INDUCING CELL DEATH BY HYPERACTIVATION OF MOTILITY NETWORKS

Abstract

The present invention provides a method of inducing cell death by hyperactivation of motility networks.

Claims

1-18. (canceled)

19. A method of treating a pathological condition in a subject comprising: administering an agent or stressor that selectively kills a cell of the subject by further activating or hyper-activating the cell, wherein the cell bears a mutated oncogene or a mutated tumor suppressor gene, wherein the gene comprises Ras, PI3K, PTEN, or another defined mutation that activates a cell migration pathway, thereby treating the pathological condition in the subject.

20. The method of claim 19, wherein the pathological condition comprises a neoplasia.

21. The method of claim 20, wherein the subject has been diagnosed with a neoplasia.

22. The method of claim 19, wherein the pathological condition comprises an inflammatory disorder.

23. The method of claim 22, wherein the subject has been diagnosed with an inflammatory disorder.

24. The method of claim 19, wherein the subject is a human or domesticated animal.

25. The method of claim 19, wherein the neoplastic cell comprises a metastatic cell or a pre-metastatic cell.

26. The method of claim 19, wherein further activating or hyper-activating the cell induces spreading and flattening of the cell.

27. The method of claim 19, wherein the cell undergoes fragmentation.

28. The method of claim 27, wherein the fragmentation comprises catastrophic fragmentation.

29. The method of claim 19, wherein the agent or stressor comprises an environmental perturbation.

30. The method of claim 29, wherein the environmental perturbation comprises a mechanical force, a temperature change, an electrical stimulus, a sound wave, osmotic shock, or other environmental change.

31. The method of claim 19, wherein the administered agent comprises a statin, a phenothiazine, an antibiotic, or an analgesic.

32. The method of claim 19, wherein the administered agent comprises a statin.

33. The method of claim 32, wherein the statin comprises pitavastatin, fluvastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, or simvastatin.

34. The method of claim 19, wherein the statin comprises pitavastatin.

35. The method of claim 19, wherein the administered agent comprises a phenothiazine.

36. The method of claim 19, wherein the administered agent comprises an antibiotic.

37. The method of claim 19, wherein the administered agent comprises an analgesic.

38. The method of claim 19, wherein the subject is a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1A and FIG. 1B show photomicrographs illustrating the chemotactic responses of Dictyostelium and human neutrophils. FIG. 1A shows Dictyostelium moving towards the cyclic adenosine monophosphate (cAMP)-filled micropipette. FIG. 1B shows HL-60 human neutrophils moving towards the peptide, fMLP (N-Formylmethionyl-leucyl-phenylalanine). Images were taken 20 minutes after pipettes were placed in fields of distributed cells. Gradients were about 20% across the lengths of the 15 μm cells.

[0048] FIG. 2 is a cartoon showing the integration of motility, polarity and directional sensing in directed migration. Motility was achieved by spontaneous extension and retraction of pseudopods every few minutes. Polarity refers to an asymmetric distribution of cytoskeletal and signaling molecules and increased sensitivity to chemoattractant at the “front”. Directional sensing can occur even in immobilized cells as evidenced by the rapid rearrangement of internal proteins in response to changes in external gradients.

[0049] FIG. 3 is a cartoon showing the optimal activation of the signal transduction network that drives cell migration. The cartoon shows cell morphology and fluorescent reporters of signal transduction (green) or cytoskeletal (orange) activity on the basal surface of cells. Decreasing signal transduction by deleting TORC2 subunits and inhibiting PI3K blocks migration. Increasing signal transduction activity by deleting PTEN (phosphatase and tensin homolog) or by expressing a constitutively active version of Ras also impairs cell migration by promoting too many projections.

[0050] FIG. 4A-FIG. 4C shows the phenotype caused by a combination of perturbations. FIG. 4A is a photomicrograph showing the expression of constitutively active RasC.sup.Q62L induced with a “tet-on” promoter in pten− cells. Cell is shown before (left) and 8 hours after (right) addition of doxycycline. Scale bar is 18 μm. FIG. 4B is a photomicrograph showing cells expressing fluorescent F-actin biosensor, GFP-LimE. Actin is recruited to the pseudopods in pten− cells whereas it is elevated globally around the perimeter in RasC.sup.Q62L/pten− cells. FIG. 4C is a cartoon showing that synergistic over activation of signal transduction and/or cytoskeletal networks lead to formation of spread cells (as compared to FIG. 3).

