Non-Small Cell Lung Cancer (NSCLC)
Lung cancer has become the number one killer among cancers worldwide (Molina, J. R. et al., Mayo Clin Proc. 2008 May; 83(5): 584-594). Only 15% of all lung cancer patients are alive five (5) years or more after diagnosis (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). The two (2) main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), the latter of which accounts for approximately 85% of all cases of lung cancer (Molina, J. R. et al., Mayo Clin Proc. 2008 May; 83(5): 584-594; Navada, S. et al., J Clin Oncol. 2006; 24(18S) suppl: 384S; Sher, T. et al., Mayo Clin Proc. 2008; 83(3): 355-367).
Risk Factors for NSCLC
The primary risk factor for lung cancer is smoking, which accounts for more than 85% of all lung cancer-related deaths (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801; Doll, R. et al., Br Med J 1976; 2: 1525-1536). The risk for lung cancer increases with the number of cigarettes smoked per day and the number of years spent smoking. In addition to the hazard of first-hand smoke, exposed nonsmokers have an increased relative risk for developing lung cancer (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801; Wald N. J. et al., Br Med J 1986; 293: 1217-1222). Radon gas, a radioactive gas that is produced by the decay of radium 226, is the second leading cause of lung cancer. The decay of this isotope leads to the production of substances that emit alpha-particles, which may cause cell damage and therefore increase the potential for malignant transformation (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801; Schrump, D. S. et al., DeVita, Hellman, and Rosenberg's Cancer: Principles & Practice of Oncology, 8th Edition. Vol. 1. Philadelphia: Lippincott Williams & Wilkins; 2008:896-946).
Asbestos, a mineral compound that breaks into small airborne shards, is a known carcinogen that increases the risk for lung cancer in people exposed to the airborne fibers, especially those who smoke (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). An estimated 3% to 4% of lung cancers are caused by asbestos exposure (Omenn, G. S. et al., Environ Health Perspect 1986; 70: 51-56). Other possible risk factors include recurring lung inflammation, lung scarring secondary to tuberculosis, family history, and exposure to other carcinogens, such as bis(chloromethyl) ether, polycyclic aromatic hydrocarbons, chromium, nickel, and organic arsenic compounds (Fraumeni, J. F., J Natl Cancer Inst 1975; 55: 1039-1046; Janerich D. T. et al., N Engl J Med 1990; 323: 632-636).
Familial clustering or aggregation of lung cancer has been reported in the past 60 years, suggesting a hereditary base to disease development (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801; Sellers, T. A. and Yang P., King R A, Rotter J I, Motulsky A G, editors. The Genetic Basis of Common Diseases. 2nd ed. New York, N.Y.: Oxford University Press; 2002. pp. 700-712; Hwang, S. J. et al., Hum Genet. 2003 August; 113(3): 238-243; Bailey-Wilson, J. E. et al., Am J Human Genet. 2004 September; 75(3): 460-474; Hung, R. J. et al., Nature. 2008; 452(7178): 633-637). An increased risk of lung cancer was found in carriers of tumor protein p53 (TP53) germline sequence variations (Hwang, S. J. et al., Hum Genet. 2003 August; 113(3): 238-243). A germline epidermal growth factor receptor (EGFR) T790M sequence variation was reported in a family with multiple cases of NSCLC (Lie, X. and Hemminki, K., Int J Cancer. 2004; 112(3): 451-457). A genome-wide linkage study of 52 extended families identified a major susceptibility locus influencing lung cancer risk at 6q23-25p (Bailey-Wilson, J. E. et al., Am J Human Genet. 2004 September; 75(3): 460-474).
Three (3) independent genetic studies have found a marker on chromosome 15 associated with lung cancer (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). In all three (3) studies, the risk was approximately 30% higher for people with 1 copy of the marker and 70-80% higher for people with two (2) copies. The region where the marker resides contains 3 genes coding for subunits of the nicotinic acetylcholine receptor, a protein on the cell surface onto which nicotine molecules latch, triggering cell change (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801; Thorgeirsson, T. E. et al., Nature. 2008; 452(7178): 638-642; Hung, R. J. et al., Nature. 2008; 452(7187): 633-637).
Staging of NSCLC
There are five stages (Stage 0 to Stage IV) in NSCLC. Stages I, II and III are further subdivided into A and B subtypes. These stages are assigned based on a Tumor, Node and Metastasis (TMN) staging system (See, cancer staging guidelines, www.NCCN.org). The TMN staging system of NSCLC is summarized in Table 1.
TABLE 1Stages 0 to IV of NSCLCTMNStage(Tumor, Nodes, Metastasis)DefinitionOccult TX, N0, M0TX = Primary tumor cannot becarcinomaassessed, or tumor proven bythe presence of malignant cellsin sputum or bronchialwashings but not visualized byimaging or bronchoscopy.N0 = No regional lymph nodemetastasis.M0 = No distant metastasis.0Tis, N0, M0Tis = Carcinoma in situ.N0 = No regional lymph nodemetastasis.M0 = No distant metastasis.IAT1a, N0, M0T1a = Tumor = 2 cm inT1b, N0, M0greatest dimension.T1b = Tumor > 2 cm but = 3cm in greatest dimension.N0 = No regional lymph nodemetastasis.M0 = No distant metastasis.IBT2a, N0, M0T2a = Tumor > 3 cm but = 5cm in greatest dimension.N0 = No regional lymph nodemetastasis.M0 = No distant metastasis.IIAT1a, N1, M0T1a = Tumor = 2 cm inT1b, N1, M0greatest dimension.T2a, N1, M0T1b = Tumor > 2 cm but = 3T2b, N0, M0cm in greatest dimension.T2a = Tumor > 3 cm but = 5cm in greatest dimension.T2b = Tumor > 5 cm but = 7cm in greatest dimension.N0 = No regional lymph nodemetastasis.N1 = Metastasis in ipsilateralperibronchial and/or ipsilateralhilar lymph nodes andintrapulmonary nodes,including involvement bydirect extension.M0 = No distant metastasis.IIBT2b, N1, M0T2b = Tumor > 5 cm but = 7T3, N0, M0cm in greatest dimension.T3 = Tumor > 7 cm or one thatdirectly invades any of thefollowing: parietal pleural(PL3) chest wall (includingsuperior sulcus tumors),diaphragm, phrenic nerve,mediastinal pleura or parietalpericardium.Tumor in the main bronchus(<2 cm distal to the carina, butwithout involvement of thecarina.Associated atelectasis orobstructive pneumonitis of theentire lung or separate tumornodule(s) in the same lobe.N0 = No regional lymph nodemetastasis.N1 = Metastasis in ipsilateralperibronchial and/or ipsilateralhilar lymph nodes andintrapulmonary nodes,including involvement bydirect extension.M0 = No distant metastasis.IIIAT1a, N2, M0T1a = Tumor = 2 cm inT1b, N2, M0 greatest dimension.T2a, N2, M0 T1b = Tumor > 2 cm but = 3T2b, N2, M0cm in greatest dimension.T3, N1, M0T2a = Tumor > 3 cm but = 5T3, N2, M0cm in greatest dimension.T4, N0, M0T2b = Tumor > 5 cm but = 7T4, N1, M0cm in greatest dimension.T3 = Tumor > 7 cm or one thatdirectly invades any of thefollowing: parietal pleural(PL3) chest wall (includingsuperior sulcus tumors),diaphragm, phrenic nerve,mediastinal pleura or parietalpericardium.Tumor in the main bronchus(<2 cm distal to the carina butwithout involvement of thecarina).Associated atelectasis orobstructive pneumonitis of theentire lung or separate tumornodule(s) in the same lobe.T4 = Tumor of any size thatinvades any of the following:mediastinum, heart, greatvessels, trachea, recurrentlaryngeal nerve, esophagus,vertebral body, carina orseparate tumor nodule(s) in adifferent ipsilateral lobe.N0 = No regional lymph nodemetastasis.N1 = Metastasis in ipsilateralperibronchial and/or ipsilateralhilar lymph nodes andintrapulmonary nodes,including involvement bydirect extension.N2 = Metastasis in ipsilateralmediastinal and/or subcarinallymph node(s).M0 = No distant metastasis.IIIBT1a, N3, M0T1a = Tumor = 2 cm inT1b, N3, M0greatest dimension.T2a, N3, M0T1b = Tumor > 2 cm but = 3T2b, N3, M0cm in greatest dimension.T3, N3, M0T2a = Tumor > 3 cm but = 5T4, N2, M0cm in greatest dimension.T4, N3, M0T2b = Tumor > 5 cm but = 7cm in greatest dimension.T3 = Tumor > 7 cm or one thatdirectly invades any of thefollowing: parietal pleural(PL3) chest wall (includingsuperior sulcus tumors),diaphragm, phrenic nerve,mediastinal pleura or parietalpericardium.Tumor in the main bronchus(<2 cm distal to the carina butwithout involvement of thecarina).Associated atelectasis orobstructive pneumonitis of theentire lung or separate tumornodule(s) in the same lobe.T4 = Tumor of any size thatinvades any of the following:mediastinum, heart, greatvessels, trachea, recurrentlaryngeal nerve, esophagus,vertebral body, carina orseparate tumor nodule(s) in adifferent ipsilateral lobe.N2 = Metastasis in ipsilateralmediastinal and/or subcarinallymph node(s).N3 = Metastasis incontralateral mediastinal,contralateral hilar, ipsilateralor contralateral scalene orsupraclavicular lymphnode(s).M0 = No distant metastasis.IVAny T, any N, M1ATX = Primary tumor cannot beAny T, any N, M1bassessed, or tumor proven bythe presence of malignant cellsin sputum or bronchialwashings but not visualized byimaging or bronchoscopy.T0 = No evidence of primarytumor.Tis = Carcinoma in situ.T1 = Tumor = 3 cm in greatestdimension, surrounded bylung or visceral pleura,without bronchoscopicevidence of invasion moreproximal than the lobarbronchus (i.e., not in the mainbronchus).T1a = Tumor = 2 cm ingreatest dimension.T1b = Tumor > 2 cm but = 3cm in greatest dimension.T2 = Tumor > 3 cm but = 7 cmor tumor with any of thefollowing features (T2 tumorswith these features areclassified T2a if = 5 cm):involves main bronchus, = 2distal to the carina, invadesvisceral pleura (PL1 or PL2)or is associated withatelectasis or obstructivepneumonitis that extends tothe hilar region but does notinvolve the entire lung.T2a = Tumor > 3 cm but = 5cm in greatest dimension.T2b = Tumor > 5 cm but = 7cm in greatest dimension.T3 = Tumor > 7 cm or one thatdirectly invades any of thefollowing: parietal pleura(PL3) chest wall (includingsuperior sulcus tumors),diaphragm, phrenic nerve,mediastinal pleura or parietalpericardium.Tumor in the main bronchus(<2 cm distal to the carina butwithout involvement of thecarina).Associated atelectasis orobstructive pneumonitis of theentire lung or separate tumornodule(s) in the same lobe.T4 = Tumor of any size thatinvades any of the following:mediastinum, heart, greatvessels, trachea, recurrentlaryngeal nerve, esophagus,vertebral body, carina orseparate tumor nodule(s) in adifferent ipsilateral lobe.NX = Regional lymph nodescannot be assessed.N0 = No regional lymph nodemetastasis.N1 = Metastasis in ipsilateralperibronchial and/or ipsilateralhilar lymph nodes andintrapulmonary nodes,including involvement bydirect extension.N2 = Metastasis in ipsilateralmediastinal and/or subcarinallymph node(s).N3 = Metastasis incontralateral mediastinal,contralateral hilar, ipsilateralor contralateral scalene orsupraclavicular lymphnode(s).M0 = No distant metastasis.M1 = Distant metastasis.M1a = Separate tumornodule(s) in a contralaterallobe tumor with pleuralnodules or malignant pleural(or pericardial) effusion.M1b = Distant metastasis.
