vector pCL-Ampho, and 3 ⁇ FuGENE HD mixed with ⁇ reduced serum solution (Opti- MEM; Invitrogen).
• Virus was collected 24 h following transfection. Before infection with virus, HCT1 16 or MCF7 cells were plated at 50,000 cells per well in a six-well plate in DMEM, 10% FCS. Infection was performed 24 h later by adding 0.5 mL DMEM, 10% FCS, 0.5 mL pooled virus, and ⁇ ⁇ , l ,000x polybrene per well. A media change was performed the following day, and cells were allowed to grow to confluency before splitting into a 10-cm plate.
• HCT1 16 or MCF7 / NLS- mCerulean cells were selected using fluorescence-activated cell sorting (Becton Dickinson FACSAria IITM) and gating on the brightest 5% cells in the Pacific blue channel.
• HCT1 16 cells tagged with NLS-mCerulean and also a doxycycline- inducible non-silencing or Rictor knockdown shRNA (hp4) construct in glass-bottom 12-well plates (MatTek Product # P12G-1.0-10-F) treated with type IV collagen.
• Multi-point serial imaging was performed using an inverted microscope fitted with a tissue culture incubator (Nikon Ti-Eclipse) every 20 minutes at 20x magnification (CFI Plan Apo 20x) for 164 hours. Both phase and fluorescent images were captured. Cells were excited with an LED (Nikon C-HGFI Intensilight HG IliumTM) and passed through a filter series (Nikon, C-FL CFP and RFP HC HISN Zero Shift Filter Set). All cell division events were tracked manually using the CFP images by recording the following
• characteristics for each cell ID based on initial frame of appearance and x/y coordinate, first frame, last frame, origin ID, progenitor IDs, and x/y coordinates for first and last frame, and end method (division, lost in tracking, lost to wash out, or lost to cell death). Each point is calculated at 20-minute intervals and only shown if there was at least one event occurring. Analysis was performed using R v2.8.0 (The R Foundation for Statistical Computing, 2008) by analyzing all division events.
• HCT 1 16 colon, MCF7 breast, MDA-MB-231 breast, A375 melanoma, and PC9 lung cancer cells carrying either doxycycline-inducible non-silencing or RICTOR-targeting shRNAs (120392, 225915) were plated in a 12- well plate at a density of 50,000 cells/per well in triplicate with doxycycline containing medium on day 1 and the cells were counted every 24 firs for 5 days. Doxycycline containing medium was replaced everyday.
• Cells were maintained at: 1) 21% oxygen, 10% fetal calf serum and 25mM D- glucose (normal condition), 2) 4% oxygen (hypoxia), 3) 1% serum (low serum), or 4) 5.56mM D-glucose (low glucose).
• HCTl 16 colon, MCF7 breast, MDA-MB-231 breast, A375 melanoma, and PC9 lung cancer cells carrying either doxycycline-inducible non-silencing or RICTOR-targeting shRNAs (120392, 225915) were plated in a 6- well plate at a density of 1,000 cells/per well in triplicate with doxycycline containing medium on day 1. Cells were allowed to grow into small colonies for 5 days and then irradiated at a dose of 2Gy. Colonies were then allowed to grow for another 2 weeks and were stained using 0.125%) Coomasie Blue. Doxycycline containing medium was replaced everyday.
• HCTl 16 colon, MCF7 breast, MDA-MB-231 breast, A375 melanoma, and PC9 lung cancer cells carrying either doxycycline-inducible non-silencing or RICTOR-targeting shRNAs (120392, 225915) were induced with Doxycycline ⁇ g/ml) for 72 hrs and then seeded onto Matrigel invasion chambers at a density of 50,000 cells per well in triplicate. Doxycycline containing medium was replaced everyday. The invasion chambers were incubated for 24hrs at 37°C and 5% C02. The chamber filters were then stained using 0.125%) Coomasie Blue and mounted onto glass slides.
• RICTOR knockdown experiments in vivo we injected 5 ⁇ 105 cells (A375, MDA-MB-231, PC9, HCTl 16, MCF7) carrying either doxycycline-inducible non-silencing or RICTOR-targeting shRNAs (120392, 225915) subcutaneously into the flanks of 5-6 week old, female nude mice (Nude/Nude). The mice were given doxycycline in water at 20 mg/ml for hairpin induction starting immediately after implantation. For induction of slow proliferators, cells were treated with AKT1/2 inhibitor and DMSO
• mice for 72 h and were harvested at 60-70%> confluence, and then counted and washed twice in PBS and resuspended in 1 : 1 Media: Matrigel (BD Biosciences).
• 5x 106, 5x 105, and 5x 104 cells respectively subcutaneously into the flanks of 5-6 week old, female nude mice.
• growing tumors were measured weekly by caliper, and mice were killed when tumors reached approximately lcm3 in size. Animal experiments were carried out under a Massachusetts General Hospital Institutional Review Board- approved protocol.

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