[0051] FIG. 5 is a series of photomicrographs showing examples of the conversion of RAM (Regulators of Adhesion and Motility) mutant to the spread cell phenotype. Tet-inducible RasC.sup.Q62L was expressed in pten−, RAM12, or RAM13 cells. Fields of cells are shown before and 20 hours after induction of RasC.sup.Q62L. More than 80% of the cells displayed a flattened phenotype after induction. Average diameters of pten−, RAM12, and RAM13 cells increased from 28.7±4.1, 27.8±5.1 and 27.8±5.0 μm before induction to 49.0±7.3, 45.5±8.6, and 46.8±7.9 μm following induction, respectively.

[0052] FIG. 6 is a series of images showing an example of a cell undergoing sparagmosis. Left, at t=0 a pten− cell expressing tet-inducible RasC.sup.Q62L displayed the typical multiple projection phenotype of a pten− cell. Middle, doxycycline was added to induce the expression of RasC.sup.Q62L and the cell adopted a spread phenotype within 8 hours. Right, after a series of ingressions, the fragmentation occurred at 27 hours. Cartoon at the bottom depicts the morphological transitions a pten− cell underwent after induction of RasC.sup.Q62L, leading to cell death. There were several cycles of cortical ingression and recovery before cell death occurred (as indicated by the recycle arrow).

[0053] FIG. 7 is a diagram showing the process of drug library screen. The initial screen included 2560 compounds from the Johns Hopkins drug library, among which 63 were found to affect the growth of Dictyostelium pten− cells. Secondary screen confirmed nine compounds with effects selective for pten− but not wild-type cells.

[0054] FIG. 8 is a series of photomicrographs showing that fluvastatin selectively affected the growth and survival of Dictyostelium pten− cells. Cells were treated with 5 μM fluvastatin or DMSO. Images were taken 17 hours (left), 41 hours (middle), and 65 hours (right) after drug treatment.

[0055] FIG. 9 is a bar graph showing statins that specifically target Dictyostelium pten− cells. Seven statins currently available on the market were tested for cytotoxicity on pten− cells. The graph quantifies growth of cells treated with 5 μM of statins or DMSO control over a three day period. Blue, orange, and yellow bars represent relative cell counts 17 hours, 41 hours, and 65 hours after drug treatment.

[0056] FIG. 10 is a series of photomicrophotographs showing cell death induced by pitavastatin in MCF-10A cells bearing a homozygous PTEN deletion. MCF-10A or pten−/− cells were treated with DMSO control or 5 μM pitavastatin. Upper and lower panels show 44 hours and 68 hours after drug treatment.

[0057] FIG. 11 are bar graphs showing that pitavastatin selectively inhibited the growth and survival of MCF-10A cells bearing a homozygous pten−/− deletion. Cells were treated with DMSO control or 5 μM drugs. Blue, orange, and yellow bars represent 20 hours, 44 hours, and 68 hours after drug treatment.

[0058] FIG. 12A and FIG. 12B are graphs showing that adding back mevalonic acid (MVA) and geranylgeranyl pyrophosphate (GGPP) blocked the toxic effects of pitavastatin on pten−/− cells. Cells were treated with increasing doses of pitavastatin in the presence or absence of MVA (FIG. 12A) and GGPP (FIG. 12B) at the indicated concentration.

DETAILED DESCRIPTION OF THE INVENTION

[0059] Directed cell migration (chemotaxis) plays an important role in many physiological processes and contributes to various pathological conditions such as cancer metastasis. Migration is mediated by a network of positive and negative feedbacks among signal transduction and cytoskeletal components. Optimal migration for a particular cell type requires that these networks operate within an appropriate physiological range. In diseases these networks can become hyper-activated, leading to aberrant migration and metastasis. Consequently current therapeutic strategies are aimed at restoring the normal physiological range and migration.