Screening for NSCLC
Computed Tomography (CT)
Computed tomography (CT) enables small lesions (in the region of 3 cm) to be detected (Read, C. et al., Primary Care Respiratory Journal (2006) 15, 332-336). Until recently, the slow scanning times and high radiation exposure of CT made it an unsuitable tool for screening. However, with the development and refinement of spiral CT, it has become possible to significantly reduce scanning times and radiation exposure. Results of CT screening have been encouraging, with approximately 80-90% of CT screen-detected bronchial carcinomasdiagnosed as early stage 1 tumors (Kaneko, M. et al., Radiology 1996; 201: 798-802; Swensen, S. J. et al., Am J Respir Crit Care Med 2002; 165: 508-513; Henschke, C. I. et al., Radiology 2006; 239(2): 586-590).
Sputum Cytometry
The diagnostic yield of sputum cytology is known to vary in relation to tumor location (Read, C. et al., Primary Care Respiratory Journal (2006) 15, 332-336). It has greatest use in the identification of central tumors and is of little or no value in the identification of peripheral cancers (Thunnissen F. B. J. M., J Clin Pathol 2003; 56: 805-810). However, it has been suggested that its sensitivity can be improved through the use of molecular genetic and immunocytochemical markers of malignancy (Marek, W. et al., Eur Respir J 2001; 18: 942-950; Xing, S. et al., Eur Respir J 2005; 25: 956-963).
Autofluorescence Bronchoscopy
Autofluorescence bronchoscopy has a high sensitivity for detection of abnormal cells (Read, C. et al., Primary Care Respiratory Journal (2006) 15, 332-336). This technique exploits the differences in the fluorescence properties of bronchial mucosa compared to mucosa of pre-invasive and invasive disease.
The best known instrument for autofluorescence bronchoscopy is the Lung Imaging Fluorescence Endoscopy (LIFE) system (Lam, S. et al., J Thorac Cardiovasc Surg 1993; 105: 1035-1040). This instrument uses a blue (442 nm) helium cadmium laser to illuminate bronchial mucosa, and the resulting fluorescence is then digitized into a real-time video image. Other devices, such as the D-light auto-fluorescence system, do not require image-intensifying cameras, but use optical filters incorporated into the bronchoscope (Leonhard, M., Diagn Therap Endosc 1999; 5: 71-75).
The majority of published studies on autofluorescence bronchoscopy have shown a significant increase in diagnostic sensitivity for dysplasia and carcinoma in situ detection (Read, C. et al., Primary Care Respiratory Journal (2006) 15, 332-3; Lam, S. et al., Chest 1998; 113: 696-702; Vermylen, P. et al., Lung Cancer 1999; 25: 161-168; Venmans B. J. et al., Diagn Therap Endosc 1999; 5: 77-84; Ikeda, N. et al., Diagn Therap Endosc 1997; 3: 197-201; Yokomise, H. et al., J Bronchol 1997; 4: 205-208; Hirsch, F. R. et al., J Natl Cancer Inst 2001; 93: 1385-1391).
Biomarkers
Several biomarkers have emerged as prognostic and predictive markers for NSCLC (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). A prognostic biomarker, which is an indicator of innate tumor aggressiveness, is a biomolecule that indicates patient survival independent of the treatment received. A predictive biomarker is a biomolecule that indicates therapeutic efficacy, i.e., an interaction exists between the biomolecule and therapy that impacts patient outcome. Among these biomarkers, evidence is strongest for EGFR, the 5′ endonuclease of the nucleotide excision repair complex (ERCC1), Kirsten rat sarcoma viral oncogene homolog (K-ras), and the regulatory subunit of ribonucleotide reductase (RRM1).
The presence of the epidermal growth factor receptor (EGFR) exon 19 deletion or exon 21 L858R mutation does not seem to be prognostic of survival for patients with NSCLC, independent of therapy (Tsao, M. S. et al., N Engl J Med 2005; 353: 133-144). However, the presence of the EGFR exon 19 deletion or exon 21 L858R mutation is predictive of treatment benefit from EGFR-TKI therapy (Sequist, L. V. et al., J Clin Oncol 2008; 26: 2442-2449; Miller, V. A., J Clin Oncol 2008; 26: 1472-1478). High ERCC1 levels are prognostic of better survival for patients with NSCLC when compared with low levels of ERCC1 expression, independent of therapy (Simon, G. R. et al., Chest 2005; 127: 978-983; Olaussen K. A. et al., N Engl J Med 2006; 355: 983-991). High levels of ERCC1 expression are also predictive of poor response to platinum-based chemotherapy (Olaussen K. A. et al., N Engl J Med 2006; 355: 983-991; Bepler, G. et al., J Clin Oncol 2006; 24: 4731-4737). The presence of K-ras mutations is prognostic of poor survival for patients with NSCLC when compared with the absence of these mutations, independent of therapy (Slebos, R. J. et al., N Engl J Med 1990; 323: 561-565). Presence of K-ras mutations is also predictive of lack of benefit from platinum/vinorelbine chemotherapy or EGFR-TKI therapy (Miller, V. A., J Clin Oncol 2008; 26: 1472-1478; Tsao, M. S. et al., J Clin Oncol 2007; 25: 5240-5247). High RRM1 levels are prognostic of better survival for patients with NSCLC compared with low levels of RRM1 expression, independent of therapy (Bepler, G. et al., J Clin Oncol 2004; 22: 1878-1885; Zheng, Z. et al., N Engl J Med 2007; 356: 800-808). High levels of RRM1 expression are also predictive of poor response to gemcitabine-based chemotherapy (Bepler, G. et al., J Clin Oncol 2008; 26(Suppl 1): Abstract 8033; Reynolds, C. et al., J Clin Oncol 2009; 27: 5808-5815).
Treatment of NSCLC
Surgery
In general, for patients with stage I or II disease, surgery provides the best chance for cure (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). The surgical procedure used depends on the extent of disease and the cardiopulmonary reserve of the patient. If anatomically appropriate and margin-negative resection can be achieved, lung-sparing anatomic resection (sleeve lobectomy) is preferred over pneumonectomy. Otherwise, lobectomy or pneumonectomy are performed if physiologically feasible (Boffa, D. J. et al., J Thorac Cardiovasc Surg 2008; 135: 247-254; Scott, W. J. et al., Chest 2007; 132: 234S-242S). Resection (including wedge resection) is preferred over ablation (i.e., radiofrequency ablation [RFA], cryotherapy, stereotactic radiation) (Scott, W. J. et al., Chest 2007; 132: 234S-242S). However, whether lung-sparing surgeries (i.e., sublobular resection), such as segmentectomy and wedge resection, are useful in patients with severely reduced pulmonary function who are otherwise not candidates for surgery is controversial (Scott, W. J. et al., Chest 2007; 132: 234S-242S; Ginsberg, R. J. et al., Ann Thorac Surg 1995; 60: 615-622; Koike, T. et al., J Thorac Cardiovasc Surg 2003; 125: 924-928).
Radiation Therapy (RT)
RT can be used as 1) an adjunct for patients with resectable NSCLC who have no contraindications for surgery; 2) the primary local treatment (i.e., definitive RT) for patients with medically inoperable or unresectable NSCLC; and/or 3) an important palliative modality for patients with incurable NSCLC (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). However, conventional RT alone only results in a median survival of 10 months and a 5-year survival rate of 5% (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801).
Chemotherapy
Many drugs are active against stage IV NSCLC. These drugs include the taxanes (paclitaxel, docetaxel), vinorelbine, etoposide, pemetrexed, the camptothecin analogs (irinotecan), and gemcitabine. Combinations using many of these drugs produce 1-year survival rates of 30% to 40% and are superior to single agents. Regimens include carboplatin/paclitaxel, cisplatin/paclitaxel, cisplatin/vinorelbine, gemcitabine/cisplatin, cisplatin/pemetrexed, and docetaxel/cisplatin (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801; Ohe, Y. et al., Ann Oncol 2007; 18: 317-323; Fossella, F. et al., J Clin Oncol 2003; 21: 3016-3024; Smit, E. F. et al., J Clin Oncol 2003; 21: 3909-3917; Zatloukal, P. et al., Lung Cancer 2004; 46: 87-98; Scagliotti, G. V. et al., J Clin Oncol 2008; 26: 3543-3551).
Although chemotherapy is appropriate for many patients with lung cancer, there is a consensus that the use of traditional chemotherapeutic agents to treat NSCLC has reached a therapeutic plateau (Molina, J. R. et al., Mayo Clin Proc 2008 May; 83(5): 584-594).
Targeted Therapy
Specific targeted therapies have been developed for treating advanced lung cancer (Sandler, A. B. et al., Clin Cancer Res 2004; 10: 4258s-4262s; Giaccone, G. et al., J Clin Oncol 2005; 23: 3235-3242). Bevacizumab is a recombinant monoclonal antibody that blocksvascular endothelial growth factor (VEGF). Erlotinib is a small molecule inhibitor of EGFR. Cetuximab is a monoclonal antibody that targets EGFR.
In 2006, the FDA approved bevacizumab for patients with unresectable, locally advanced, recurrent, or metastatic nonsquamous NSCLC (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). It has been recommended that bevacizumab be used in combination with paclitaxel and carboplatin for select patients with advanced nonsquamous NSCLC (Sandler, A. et al., N Engl J Med 2006; 355: 2542-2550). To undergo treatment with bevacizumab and chemotherapy, patients must have nonsquamous NSCLC and no history of hemoptysis. Any regimen with a high risk for thrombocytopenia (i.e., possible bleeding) should be used with caution when combined with bevacizumab (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801).
In 2004, the FDA approved Erlotinib for the treatment of patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). However, erlotinib can also be given as first-line therapy in patients with advanced or metastatic NSCLC who have a known active EGFR mutation or gene amplification (Sequist, L. V. et al., Oncologist 2007; 12: 90-98; N Eng J Med 2009; 361: 947-957; Inoue, A. et al., J Clin Oncol 2009; 27: 1394-1400).
A recent phase III randomized trial assessed cisplatin/vinorelbine with or without cetuximab for patients with advanced NSCLC (Pirker, R. et al., Lancet 2009; 373: 1525-1531). The results indicated that adding cetuximab slightly increased overall survival (11.3 vs. 10.1 months; P=0.04) (Pirker, R. et al., Lancet 2009; 373: 1525-1531; Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801).
However, there is accumulating evidence that NSCLC acquires resistance to these specific targeted therapies (e.g., erlotinib and gefitinib) and how cancers such as NSCLC become resistant to, for example, EGFR inhibitors (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801). The most common known mechanism is the acquisition of a secondary mutation in EGFR, known as T790M, which renders the kinase resistant to erlotinib and gefitinib (Nguyen, K. S. et al., Clin Lung Cancer 2009; 10: 281-289; Gazdar, A. F., Oncogene 2009; 28(Suppl 1): S24-S31). Amplification of the MET oncogene is another validated resistance mechanism (Ettinger, D. S. et al., J Natl Compr Canc Netw 2010; 8: 740-801).
DNA Damage-Signaling in Human Tumors
DNA damage signaling and checkpoint control pathways are among the most commonly mutated networks in human tumors (Morandell, S. et al., Cell Reports 5, 868-877, Nov. 27, 2013; Negrini, S. et al., Nat Rev Mol Cell Biol 2010; 11: 220-228). The three major DNA damage-responsive cell cycle checkpoints are the G1/S checkpoint, intra S-phase checkpoint, and the G2/M checkpoint. In response to DNA damage, eukaryotic cells activate complex signaling networks that mediate DNA repair and cell cycle arrest or, if the damage is extensive, trigger apoptosis (Ciccia, A. and Elledge, S. J., Mol Cell 2010; 40: 179-204). Three canonical protein kinase pathways in both normal and cancer cells arrest the cell cycle in response to damaged DNA: the Ataxia-Telangiectasia and Rad-3 related through Chk1 (ATR-Chk1) pathway, the Ataxia-Telangiectasia mutated through Chk2 (ATM-Chk2) pathway, and the stress-activated protein kinases p38 mitogen-activated protein kinase (MAPK) and its substrate MAPKAP kinase-2 (MK2) (Morandell, S. et al., Cell Reports 5, 868-877, Nov. 27, 2013). The ATM/Chk2 pathway responds primarily to DNA double strand breaks, while the ATRpChk1 pathway is activated by bulky DNA lesions, and following replication fork collapse during S-phase. In contrast to the DNA damage-specific activation of Chk1 and Chk2, the p38MAPK pathway is a general stress-activated kinase pathway that responds to various cellular stimuli, including cytokines, hyperosmolarity, and UV irradiation.
Tumor suppressor protein p53 is a major downstream effector of the aforementioned DNA damage kinase pathways. In normal cells, p53-dependent signaling results in G1 arrest, mainly mediated by transcriptional upregulation of p21. P21 also appears to play a role in sustaining the G2 checkpoint after gamma-irradiation. If DNA damage is extensive, however, p53-dependent pathways target the damaged cell for apoptotic cell death. The MK2 pathway is critical for arresting the cell cycle after genotoxic stress, including cisplatin-induced DNA crosslinks and topoisomerase-inhibitor-induced DNA strand breaks only in tumor cells that lack functional p53 (Manke, I. A. et al., Mol Cell 2005; 17: 37-48; Reinhardt, H. C. et al., Cancer Cell 2007; 11: 175-189). Both the ATRChk1 pathway and the p38-MK2 pathway are required for effective cell-cycle checkpoint function in the absence of p53 (Reinhardt, H. C. et al., Mol Cell 2010; 40: 34-49).
Recently, Morandell et al. (Cell Reports 5, 868-877, Nov. 27, 2013) showed that, in response to genotoxic chemotherapy, MK2 is essential for the survival of NSCLC tumor cells that lack functional p53 but is dispensable in p53-proficient cells. MK2 was found to specifically sensitize p53-deficient tumors to the DNA-damaging agent cisplatin but had no effect on the treatment response of p53-proficient cancer cells. This suggests a potential for enhancement chemosensitization of p53-deficient tumors to DNA-damaging chemotherapy in vivo through synthetic lethality between p53 and MK2 (Morandell, S. et al., Cell Reports 5, 868-877, Nov. 27, 2013).
Kinases
Kinases are a ubiquitous group of enzymes that catalyze the phosphoryl transfer reaction from a phosphate donor (usually adenosine-5′-triphosphate (ATP)) to a receptor substrate. A classification of all available kinase sequences (approximately 60,000 sequences) indicates kinases can be grouped into 25 families of homologous (meaning derived from a common ancestor) proteins. These kinase families are assembled into 12 fold groups based on similarity of structural fold. Further, 22 of the 25 families (approximately 98.8% of all sequences) belong to 10 fold groups for which the structural fold is known. Of the other 3 families, polyphosphate kinase forms a distinct fold group, and the 2 remaining families are both integral membrane kinases and comprise the final fold group. These fold groups not only include some of the most widely spread protein folds, such as Rossmann-like fold (three or more parallel β strands linked by two α helices in the topological order β-α-β-α-β), ferredoxin-like fold (a common α-β protein fold with a signature βαββαβ secondary structure along its backbone), TIM-barrel fold (meaning a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone), and antiparallel β-barrel fold (a beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last), but also all major classes (all α, all β, α+β, α/β) of protein structures. Within a fold group, the core of the nucleotide-binding domain of each family has the same architecture, and the topology of the protein core is either identical or related by circular permutation. Homology between the families within a fold group is not implied.
Group I (23,124 sequences) kinases incorporate protein S/T-Y kinase, atypical protein kinase, lipid kinase, and ATP grasp enzymes and further comprise the protein S/T-Y kinase, and atypical protein kinase family (22,074 sequences). These kinases include: choline kinase (EC 2.7.1.32); protein kinase (EC 2.7.137); phosphorylase kinase (EC 2.7.1.38); homoserine kinase (EC 2.7.1.39); I-phosphatidylinositol 4-kinase (EC 2.7.1.67); streptomycin 6-kinase (EC 2.7.1.72); ethanolamine kinase (EC 2.7.1.82); streptomycin 3′-kinase (EC 2.7.1.87); kanamycin kinase (EC 2.7.1.95); 5-methylthioribose kinase (EC 2.7.1.100); viomycin kinase (EC 2.7.1.103); [hydroxymethylglutaryl-CoA reductase (NADPH2)] kinase (EC 2.7.1.109); protein-tyrosine kinase (EC 2.7.1.112); [isocitrate dehydrogenase (NADP+)] kinase (EC 2.7.1.116); [myosin light-chain] kinase (EC 2.7.1.117); hygromycin-B kinase (EC 2.7.1.119); calcium/calmodulin-dependent protein kinase (EC 2.7.1.123); rhodopsin kinase (EC 2.7.1.125); [beta-adrenergic-receptor] kinase (EC 2.7.1.126); [myosin heavy-chain] kinase (EC 2.7.1.129); [Tau protein] kinase (EC 2.7.1.135); macrolide 2′-kinase (EC 2.7.1.136); I-phosphatidylinositol 3-kinase (EC 2.7.1.137); [RNA-polymerase]-subunit kinase (EC 2.7.1.141); phosphatidylinositol-4,5-bisphosphate 3-kinase (EC 2.7.1.153); and phosphatidylinositol-4-phosphate 3-kinase (EC 2.7.1.154). Group I further comprises the lipid kinase family (321 sequences). These kinases include: I-phosphatidylinositol-4-phosphate 5-kinase (EC 2.7.1.68); I D-myo-inositol-triphosphate 3-kinase (EC 2.7.1.127); inositol-tetrakisphosphate 5-kinase (EC 2.7.1.140); I-phosphatidylinositol-5-phosphate 4-kinase (EC 2.7.1.149); I-phosphatidylinositol-3-phosphate 5-kinase (EC 2.7.1.150); inositol-polyphosphate multikinase (EC 2.7.1.151); and inositol-hexakiphosphate kinase (EC 2.7.4.21). Group I further comprises the ATP-grasp kinases (729 sequences) which include inositol-tetrakisphosphate I-kinase (EC 2.7.1.134); pyruvate, phosphate dikinase (EC 2.7.9.1); and pyruvate, water dikinase (EC 2.7.9.2).