[0060] In contrast, this invention is based, in part, upon the surprising discovery that hyper-activations of motility networks are themselves stresses or act to make cells more sensitive to external stresses which can then induce cell death. Methods of hyper-activation include, but are not limited to, combinations of activations of oncogenes and inactivation of tumor suppressor genes. External stressors include, but are not limited to, statins. Observations in such diverse cells as soil amoebae and pre-metastatic cells demonstrate that this sensitivity is a fundamental property of eukaryotic cells. This property can be exploited to selectively kill hyper-activated cells such as certain metastatic cancer cells and certain inflammatory cells.

BACKGROUND

[0061] Directed cell migration (chemotaxis) plays a fundamental role in many physiological processes and contributes to various pathological conditions such as cancer metastasis (Theveneau E, Mayor R. 2012. Developmental Biology 366 (1):34-54, Richardson B E, Lehmann R. 2010. Nat Rev Mol Cell Biol 11 (1):37-49, and Sixt M. 2011. Immunol Lett 138 (1):32-34). During embryogenesis chemoattractants and mechanical forces guide primordial germ cells to proper locations, mediate the formation of organs, and control the wiring of the nervous system. In the adult, directed migration is critical for immune cell trafficking, wound healing, and stem cell homing to niches. It is also involved in the pathology of numerous diseases (Kolaczkowska E, Kubes P. 2013. Nat Rev Immunol 13(3):159-175, Zernecke A, Weber C. 2010. Cardiovasc Res 86(2):192-201, Bravo-Cordero J J, et al., 2012. Curr Opin Cell Biol 24 (2):277-283 and Roussos E T, et al., 2011. Nat Rev Cancer 11 (8):573-587). The migration of immune cells contributes to inflammatory disorders such as asthma, arthritis, and vascular disease. In cancer metastasis, cells escape the primary tumor, circumventing surgical intervention. These cells can enter the circulation and then migrate out to new sites in target organs. Like other fundamental cell biological processes, networks of signal transduction and cytoskeletal pathways are involved and therapeutic approaches directed at multiple targets are likely to be more effective. A systems approach to understanding and controlling directed migration is the most effective for ultimately designing unique, combined therapeutic strategies.

Dictyostelium as a Discovery Vehicle

[0062] Dictyostelium is a soil-dwelling amoeba, known for its remarkable life cycle consisting of a unicellular and multicellular phase. It is present in most terrestrial ecosystems as a normal and often abundant component of the soil microflora, and plays an important role in the maintenance of balanced bacterial populations in soils. The unicellular phase consists of solitary amoebae feeding on bacteria and reproducing by binary fission. When challenged by starvation, the amoebae collect into aggregates and develop into multicellular fruiting bodies. Its complex life cycle, ease of maintenance, and genetic and cell biological accessibility makes Dictyostelium an idea model organism for laboratory studies of a wide range of biological problems including signal transduction, chemotaxis, and cellular differentiation.

Chemotaxis is a Fundamental Cellular Process

[0063] Chemotaxis is a vital process in which organisms efficiently respond to the changes in the chemical composition of their environment, moving toward chemically favorable environments while avoiding unfavorable environments. It is a major process in normal physiology and in the pathogenesis of many diseases. The classic view of chemotaxis implies that motility arises from the activity of the actin/myosin cytoskeletal network and that a short, linear signal transduction pathway proceeding from chemoattractant receptors to Rac family GTPases to actin polymerization guides the process. However, it is now recognized that Ras proteins, phosphatidylinositol 3, 4, 5 phosphate (PIP3), TORC2, and components of other signal transduction pathways are important, conserved intermediates (Cai, H., et al., 2010. J. Cell Biol. 190:233-245, Lee S, et al., 1999. Mol Biol Cell 10(9):2829-2845, Kamimura Y, et al., 2008. Current Biology 18 (14):1034-1043, Bosgraaf L, et al., 2008. J Cell Sci 121(21):3589-3597, Liu L, et al., 2010. Developmental Cell 19 (6):845-857, Chen L, et al., 2003. Mol Biol Cell. 14(12), 5028-5037 and Iijima, M and Devreotes, P N. 2002. Cell. 109, 599-610). Furthermore, the prevailing view of chemotaxis implies that the role of signal transduction is merely to guide an autonomously active motile machinery. However, a new paradigm for motility and chemotaxis is emerging that signal transduction pathways control cell morphology and random motility not just guidance (Iijima, M and Devreotes, P N. 2002. Cell. 109, 599-610, Sasaki A T, et al., 2007. J Cell Bio. 178(2):185-191, and Postma M, et al., 2004. J Cell Sci. June 15; 117(Pt 14):2925-35).