Group II (17,071 sequences) kinases incorporate the Rossman-like kinases. Group II comprises the P-loop kinase family (7,732 sequences). These include gluconokinase (EC 2.7.1.12); phosphoribulokinase (EC 2.7.1.19); thymidine kinase (EC 2.7.1.21); ribosylnicotinamide kinase (EC 2.7.1.22); dephospho-CoA kinase (EC 2.7.1.24); adenylylsulfate kinase (EC 2.7.1.25); pantothenate kinase (EC 2.7.1.33); protein kinase (bacterial) (EC 2.7.1.37); uridine kinase (EC 2.7.1.48); shikimate kinase (EC 2.7.1.71); deoxycytidine kinase (EC 2.7.1.74); deoxyadenosine kinase (EC 2.7.1.76); polynucleotide 5′-hydroxyl-kinase (EC 2.7.1.78); 6-phosphofructo-2-kinase (EC 2.7.1.105); deoxyguanosine kinase (EC 2.7.1.113); tetraacyldisaccharide 4′-kinase (EC 2.7.1.130); deoxynucleoside kinase (EC 2.7.1.145); adenosylcobinamide kinase (EC 2.7.1.156); polyphosphate kinase (EC 2.7.4.1); phosphomevalonate kinase (EC 2.7.4.2); adenylate kinase (EC 2.7.4.3); nucleoside-phosphate kinase (EC 2.7.4.4); guanylate kinase (EC 2.7.4.8); thymidylate kinase (EC 2.7.4.9); nucleoside-triphosphate-adenylate kinase (EC 2.7.4.10); (deoxy)nucleoside-phosphate kinase (EC 2.7.4.13); cytidylate kinase (EC 2.7.4.14); and uridylate kinase (EC 2.7.4.22). Group II further comprises the phosphoenolpyruvate carboxykinase family (815 sequences). These enzymes include protein kinase (HPr kinase/phosphatase) (EC 2.7.1.37); phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32); and phosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49). Group II further comprises the phosphoglycerate kinase (1,351 sequences) family. These enzymes include phosphoglycerate kinase (EC 2.7.2.3) and phosphoglycerate kinase (GTP) (EC 2.7.2.10). Group II further comprises the aspartokinase family (2,171 sequences). These enzymes include carbamate kinase (EC 2.7.2.2); aspartate kinase (EC 2.7.2.4); acetylglutamate kinase (EC 2.7.2.8 1); glutamate 5-kinase (EC 2.7.2.1) and uridylate kinase (EC 2.7.4.). Group II further comprises the phosphofructokinase-like kinase family (1,998 sequences). These enzymes include 6-phosphofrutokinase (EC 2.7.1.11); NAD(+) kinase (EC 2.7.1.23); I-phosphofructokinase (EC 2.7.1.56); diphosphate-fructose-6-phosphate I-phosphotransferase (EC 2.7.1.90); sphinganine kinase (EC 2.7.1.91); diacylglycerol kinase (EC 2.7.1.107); and ceramide kinase (EC 2.7.1.138). Group II further comprises the ribokinase-like family (2,722 sequences). These enzymes include: glucokinase (EC 2.7.1.2); ketohexokinase (EC 2.7.1.3); fructokinase (EC 2.7.1.4); 6-phosphofructokinase (EC 2.7.1.11); ribokinase (EC 2.7.1.15); adenosine kinase (EC 2.7.1.20); pyridoxal kinase (EC 2.7.1.35); 2-dehydro-3-deoxygluconokinase (EC 2.7.1.45); hydroxymethylpyrimidine kinase (EC 2.7.1.49); hydroxyethylthiazole kinase (EC 2.7.1.50); I-phosphofructokinase (EC 2.7.1.56); inosine kinase (EC 2.7.1.73); 5-dehydro-2-deoxygluconokinase (EC 2.7.1.92); tagatose-6-phosphate kinase (EC 2.7.1.144); ADP-dependent phosphofructokinase (EC 2.7.1.146); ADP-dependent glucokinase (EC 2.7.1.147); and phosphomethylpyrimidine kinase (EC 2.7.4.7). Group II further comprises the thiamin pyrophosphokinase family (175 sequences) which includes thiamin pyrophosphokinase (EC 2.7.6.2). Group II further comprises the glycerate kinase family (107 sequences) which includes glycerate kinase (EC 2.7.1.31).
Group III kinases (10,973 sequences) comprise the ferredoxin-like fold kinases. Group III further comprises the nucleoside-diphosphate kinase family (923 sequences). These enzymes include nucleoside-diphosphate kinase (EC 2.7.4.6). Group III further comprises the HPPK kinase family (609 sequences). These enzymes include 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2.7.6.3). Group III further comprises the guanido kinase family (324 sequences). These enzymes include guanidoacetate kinase (EC 2.7.3.1); creatine kinase (EC 2.7.3.2); arginine kinase (EC 2.7.3.3); and lombricine kinase (EC 2.7.3.5). Group III further comprises the histidine kinase family (9,117 sequences). These enzymes include protein kinase (histidine kinase) (EC 2.7.1.37); [pyruvate dehydrogenase (lipoamide)] kinase (EC 2.7.1.99); and [3-methyl-2-oxybutanoate dehydrogenase(lipoamide)] kinase (EC 2.7.1.115).
Group IV kinases (2,768 sequences) incorporate ribonuclease H-like kinases. These enzymes include hexokinase (EC 2.7.1.1); glucokinase (EC 2.7.1.2); fructokinase (EC 2.7.1.4); rhamnulokinase (EC 2.7.1.5); mannokinase (EC 2.7.1.7); gluconokinase (EC 2.7.1.12); L-ribulokinase (EC 2.7.1.16); xylulokinase (EC 2.7.1.17); erythritol kinase (EC 2.7.1.27); glycerol kinase (EC 2.7.1.30); pantothenate kinase (EC 2.7.1.33); D-ribulokinase (EC 2.7.1.47); L-fucolokinase (EC 2.7.1.51); L-xylulokinase (EC 2.7.1.53); allose kinase (EC 2.7.1.55); 2-dehydro-3-deoxygalactonokinase (EC 2.7.1.58); N-acetylglucosamine kinase (EC 2.7.1.59); N-acylmannosamine kinase (EC 2.7.1.60); polyphosphate-glucose phosphotransferase (EC 2.7.1.63); beta-glucoside kinase (EC 2.7.1.85); acetate kinase (EC 2.7.2.1); butyrate kinase (EC 2.7.2.7); branched-chain-fatty-acid kinase (EC 2.7.2.14); and propionate kinase (EC 2.7.2.15).
Group V kinases (1,119 sequences) incorporate TIM β-barrel kinases. These enzymes include pyruvate kinase (EC 2.7.1.40).
Group VI kinases (885 sequences) incorporate GHMP kinases. These enzymes include galactokinase (EC 2.7.1.6); mevalonate kinase (EC 2.7.1.36); homoserine kinase (EC 2.7.1.39); L-arabinokinase (EC 2.7.1.46); fucokinase (EC 2.7.1.52); shikimate kinase (EC 2.7.1.71); 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythriol kinase (EC 2.7.1.148); and phosphomevalonate kinase (EC 2.7.4.2).
Group VII kinases (1,843 sequences) incorporate AIR synthetase-like kinases. These enzymes include thiamine-phosphate kinase (EC 2.7.4.16) and selenide, water dikinase (EC 2.7.9.3).
Group VIII kinases (565 sequences) incorporate riboflavin kinases (565 sequences). These enzymes include riboflavin kinase (EC 2.7.1.26).
Group IX kinases (197 sequences) incorporate dihydroxyacetone kinases. These enzymes include glycerone kinase (EC 2.7.1.29).
Group X kinases (148 sequences) incorporate putative glycerate kinases. These enzymes include glycerate kinase (EC 2.7.1.31).
Group XI kinases (446 sequences) incorporate polyphosphate kinases. These enzymes include polyphosphate kinases (EC 2.7.4.1).
Group XII kinases (263 sequences) incorporate integral membrane kinases. Group XII comprises the dolichol kinase family. These enzymes include dolichol kinases (EC 2.7.1.108). Group XII further comprises the undecaprenol kinase family. These enzymes include undecaprenol kinases (EC 2.7.1.66).
Kinases play indispensable roles in numerous cellular metabolic and signaling pathways, and are among the best-studied enzymes at the structural, biochemical, and cellular level. Despite the fact that all kinases use the same phosphate donor (in most cases, ATP) and catalyze apparently the same phosphoryl transfer reaction, they display remarkable diversity in their structural folds and substrate recognition mechanisms. This probably is due largely to the diverse nature of the structures and properties of their substrates.
Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinases (MK2 and MK3)
Different groups of MAPK-activated protein kinases (MAP-KAPKs) have been defined downstream of mitogen-activated protein kinases (MAPKs). These enzymes transduce signals to target proteins that are not direct substrates of the MAPKs and, therefore, serve to relay phosphorylation-dependent signaling with MAPK cascades to diverse cellular functions. One of these groups is formed by the three MAPKAPKs: MK2, MK3 (also known as 3pK), and MK5 (also designated PRAK). Mitogen-activated protein kinase-activated protein kinase 2 (also referred to as “MAPKAPK2”, “MAPKAP-K2”, “MK2”) is a kinase of the serine/threonine (Ser/Thr) protein kinase family. MK2 is highly homologous to MK3 (approximately 75% amino acid identity). The kinase domains of MK2 and MK3 are most similar (approximately 35% to 40% identity) to calcium/calmodulin-dependent protein kinase (CaMK), phosphorylase b kinase, and the C-terminal kinase domain (CTKD) of the ribosomal S6 kinase (RSK) isoforms. The MK2 gene encodes two alternatively spliced transcripts of 370 amino acids (MK2A) and 400 amino acids (MK2B). The MK3 gene encodes one transcript of 382 amino acids. The MK2- and MK3 proteins are highly homologous, yet MK2A possesses a shorter C-terminal region. The C-terminus of MK2B contains a functional bipartite nuclear localization sequence (NLS) (Lys-Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 21) that is not present in the shorter MK2A isoform, indicating that alternative splicing determines the cellular localization of the MK2 isoforms. MK3 possesses a similar nuclear localization sequence. The nuclear localization sequence found in both MK2B and MK3 encompasses a D domain (Leu-Leu-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 22), which was shown to mediate the specific interaction of MK2B and MK3 with p38α and p38β. MK2B and MK3 also possess a functional nuclear export signal (NES) located N-terminal to the NLS and D domain. The NES in MK2B is sufficient to trigger nuclear export following stimulation, a process which may be inhibited by leptomycin B. The sequence N-terminal to the catalytic domain in MK2 and MK3 is proline rich and contains one (MK3) or two (MK2) putative Src homology 3 (SH3) domain-binding sites, which studies have shown, for MK2, to mediate binding to the SH3 domain of c-Abl in vitro. Recent studies suggest that this domain is involved in MK2-mediated cell migration.
MK2B and MK3 are located predominantly in the nucleus of quiescent cells while MK2A is present in the cytoplasm. Both MK2B and MK3 are rapidly exported to the cytoplasm via a chromosome region maintenance protein (CRM1)-dependent mechanism upon stress stimulation. Nuclear export of MK2B appears to be mediated by kinase activation, as phosphomimetic mutation of Thr334 within the activation loop of the kinase enhances the cytoplasmic localization of MK2B. Without being limited by theory, it is thought that MK2B and MK3 may contain a constitutively active nuclear localization signal (NLS) and a phosphorylation-regulated nuclear export signal (NES).
MK2 and MK3 appear to be expressed ubiquitously, with increased relative expression in the heart, lungs, kidney, reproductive organs (mammary and testis), skin and skeletal muscle tissues, as well as in immune-related cells such as white blood cells/leukocytes and dendritic cells.
Activation of MK2 and MK3 Activity
Various activators of p38α and p38β potently stimulate MK2 and MK3 activity. p38 mediates the in vitro and in vivo phosphorylation of MK2 on four proline-directed sites: Thr25, Thr222, Ser272, and Thr334. Of these sites, only Thr25 is not conserved in MK3. Without being limited by theory, while the function of phosphorylated Thr25 is unknown, its location between the two SH3 domain-binding sites suggests that it may regulate protein-protein interactions. Thr222 in MK2 (Thr201 in MK3) is located in the activation loop of the kinase domain and has been shown to be essential for MK2 and MK3 kinase activity. Thr334 in MK2 (Thr313 in MK3) is located C-terminal to the catalytic domain and is essential for kinase activity. The crystal structure of MK2 has been resolved and, without being limited by theory, suggests that Thr334 phosphorylation may serve as a switch for MK2 nuclear import and export. Phosphorylation of Thr334 also may weaken or interrupt binding of the C terminus of MK2 to the catalytic domain, exposing the NES and promoting nuclear export.
Studies have shown that while p38 is capable of activating MK2 and MK3 in the nucleus, experimental evidence suggests that activation and nuclear export of MK2 and MK3 are coupled by a phosphorylation-dependent conformational switch that also dictates p38 stabilization and localization, and the cellular location of p38 itself is controlled by MK2 and possibly MK3. Additional studies have shown that nuclear p38 is exported to the cytoplasm in a complex with MK2 following phosphorylation and activation of MK2. The interaction between p38 and MK2 may be important for p38 stabilization since studies indicate that p38 levels are low in MK2-deficient cells and expression of a catalytically inactive MK2 protein restores p38 levels.
Substrates and Functions
MK2 shares many substrates with MK3. Both enzymes have comparable substrate preferences and phosphorylate peptide substrates with similar kinetic constants. The minimum sequence required for efficient phosphorylation by MK2 was found to be Hyd-Xaa-Arg-Xaa-Xaa-pSer/pThr (SEQ ID NO: 22), where Hyd is a bulky, hydrophobic residue.
Accumulating evidence has shown that MK2 phophorylates a variety of proteins, which include, but are not limited to, 5-Lipooxygenase (ALOX5), Cell Division Cycle 25 Homolog B (CDC25B), Cell Division Cycle 25 Homolog C (CDC25C), Embryonic Lethal, Abnormal Vision, Drosophila-Like 1 (ELAVL1), Heterogeneous Nuclear Ribonucleoprotein A0 (HNRNPA0), Heat Shock Factor protein 1 (HSF1), Heat Shock Protein Beta-1 (HSPB1), Keratin 18 (KRT18), Keratin 20 (KRT20), LIM domain kinase 1 (LIMK1), Lymphocyte-specific protein 1 (LSP1), Polyadenylate-Binding Protein 1 (PABPC1), Poly(A)-specific Ribonuclease (PARN), CAMP-specific 3′,5′-cyclic Phosphodiesterase 4A (PDE4A), RCSD domain containing 1 (RCSD1), Ribosomal protein S6 kinase, 90 kDa, polypeptide 3 (RPS6KA3), TGF-beta activated kinase 1/MAP3K7 binding protein 3 (TAB3), and Tristetraprolin (TTP/ZFP36).
Heat-Shock Protein Beta-1 (also termed HSPB1 or HSP27) is a stress-inducible cytosolic protein that is ubiquitously present in normal cells and is a member of the small heat-shock protein family. The synthesis of HSPB1 is induced by heat shock and other environmental or pathophysiologic stresses, such as UV radiation, hypoxia and ischemia. Besides its putative role in thermoresistance, HSPB1 is involved in the survival and recovery of cells exposed to stressful conditions.
Experimental evidence supports a role for p38 in the regulation of cytokine biosynthesis and cell migration. The targeted deletion of the mk2 gene in mice suggested that although p38 mediates the activation of many similar kinases, MK2 seems to be the key kinase responsible for these p38-dependent biological processes. Loss of MK2 leads (i) to a defect in lipopolysaccharide (LPS)-induced synthesis of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and gamma interferon (IFN-γ) and (ii) to changes in the migration of mouse embryonic fibroblasts, smooth muscle cells, and neutrophils.
Consistent with a role for MK2 in inflammatory and immune responses, MK2-deficient mice showed increased susceptibility to Listeria monocytogenes infection and reduced inflammation-mediated neuronal death following focal ischemia. Since the levels of p38 protein also are reduced significantly in MK2-deficient cells, it was necessary to distinguish whether these phenotypes were due solely to the loss of MK2. To achieve this, MK2 mutants were expressed in MK2-deficient cells, and the results indicated that the catalytic activity of MK2 was not necessary to restore p38 levels but was required to regulate cytokine biosynthesis.
Knockout or knockdown studies of MK2 provide strong support that activated MK2 enhances stability of IL-6 mRNA through phosphorylation of proteins interacting with the AU-rich 3′ untranslated region of IL-6 mRNA. In particular, it has been shown that MK2 is principally responsible for phosphorylation of hnRNPA0, an mRNA-binding protein that stabilizes IL-6 RNA. In addition, several additional studies investigating diverse inflammatory diseases have found that levels of pro-inflammatory cytokines, such as IL-6, IL-1β, TNF-α and IL-8, are increased in induced sputum from patients with stable chronic obstructive pulmonary disease (COPD) or from the alveolar macrophages of cigarette smokers (Keatings V. et al, Am J Resp Crit Care Med, 1996, 153:530-534; Lim, S. et al., J Respir Crit Care Med, 2000, 162:1355-1360).
Regulation of mRNA Translation.
Previous studies using MK2 knockout mice or MK2-deficient cells have shown that MK2 increases the production of inflammatory cytokines, including TNF-α, IL-1, and IL-6, by increasing the rate of translation of its mRNA. No significant reductions in the transcription, processing, and shedding of TNF-α could be detected in MK2-deficient mice. The p38 pathway is known to play an important role in regulating mRNA stability, and MK2 represents a likely target by which p38 mediates this function. Studies utilizing MK2-deficient mice indicated that the catalytic activity of MK2 is necessary for its effects on cytokine production and migration, suggesting that, without being limited by theory, MK2 phosphorylates targets involved in mRNA stability. Consistent with this, MK2 has been shown to bind and/or phosphorylate the heterogeneous nuclear ribonucleoprotein (hnRNP) A0, tristetraprolin (TTP), the poly(A)-binding protein PABP1, and HuR, a ubiquitously expressed member of the ELAV (Embryonic-Lethal Abnormal Visual in Drosophila melanogaster) family of RNA-binding protein. These substrates are known to bind or copurify with mRNAs that contain AU-rich elements in the 3′ untranslated region, suggesting that MK2 may regulate the stability of AU-rich mRNAs such as TNF-α. It currently is unknown whether MK3 plays a similar role, but LPS treatment of MK2-deficient fibroblasts completely abolished hnRNP A0 phosphorylation, suggesting that MK3 is not able to compensate for the loss of MK2.
MK3 participates with MK2 in phosphorylation of the eukaryotic elongation factor 2 (eEF2) kinase. eEF2 kinase phosphorylates and inactivates eEF2. eEF2 activity is critical for the elongation of mRNA during translation, and phosphorylation of eEF2 on Thr56 results in the termination of mRNA translation. MK2 and MK3 phosphorylation of eEF2 kinase on Ser377 suggests that these enzymes may modulate eEF2 kinase activity and thereby regulate mRNA translation elongation.
Transcriptional Regulation by MK2 and MK3
Nuclear MK2, similar to many MKs, contributes to the phosphorylation of cAMP response element binding (CREB), Activating Transcription Factor-1 (ATF-1), serum response factor (SRF), and transcription factor ER81. Comparison of wild-type and MK2-deficient cells revealed that MK2 is the major SRF kinase induced by stress, suggesting a role for MK2 in the stress-mediated immediate-early response. Both MK2 and MK3 interact with basic helix-loop-helix transcription factor E47 in vivo and phosphorylate E47 in vitro. MK2-mediated phosphorylation of E47 was found to repress the transcriptional activity of E47 and thereby inhibit E47-dependent gene expression, suggesting that MK2 and MK3 may regulate tissue-specific gene expression and cell differentiation.