[0064] Knowledge of the signal transduction networks that regulate cell migration is still evolving, but it already has significant implications. Many important oncogenes and tumor suppressors, such as Ras, PI3K, and PTEN, are involved. Ras is a superfamily of small G proteins (guanosine-nucleotide-binding proteins) which are ubiquitously expressed in all cell linages and organs and functions as a binary signaling switch with select on and off states. The human oncogenic members of the Ras family have been reviewed (Rojas, et al., J Cell Biol 196, no. 2 189-201, and Karnoub, et al., Nat Rev Mol Cell Biol. 2008, July 9(7): 517-531, both of which are incorporated herein by reference), and in general they regulate cell proliferation, differentiation, morphology, and apoptosis. PI3K are a family of enzymes also involved in cell growth and survival as well as motility. PI3Ks phosphorylate the 3-position hydroxyl group of the inositol ring of phosphatidylinositol-4, 5-trisphosphate (PIP.sub.2) to form phosphatidylinositol-3, 4, 5-trisphosphate (PIP.sub.3). PTEN is a phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase that negatively regulates PI3K signaling by dephosphorylating the PIP3. In many cancers, oncogenic PIK3CA is activated or tumor suppressor PTEN is inactivated and this pathway is overactive, thus reducing apoptosis and allowing proliferation. Orthologs of Ras, PI3K, PTEN and many other components involved in chemotaxis have been identified in many metazoans and strong orthologs are present in Dictyostelium.

Statins are Well Established Drugs

[0065] Statins are a class of cholesterol lowering drugs that inhibit the enzyme HMG-CoA reductase which plays a central role in the production of cholesterol. High cholesterol levels have been associated with cardiovascular disease (CVD), and statins have been found to prevent cardiovascular disease and mortality in those who are at high risk. Statins act by competitively inhibiting HMG-CoA reductase, the first committed enzyme of the mevalonate pathway. Because statins are similar in structure to HMG-CoA on a molecular level, they fit into the enzyme's active site and compete with the native substrate (HMG-CoA). This competition reduces the rate by which HMG-CoA reductase is able to produce mevalonate, the next molecule in the cascade that eventually produces cholesterol. A variety of natural statins are produced by Penicillium and Aspergillus fungi as secondary metabolites. These natural statins probably function to inhibit HMG-CoA reductase enzymes in bacteria and fungi that compete with the producer.

Example 1: Mechanisms of Migration and Chemotaxis Discovered in Dictyostelium are Operative in Human Leukocytes

[0066] Many of the mechanisms of cell migration and chemotaxis were initially discovered in one of the most extensively studied model systems, Dictyostelium, and later found to be operative in higher eukaryotic cells (FIG. 1A and FIG. 1B). Dictyostelium cells, human leukocytes, and metastasizing cancer cells displayed “amoeboid” movement, rhythmically extending pseudopods which intermittently pulled the cells forward whereas the “mesenchymal” motility of fibroblasts was characterized by the gliding lamellipodia (Friedl P, et al., 1998. Microsc Res Tech 43(5):369-378). Guidance signals for migrating cells acted through GPCRs, RTKs, and other receptor classes or were derived from substrate stiffness, electric fields, or light or temperature gradients. Nevertheless, many of the signal transduction and cytoskeletal events that occurred selectively on the leading and trailing edges of migrating cells were the same. During directed migration, integration of motility, polarity and directional sensing was observed, although each of these processes was also observed separately (FIG. 2). For example, cells moved randomly by spontaneously extending pseudopodia in the absence of a chemoattractant gradient. Cells had a stable axis of polarity and were more sensitive to the chemoattractant at the front which allowed them to migrate efficiently. However, less polarized cells still migrated directionally and reacted more quickly to shifting cues Immobilized cells maintained their ability to sense directional cues by localizing proteins toward or away from the gradient. The polarity of static cytoskeleton in the immotile cells also retained polarized sensitivity. These and other similarities indicated that common mechanisms evolved in the earliest eukaryotic cells and have been remarkably conserved (Artemenko, Y., et al., P. N. 2014. Cell Mol Life Sci. October; 71(19):3711-47). Research in Dictyostelium and other model organisms continues to contribute to the understanding of migration and chemotaxis.