Other Targets of MK2 and MK3
Several other MK2 and MK3 substrates also have been identified, reflective of the diverse functions of MK2 and MK3 in several biological processes. The scaffolding protein 14-3-3ζ is a physiological MK2 substrate. Studies indicate that 14-3-3ζ interacts with a number of components of cell signaling pathways, including protein kinases, phosphatases, and transcription factors. Additional studies have shown that MK2-mediated phosphorylation of 14-3-3ζ on Ser58 compromises its binding activity, suggesting that MK2 may affect the regulation of several signaling molecules normally regulated by 14-3-3ζ.
Additional studies have shown that MK2 also interacts with and phosphorylates the p16 subunit of the seven-member Arp2 and Arp3 complex (p16-Arc) on Ser77. p16-Arc has roles in regulating the actin cytoskeleton, suggesting that MK2 may be involved in this process. Further studies have shown that the small heat shock protein HSPB1, lymphocyte-specific protein LSP-1, and vimentin are phosphorylated by MK2. HSPB1 forms large oligomers which may act as molecular chaperones and protect cells from heat shock and oxidative stress. Upon phosphorylation, HSPB1 loses its ability to form large oligomers and is unable to block actin polymerization, suggesting that MK2-mediated phosphorylation of HSPB1 serves a homeostatic function aimed at regulating actin dynamics that otherwise would be destabilized during stress. MK3 also was shown to phosphorylate HSPB1 in vitro and in vivo, but its role during stressful conditions has not yet been elucidated.
It was also shown that HSPB1 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-kappa B (NF-κB) by degrading its main inhibitor, I kappa B-alpha (IκB-alpha), and it was shown that overexpresion of HSPB1 increases NF-kappaB (NF-κB) nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, TNF-alpha, and Interleukin-1 beta (IL-1β). Additionally, previous studies have suggested that HSPB1, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I kappa B-alpha (IκB-alpha); and that this function of HSPB1 accounts for its anti-apoptotic properties through the enhancement of NF-kappa B (NF-κB) activity (Parcellier, A. et al., Mol Cell Biol, 23(16): 5790-5802, 2003).
MK2 and MK3 also may phosphorylate 5-lipoxygenase. 5-lipoxygenase catalyzes the initial steps in the formation of the inflammatory mediators, leukotrienes. Tyrosine hydroxylase, glycogen synthase, and Akt also were shown to be phosphorylated by MK2.
MK2 phosphorylates the tumor suppressor protein tuberin on Ser1210, creating a docking site for 14-3-3ζ. Tuberin and hamartin normally form a functional complex that negatively regulates cell growth by antagonizing mTOR-dependent signaling, suggesting that p38-mediated activation of MK2 may regulate cell growth by increasing 14-3-3ζ binding to tuberin.
Accumulating evidence has suggested that the reciprocal crosstalk between the p38 MAPK-pathway and signal transducer and activator of transcription 3 (STAT3)-mediated signal-transduction forms a critical axis successively activated in lipopolysaccharide (LPS) challenge models. It was shown that the balanced activation of this axis is essential for both induction and propagation of the inflammatory macrophage response as well as for the control of the resolution phase, which is largely driven by IL-10 and sustained STAT3 activation (Bode, J. et al., Cellular Signalling, 24: 1185-1194, 2012). In addition, another study has shown that MK2 controls LPS-inducible IFNβ gene expression and subsequent IFNβ-mediated activation of STAT3 by neutralizing negative regulatory effects of MK3 on LPS-induced p65 and IRF3-mediated signaling. In mk2/3 knockout macrophages, IFNβ-dependent STAT3 activation occurred independently from IL-10, because, in contrast to IFNβ-, impaired IL-10 expression is not restored upon additional deletion of MK3 in mk2/3 knockout macrophages (Ehlting, C. et al., J. Biol. Chem., 285(27): 24113-24124).
Kinase Inhibition
The eukaryotic protein kinases constitute one of the largest superfamilies of homologous proteins that are related by virtue of their catalytic domains. Most related protein kinases are specific for either serine/threonine or tyrosine phosphorylation. Protein kinases play an integral role in the cellular response to extracellular stimuli. Thus, stimulation of protein kinases is considered to be one of the most common activation mechanisms in signal transduction systems. Many substrates are known to undergo phosphorylation by multiple protein kinases, and a considerable amount of information on primary sequence of the catalytic domains of various protein kinases has been published. These sequences share a large number of residues involved in ATP binding, catalysis, and maintenance of structural integrity. Most protein kinases possess a well conserved 30-32 kDa catalytic domain.
Studies have attempted to identify and utilize regulatory elements of protein kinases. These regulatory elements include inhibitors, antibodies, and blocking peptides.
Inhibitors
Enzyme inhibitors are molecules that bind to enzymes thereby decreasing enzyme activity. The binding of an inhibitor may stop a substrate from entering the active site of the enzyme and/or hinder the enzyme from catalyzing its reaction (as in inhibitors directed at the ATP biding site of the kinase). Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically (e.g., by modifying key amino acid residues needed for enzymatic activity) so that it no longer is capable of catalyzing its reaction. In contrast, reversible inhibitors bind non-covalently, and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.
Enzyme inhibitors often are evaluated by their specificity and potency. The term “specificity” as used in this context refers to the selective attachment of an inhibitor or its lack of binding to other proteins. The term “potency” as used herein refers to an inhibitor's dissociation constant, which indicates the concentration of inhibitor needed to inhibit an enzyme.
Inhibitors of protein kinases have been studied for use as a tool in protein kinase activity regulation. Inhibitors have been studied for use with, for example, cyclin-dependent (Cdk) kinase, MAP kinase, serine/threonine kinase, Src Family protein tyrosine kinase, tyrosine kinase, calmodulin (CaM) kinase, casein kinase, checkpoint kinase (Chk1), glycogen synthase kinase 3 (GSK-3), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase 1 (MEK), myosin light chain kinase (MLCK), protein kinase A, Akt (protein kinase B), protein kinase C, protein kinase G, protein tyrosine kinase, Raf kinase, and Rho kinase.
Small-Molecule MK2 Inhibitors
While individual inhibitors that target MK2 with at least modest selectivity with respect to other kinases have been designed, it has been difficult to create compounds with favorable solubility and permeability. As a result, there are relatively few biochemically efficient MK2 inhibitors that have advanced to in vivo pre-clinical studies (Edmunds, J. and Talanian, MAPKAP Kinase 2 (MK2) as a Target for Anti-inflammatory Drug Discovery. In Levin, J and Laufer, S (Ed.), RSC Drug Discovery Series No. 26, p 158-175, the Royal Society of Chemistry, 2012; incorporated by reference in its entirety).
The majority of disclosed MK2 inhibitors are classical type I inhibitors as revealed by crystallographic or biochemical studies. As such, they bind to the ATP site of the kinase and thus compete with intra-cellular ATP (estimated concentration 1 mM-5 mM) to inhibit phosphorylation and activation of the kinase. Representative examples of such small-molecule MK2 inhibitors include, but are not limited to,

Blocking Peptides
A peptide is a chemical compound composed of a chain of two or more amino acids whereby the carboxyl group of one amino acid in the chain is linked to the amino group of the other via a peptide bond. Peptides have been used inter alia in the study of protein structure and function. Synthetic peptides may be used inter alia as probes to see where protein-peptide interactions occur. Inhibitory peptides may be used inter alia in clinical research to examine the effects of peptides on the inhibition of protein kinases, cancer proteins and other disorders.
The use of several blocking peptides has been studied. For example, extracellular signal-regulated kinase (ERK), a MAPK protein kinase, is essential for cellular proliferation and differentiation. The activation of MAPKs requires a cascade mechanism whereby MAPK is phosphorylated by an upstream MAPKK (MEK) which then, in turn, is phosphorylated by a third kinase MAPKKK (MEKK). The ERK inhibitory peptide functions as a MEK decoy by binding to ERK.
The synthetic peptide autocamtide-2 related inhibitory peptide (AIP), is a highly specific and potent inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII). AIP is a non-phosphorylatable analog of autocamtide-2, a highly selective peptide substrate for CaMKII. AIP inhibits CaMKII with an IC50 of 100 nM (IC50 is the concentration of an inhibitor required to obtain 50% inhibition). The AIP inhibition is non-competitive with respect to syntide-2 (CaMKII peptide substrate) and ATP but competitive with respect to autocamtide-2. The inhibition is unaffected by the presence or absence of Ca2+/calmodulin. CaMKII activity is inhibited completely by AIP (1 μM) while PKA, PKC and CaMKIV are not affected.
Cell division protein kinase 5 (Cdk5) inhibitory peptide (CIP) is another blocking peptide. Cdk5 phosphorylates the microtubule protein tau at Alzheimer's Disease-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to amyloid β peptides. Upon neuronal infections with CIP, CIPs selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. The reasons for the specificity demonstrated by CIP are not fully understood.
Additional blocking peptides have been studied for inhibiting extracellular-regulated kinase 2 (ERK2), ERK3, p38/HOG1, protein kinase C, casein kinase II, Ca2+/calmodulin kinase IV, casein kinase II, Cdk4, Cdk5, DNA-dependent protein kinase (DNA-PK), serine/threonine-protein kinase PAK3, phosphoinositide (PI)-3 kinase, PI-5 kinase, PSTAIRE (the cdk highly conserved sequence), ribosomal S6 kinase, GSK-4, germinal center kinase (GCK), SAPK (stress-activated protein kinase), SEK1 (stress signaling kinase), and focal adhesion kinase (FAK).
Protein Substrate-Competitive Inhibitors
Most of the protein kinase inhibitors developed to date are ATP competitors. This type of molecule competes for the ATP binding site of the kinase and often shows off-target effects due to serious limitations in its specificity. The low specificity of these inhibitors is due to the fact that the ATP binding site is highly conserved among diverse protein kinases. Non-ATP competitive inhibitors, on the other hand, such as substrate competitive inhibitors, are expected to be more specific as the substrate binding sites have a certain degree of variability among the various protein kinases.
Although substrate competitive inhibitors usually have a weak binding interaction with the target enzyme in vitro, studies have shown that chemical modifications can improve the specific biding affinity and the in vivo efficacy of substrate inhibitors (Eldar-Finkelman, H. et al., Biochim, Biophys. Acta, 1804(3):598-603, 2010). In addition, substrate competitive inhibitors show better efficacy in cells than in cell-free conditions in many cases (van Es, J. et al., Curr. Opin. Gent. Dev. 13:28-33, 2003).
In an effort to enhance specificity and potency in protein kinase inhibition, bisubstrate inhibitors also have been developed. Bisubstrate inhibitors, which consist of two conjugated fragments, each targeted to a different binding site of a bisubstrate enzyme, form a special group of protein kinase inhibitors that mimic two natural substrates/ligands and that simultaneously associate with two regions of given kinases. The principle advantage of bisubstrate inhibitors is their ability to generate more interactions with the target enzyme that could result in improved affinity and selectivity of the conjugates, when compared with single-site inhibitors. Examples of bisubstrate inhibitors include, but are not limited to, nucleotide-peptide conjugates, adenosine derivative-peptide conjugates, and conjugates of peptides with potent ATP-competitive inhibitors.
Protein Transduction Domains (PTD)/Cell Permeable Proteins (CPP)
The plasma membrane presents a formidable barrier to the introduction of macromolecules into cells. For nearly all therapeutics to exert their effects, at least one cellular membrane must be traversed. Traditional small molecule pharmaceutical development relies on the chance discovery of membrane permeable molecules with the ability to modulate protein function. Although small molecules remain the dominant therapeutic paradigm, many of these molecules suffer from lack of specificity, side effects, and toxicity. Information-rich macromolecules, which have protein modulatory functions far superior to those of small molecules, can be created using rational drug design based on molecular, cellular, and structural data. However, the plasma membrane is impermeable to most molecules of size greater than 500 Da. Therefore, the ability of cell penetrating peptides, such as the basic domain of Trans-Activator of Transcription (Tat), to cross the cell membrane and deliver macromolecular cargo in vivo, can greatly facilitate the rational design of therapeutic proteins, peptides, and nucleic acids.
Protein transduction domains (PTDs) are a class of peptides capable of penetrating the plasma membrane of mammalian cells and of transporting compounds of many types and molecular weights across the membrane. These compounds include effector molecules, such as proteins, DNA, conjugated peptides, oligonucleotides, and small particles such as liposomes. When PTDs are chemically linked or fused to other proteins, the resulting fusion peptides still are able to enter cells. Although the exact mechanism of transduction is unknown, internalization of these proteins is not believed to be receptor-mediated or transporter-mediated. PTDs are generally 10-16 amino acids in length and may be grouped according to their composition, such as, for example, peptides rich in arginine and/or lysine.
The use of PTDs capable of transporting effector molecules into cells has become increasingly attractive in the design of drugs as they promote the cellular uptake of cargo molecules. These cell-penetrating peptides, generally categorized as amphipathic (meaning having both a polar and a nonpolar end) or cationic (meaning of or relating to containing net positively charged atoms) depending on their sequence, provide a non-invasive delivery technology for macromolecules. PTDs often are referred to as “Trojan peptides”, “membrane translocating sequences”, or “cell permeable proteins” (CPPs). PTDs also may be used to assist novel HSPB1 kinase inhibitors to penetrate cell membranes. (see, e.g., U.S. Pat. No. 8,536,303, and U.S. Pat. No. 8,741,849, the contents of each of which are incorporated by reference in their entirety herein).
Viral PTD Containing Proteins
The first proteins to be described as having transduction properties were of viral origin. These proteins still are the most commonly accepted models for PTD action. The HIV-1 Transactivator of Transcription (Tat) and HSV-1 VP 22 protein are the best characterized viral PTD containing proteins.
Tat (HIV-1 trans-activator gene product) is an 86-amino acid polypeptide, which acts as a powerful transcription factor of the integrated HIV-1 genome. Tat acts on the viral genome, stimulating viral replication in latently infected cells. The translocation properties of the Tat protein enable it to activate quiescent infected cells, and it may be involved in priming of uninfected cells for subsequent infection by regulating many cellular genes, including cytokines. The minimal PTD of Tat is the 9 amino acid protein sequence RKKRRQRRR (TAT49-57; SEQ ID NO: 20). Studies utilizing a longer fragment of Tat demonstrated successful transduction of fusion proteins up to 120 kDa. The addition of multiple Tat-PTDs as well as synthetic Tat derivatives has been demonstrated to mediate membrane translocation. Tat PTD containing fusion proteins have been used as therapeutic moieties in experiments involving cancer, transporting a death-protein into cells, and disease models of neurodegenerative disorders.
The mechanism used by transducing peptides to permeate cell membranes has been the subject of considerable interest in recent years, as researchers have sought to understand the biology behind transduction. Early reports that Tat transduction occurred by a nonendocytic mechanism have largely been dismissed as artifactual though other cell-penetrating peptides might be taken up by way of direct membrane disruption. The recent findings that transduction of Tat and other PTDs occurs by way of macropinocytosis, a specialized form of endocytosis, has created a new paradigm in the study of these peptides. Enhanced knowledge of the mechanism of transduction helped improve transduction efficiency with the ultimate goal of clinical success (Snyder E. and Dowdy, S., Pharm Res., 21(3):389-393, 2004).
The current model for Tat-mediated protein transduction is a multistep process that involves binding of Tat to the cell surface, stimulation of macropinocytosis, uptake of Tat and cargo into macropinosomes, and endosomal escape into the cytoplasm. The first step, binding to the cell surface, is thought to be through ubiquitous glycan chains on the cell surface. Stimulation of macropinocytosis by Tat occurs by an unknown mechanism that might include binding to a cell surface protein or might occur by way of proteoglycans or glycolipids. Uptake by way of macropinocytosis, a form of fluid phase endocytosis used by all cell types, is required for Tat and polyarginine transduction. The final step in Tat transduction is escape from macropinosomes into the cytoplasm; this process is likely to be dependent on the pH drop in endosomes that, along with other factors, facilitates a perturbation of the membrane by Tat and release of Tat and its cargo (i.e. peptide, protein or drug etc.) to the cytoplasm (Snyder E. and Dowdy, S., Pharm Res., 21(3):389-393, 2004).
VP22 is the HSV-1 tegument protein, a structural part of the HSV virion. VP22 is capable of receptor independent translocation and accumulates in the nucleus. This property of VP22 classifies the protein as a PTD containing peptide. Fusion proteins comprising full length VP22 have been translocated efficiently across the plasma membrane.
Homeoproteins with Intercellular Translocation Properties
Homeoproteins are highly conserved, transactivating transcription factors involved in morphological processes. They bind to DNA through a specific sequence of 60 amino acids. The DNA-binding homeodomain is the most highly conserved sequence of the homeoprotein. Several homeoproteins have been described as exhibiting PTD-like activity; they are capable of efficient translocation across cell membranes in an energy-independent and endocytosis-independent manner without cell type specificity.
The Antennapedia protein (Antp) is a trans-activating factor capable of translocation across cell membranes; the minimal sequence capable of translocation is a 16 amino acid peptide corresponding to the third helix of the protein's homeodomain (HD). The internalization of this helix occurs at 4° C., suggesting that this process is not endocytosis dependent. Peptides of up to 100 amino acids produced as fusion proteins with AntpHD penetrate cell membranes.
Other homeodomains capable of translocation include Fushi tarazu (Ftz) and Engrailed (En) homeodomain. Many homeodomains share a highly conserved third helix.
Human PTDs
Human PTDs may circumvent potential immunogenicity issues upon introduction into a human patient. Peptides with PTD sequences include: Hoxa-5, Hox-A4, Hox-B5, Hox-B6, Hox-B7, HOX-D3, GAX, MOX-2, and FtzPTD. These proteins all share the sequence found in AntpPTD. Other PTDs include Islet-1, Interleukin-1 (IL-1), Tumor Necrosis Factor (TNF), and the hydrophobic sequence from Kaposi-fibroblast growth factor or Fibroblast Growth Factor-4 (FGF-4) signal peptide, which is capable of energy-, receptor-, and endocytosis-independent translocation. Unconfirmed PTDs include members of the Fibroblast Growth Factor (FGF) family. FGFs are polypeptide growth factors that regulate proliferation and differentiation of a wide variety of cells. Several publications have reported that basic fibroblast growth factor (FGF-2) exhibits an unconventional internalization similar to that of VP-22, Tat, and homeodomains. It has also been reported that acidic FGF (FGF-1) translocated cell membranes at temperatures as low as 4° C. However, no conclusive evidence exists about the domain responsible for internalization or the translocation properties of fusion proteins (Beerens, A. et al., Curr Gene Ther., 3(5):486-494, 2003).
Synthetic PTDs
Several peptides have been synthesized in an attempt to create more potent PTDs and to elucidate the mechanisms by which PTDs transport proteins across cell membranes. Many of these synthetic PTDs are based on existing and well documented peptides, while others are selected for their basic residues and/or positive charges, which are thought to be crucial for PTD function. A few of these synthetic PTDs showed better translocation properties than the existing ones (Beerens, A. et al., Curr Gene Ther., 3(5):486-494, 2003). Exemplary Tat-derived synthetic PTDs include, for example, but are not limited to, WLRRIKAWLRRIKA (SEQ ID NO: 12); WLRRIKA (SEQ ID NO: 13); YGRKKRRQRRR (SEQ ID NO: 14); WLRRIKAWLRRI (SEQ ID NO: 15); FAKLAARLYR (SEQ ID NO: 16); KAFAKLAARLYR (SEQ ID NO: 17); and HRRIKAWLKKI (SEQ ID NO: 18).