Example 2: An Optimal Amount of Spontaneous Activation of the Signal Transduction Network Acts as the “Pacemaker” for Cell Migration and Defects that Cause Excessive Cell Spreading are Additive

[0067] The essential roles of signal transduction events in motility and the optimal activation of the signal transduction network to drive cell migration were evaluated (FIG. 3). In amoeboid cells, such as Dictyostelium and human neutrophils, the signal transduction network was excitable and “fired” stochastically every few minutes, initiating waves of activity that propagate along the cell surface even in the absence of the cytoskeleton (Xiong Y et al., 2010 Proc Natl Acad Sci 107(40), 17079; Huang et al., 2013 Nat Cell Biol 15(11), 1307; Asano Y et al., 2008, 65(12) 923; Gerisch G. et al. 2012, Biophys J. 103(6), 1170; Weiner, O D et al., 2007 PLoS Biol., 5(9); Arai Y et al., 2010 Proc Natl Acad Sci 107(27), 12399; Taniguchi D et al., 2013 Proc Natl Acad Sci, 110(13), 5016). Without input from the signal transduction network, the cytoskeletal network displayed oscillations at local regions which caused only weak undulations on the cell perimeter (Huang et al., 2013 Nat Cell Biol 15(11), 1307).

[0068] The coupling of the signal transduction and cytoskeletal networks leads to pseudopod extension. Thus, an optimal amount of spontaneous activation of the signal transduction network acted as the “pacemaker” for cell migration (FIG. 3). If several signal transduction pathways were blocked simultaneously, e.g. deleting TORC2 subunits and inhibiting PI3K, cytoskeletal oscillations still occurred, but cells did not extend pseudopods or migrate (Huang et al., 2013 Nat Cell Biol 15(11), 1307). However, if signal transduction events were so elevated that they overrode polarity, pseudopods were extended simultaneously along the perimeter and the cell also did not move (Cai H et al., 2010 J. Cell Biol. 190, 233; Iijima M et al., 2002 Cell 109, 599). For example, deleting PTEN or expressing a constitutively active version of Ras impaired cell migration by promoting too many projections.

[0069] Research into the parallel pathways of the signal transduction network led to the discovery that defects that cause excessive cell spreading were additive. Ras/TORC2 and PI3K pathways were constitutively activated by expressing RasC.sup.Q62L in cells lacking PTEN. As noted earlier, RasC.sup.Q62L expression or PTEN depletion each caused an increase in lateral pseudopod formation (FIG. 3); however, the combination yielded a phenotype much stronger than that caused by either individual perturbation. The combination of the two perturbations produced extremely spread and flattened cells, with the entire perimeter consisting of a continuous “pseudopod” (FIG. 4).

Example 3: Signals from Multiple Pathways Impinging on the Cytoskeleton are Integrated to Generate the Flattened Phenotype

[0070] The phenotype did not depend specifically on the RasC/TORC2 or PI3K pathways. Rather, signals from multiple pathways impinging on the cytoskeleton can be integrated to generate the phenotype. RAM (Regulator of Adhesion and Motility) mutants were isolated in a screen for regulators of cell morphology and migration. Mutant cells were more spread and adhered more strongly than wild-type cells. Most of the mutants also displayed strong defects in cell motility and chemotaxis. When constitutively active RasC.sup.Q62L was expressed in the RAM mutants, these cells also formed extremely spread cells like those seen in the pten− cell background (FIG. 5). In another example, Rap1 is a small GTPase that controls cell adhesion in a variety of cell types. When constitutively active Rap1A.sup.G12V was expressed in pten− or RAM cells, a similar phenotype was observed.

Example 4: Hyper-Activation of the Signal Transduction Network Results in Cell Fragmentation and Death

[0071] The expression of RasC.sup.Q62L in pten− cells maintained for an additional 16-28 hours resulted in cells that underwent a catastrophic fragmentation and death (FIG. 6). It was verified that 98% of the induced cells were dead by Trypan Blue staining and their failure to form foci on re-plating. The surviving 2% of cells were not flattened, indicating that they lost expression of RasC.sup.Q62L. This observed mode of cell death has not been elucidated before in either Dictyostelium or in mammalian cells. This mechanism was named “sparagmosis” from the Greek sparasso, meaning “tear, rend, or pull to pieces.” Other pairwise combinations of perturbations that generated flattened cells such as expression of RasC.sup.Q62L in RAM mutants or expression of Rap1A.sup.G12V in pten− also led to similar cell death by fragmentation.

Example 5: Screen to Identify Compounds that Selectively Kill pten− Cells

[0072] The observation that further activation of the signal transduction network in pten-cells resulted in cell death prompted screening for small molecules that specifically targeted cells with PTEN mutation. Since PTEN is one of the most commonly mutated tumor suppressors in human cancer, the molecules identified in the screen will provide a new way of confronting metastatic and pre-metastatic cancer cells.

[0073] The John Hopkins Drug Library contains 2560 drugs that are either FDA approved, have been proven in other countries for treating human diseases, or have entered phase II clinical trials. In a phenotypic screen using the library, nine compounds were found to selectively slow down the growth of or kill pten− cells (FIG. 7). These compounds fell into several classes: certain phenothiazines (promazine, chlorpromazine, and thioridazine), certain antibiotics (polymyxin and chloroxine), certain statins (fluvastatin and pitavastatin), and others (benfluorex and phenazopyridine). All of the hits were validated independently with purchased compounds and repeating the experiments. Importantly, these drugs had little or no effect on the morphology and growth of wild type cells.

[0074] In particular, the two statins, fluvastatin (trade name Lescol) and pitavastatin (Livalo) displayed high cytotoxicity for Dictyostelium pten− cells (FIG. 8, fluvastatin shown as an example). Compared to cells treated with DMSO control, which continued to grow over a three day period, pten− cells treated with fluvastatin and pitavastatin grew much slower. Cell death started to occur around 40 hours after drug treatment and a marked increase was observed in the following 24 hours. In contrast, wild type cells treated with the statins showed no significant growth defect.

[0075] Statins are inhibitors of HMG-CoA reductase, the rate-controlling enzyme of the mevalonate pathway. In addition to fluvastatin and pitavastatin, the other statins currently available on the market include atorvastatin (Lipitor), lovastatin (Mevacor, Altocor), pravastatin (Pravachol), rosuvastatin (Crestor), and simvastatin (Zocor). All the other five statins were tested for their cytotoxicity on pten− cells (FIG. 9). Fluvastatin and pitavastatin displayed higher efficacy compared to all other statins. Cell death was also observed with simvastatin treatment, whereas the other four statins were well tolerated.

Example 6: Statins Induced Cell Death in Pre-Metastatic Cells and the Effects were Mediated Through the Geranylgeranyl Pathway

[0076] To test whether the hits from the drug screen targeted human pre-metastatic cells, the growth of the human mammary epithelial cell lines MCF-10A, MCF-10A with homozygous pten deletion (pten−/−), or MCF10-A harboring oncogenic PIK3CA mutations under compound treatment were measured. At 5 μM, pitavastatin resulted in marked reduction in cell growth as well as extensive cell death in pten−/− as well as PIK3CA knock-in cells but not MCF-10A cells (FIG. 10 and FIG. 11, pten−/− shown as an example).

[0077] The use of statins is well established in the clinic to treat hypercholesterolemia by targeting HMG-CoA reductase that is responsible for cholesterol production. The mevalonate pathway is also the source of other biologically active metabolites such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are critical for posttranslational modifications of Ras and RhoA, respectively. To examine whether the cytotoxic effects of pitavastatin were due to decreased production of cholesterol or some other metabolites, add-back experiments were performed. Supplementation of either mevalonic acid (MVA), the enzymatic product of HMG-CoA reductase, or GGPP, but not cholesterol, completely blocked the effects of pitavastatin (FIG. 12). This observation revealed a distinct antitumorigenic mechanism of pitavastatin, which was segregated from its cholesterol lowing ability. It also indicated the use of statins as a therapeutic for tumor bearing mutations in PTEN or PI3K.

OTHER EMBODIMENTS

[0078] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0079] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.