Compositions Comprising PTDs Fused to MK2 Inhibitor Peptide Therapeutic Domains (TD)
Several MK2 inhibitor peptides (TD) have been synthesized, fused to synthetic PTDs and the use of compositions comprising these fused polypeptides has been studied. These polypeptides include, but are not limited to, YARAAARQARAKALARQLGVAA (SEQ ID NO: 1; MMI-0100), YARAAARQARAKALNRQLGVA (SEQ ID NO: 19; MMI-0200), FAKLAARLYRKALARQLGVAA (SEQ ID NO: 3; MMI-0300), KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4; MMI-0400), HRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 7; MMI-0500), YARAAARDARAKALNRQLAVAA (SEQ ID NO: 23; MMI-0600), and YARAAARQARAKALNRQLAVA (SEQ ID NO: 24; MMI-0600-2). Both in vitro and in vivo studies have shown that these polypeptides can be useful in the treatment of various diseases, disorders and conditions. These include, without limitation, hyperplasia and neoplasm (U.S. Pat. Nos. 8,536,303 and 8,741,849) inflammatory disorders (U.S. application Ser. No. 12/634,476 and U.S. application Ser. No. 13/934,933), adhesions (U.S. application Ser. No. 12/582,516), failure of a vascular graft due to neospasm (U.S. application Ser. No. 13/114,872), improving neurite outgrowth (U.S. application Ser. No. 12/844,815), cutaneous scarring (U.S. application Ser. No. 13/829,876), coronary artery bypass vascular graft failure (U.S. application Ser. No. 13/700,087) and interstitial lung disease and pulmonary fibrosis (U.S. application Ser. No. 13/445,759).
Peptide compositions present a number of particular challenges to formulation scientists (R. W. Payne and M. C. Manning, “Peptide formulation: challenges and strategies,” Innovations in Pharmaceutical Technology, 64-68 (2009)). First, since peptides do not have a globular structure that can sequester reactive groups, the side chains of nearly all residues in a peptide are fully solvent exposed, and can exhibit chemical degradation through hydrolytic reactions, for example, oxidation and deamidation. Second, the conformation in aqueous solution may have little similarity to the structure found when bound to a receptor. Third, many peptides tend to be monomeric at very low concentration, but may self-assemble as the concentration is increased and behave as if in a highly associated state, but these structures are too transient or fluxional to provide any increase in long-term stability. Fourth, the propensity of peptides to self-associate is connected with their physical instability, meaning their likelihood of forming aggregates. Moreover, excipients present in a peptide formulation can chemically degrade, interact with various surfaces during manufacturing, interact with the container or closure, or interact with the peptide itself, thereby negatively affecting critical properties of the preparation (Lars Hovgaard, and Sven Frokjaer, “Pharmaceutical Formulation Development of Peptides and Proteins, 2nd Ed., CRC Press (2012) pp. 212-213).
TrkB is Highly Expressed in NSCLC
Aberrant regulation in the invasion of cancer cells is closely associated with their metastatic potential. (Zhang, S. et al., “TrkB is highly expressed in NSCLC and mediates BDNF-induced activation of Pyk2 signaling and the invasion of A549 cells,” BMC Cancer 2010, 10: 43). Tropomysin-related kinase B (TrkB), a member of the Trk family, functions as a receptor tyrosine kinase and is considered to facilitate tumor metastasis. Id. Brain-derived neurotrophic factor (BDNF), the primary ligand binding to TrkB, results in the regulation of various cellular activities in neuroblastoma, such as cell differentiation, apoptosis, and invasion. Id. TrkB is up-regulated in a variety of primary human tumors, including neuroblastoma and ovarian cancer. Id. Enhanced TrkB signaling promotes cell survival in an anchorage dependent manner. Id. When activated by BDNF, TrkB leads to the activation of downstream signaling molecules, such as phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), which induces the differential regulation of apoptosis and metastasis. Id.
Proline-rich tyrosine kinase 2 (Pyk2) is an extensively expressed non-receptor tyrosine kinase that integrates signals from receptor tyrosine kinases and intracellular signaling molecules in such essential cellular processes as cell differentiation, proliferation and migration. Id. Pyk2 is rapidly tyrosine phosphorylated in response to various extracellular signals, and activated Pyk2 signaling promotes cell survival and migration in an anchorage-independent manner. Id. The tyrosine 402 (Tyr402) of Pyk2 serves as the primary autophosphorylation site essential for Pyk2 activity and function. Id. High activity of Tyr402 is found in tumor cells with a more invasive and metastatic phenotype. Id.
High expression of TrkB has been reported in NSCLC, particularly correlated with lymph node metastasis and TNM stage. Id. TrkB-siRNA interrupted BDNF-promoted Pyk2 and extracellular regulating kinase (ERK) activation and invasion of lung adenocarcinoma A549 cells. Id. Moreover, Pyk2-siRNA inhibited BDNF-associated ERK phosphorylation and cell invastion. Id. The investigators considered these data as evidence that TrkB/Pyk2/ERK signaling mediates BDNF-induced invasion of A549 cells, and suggested that suppression of TrkB may provide a helpful target for inhibitory therapies of metastasis in NSCLC. Id.
The described invention offers an approach to treat non-small cell lung carcinoma using cell-penetrating, peptide-based inhibitors of MK2, that may be effective to overcome tumor chemoresistance, to enhance tumor chemosensitivity and to slow progression of the non-small cell lung cancer solid tumor comprising the population of tumor cells.


The present invention relates generally to medical devices and more particularly to delivery systems for self-expandable medical devices.
Self-expanding medical devices are used by physicians to treat numerous conditions using minimally invasive procedures. Examples of self-expanding medical devices include stents, stent-grafts, filters, valves, etc. Typically, self-expanding medical devices are made from an elastic structure that may be compressed into a low profile state that can be passed through vessels in a patient with minimal trauma. Once at the desired treatment site, the self-expanding medical device is released and self-expands like a spring until it contacts a tissue wall which prevents further expansion. Common materials that are used in self-expanding medical devices include nitinol and stainless steel, although other materials are also possible.
One type of self-expanding medical device that has become especially common is intraluminal stents. Stents are used to treat various organs, such as the vascular system, colon, biliary tract, urinary tract, esophagus, trachea and the like. For example, stents are commonly used to treat blockages, occlusions, narrowing ailments and other similar problems that restrict flow through a passageway. One area where stents are commonly used for treatment involves implanting an endovascular stent into the vascular system in order to improve or maintain blood flow through narrowed arteries. However, stents are also used in other treatments as well, such as the treatment of aneurysms. Stents have been shown to be useful in treating various vessels throughout the vascular system, including both coronary vessels and peripheral vessels (e.g., carotid, brachial, renal, iliac and femoral). In addition, stents have been used in other body vessels as well, such as the digestive tract.
The use of stents in coronary and peripheral vessels has drawn particular attention from the medical community because of the growing number of people each year that suffer from vasculature problems associated with stenosis (i.e., narrowing of a vessel). This has led to an increased demand for medical procedures to treat such problems. The widespread frequency of heart problems and other vasculature problems may be due to a number of societal changes, including the tendency of people to exercise less and the prevalence of unhealthy diets, in conjunction with the fact that people generally have longer life spans now than previous generations. Stents have become a popular alternative for treating vascular stenosis because stenting procedures are considerably less invasive than conventional procedures. For example, stenosis of the coronary arteries was traditionally treated with bypass surgery. In general, bypass surgery involves splitting the chest bone to open the chest cavity and grafting a replacement vessel onto the heart to bypass the blocked, or stenosed, artery. However, coronary bypass surgery is a very invasive procedure that is risky and requires a long recovery time for the patient. Vascular stents are also being more widely used to treat many different peripheral arteries due to the minimally invasive nature of stenting procedures. To address the growing demand for minimally invasive medical procedures for the treatment of coronary arteries, peripheral arteries and other passageway problems, the medical community has begun to turn away from conventional invasive procedures like bypass surgery and increasingly the treatment of choice now involves a variety of stenting procedures.
Many different types of stents and stenting procedures are possible. In general, however, stents are typically designed as tubular support structures that may be inserted percutaneously and transluminally through a body passageway. Traditionally, stents are made from a metal or other synthetic material with a series of radial openings extending through the support structure of the stent to facilitate compression and expansion of the stent. Although stents may be made from many types of materials, including non-metallic materials, common examples of metallic materials that may be used to make stents include stainless steel, nitinol, cobalt-chrome alloys, amorphous metals, tantalum, platinum, gold and titanium. Typically, stents are implanted within a passageway by positioning the stent within the area to be treated and then expanding the stent from a compressed diameter to an expanded diameter. The ability of the stent to expand from a compressed diameter makes it possible to thread the stent to the area to be treated through various narrow body passageways while the stent is in the compressed diameter. Once the stent has been positioned and expanded at the area to be treated, the tubular support structure of the stent contacts and radially supports the inner wall of the passageway. As a result, the implanted stent mechanically prevents the passageway from narrowing and keeps the passageway open to facilitate fluid flow through the passageway.
Stents can generally be characterized as either balloon-expandable or self-expanding. Traditionally, balloon-expandable stents have been used most often in coronary vessels than in peripheral vessels because of the deformable nature of these stents. One reason for this is that peripheral vessels tend to experience frequent traumas from external sources (e.g., impacts to a person's arms, legs, etc.) which are transmitted through the body's tissues to the vessel. In the case of peripheral vessels, there is an increased risk that an external trauma could cause a balloon-expandable stent to plastically deform in unexpected ways with potentially severe and/or catastrophic results. However, in the case of coronary vessels, this risk is minimal since coronary vessels rarely experience traumas transmitted from external sources.
Self-expanding stents are increasingly used and accepted by physicians for treating a variety of ailments. Self-expanding stents are usually made of shape memory materials or other elastic materials that act like a spring. Typical metals used in this type of stent include nitinol and 304 stainless steel. A common procedure for implanting a self-expanding stent involves a two-step process. First, the narrowed vessel portion to be treated is dilated with a balloon but without a stent mounted on the balloon. Second, a stent is implanted into the dilated vessel portion. To facilitate stent implantation, the stent is installed on the end of an inner catheter in a compressed, small diameter state and is usually retained in the small diameter by inserting the stent into an outer sheath at the end of the catheter. The stent is then guided to the balloon-dilated portion and is released from the inner catheter by pulling the outer sheath away from the stent. Once released from the outer sheath, the stent radially springs outward to an expanded diameter until the stent contacts and presses against the vessel wall. Traditionally, self-expanding stents have been more commonly used in peripheral vessels than in coronary vessels due to the shape memory characteristic of the metals that are used in these stents. One advantage of self-expanding stents for peripheral vessels is that traumas from external sources do not permanently deform the stent. Instead, the stent may temporarily deform during an unusually harsh trauma but will spring back to its expanded state once the trauma is relieved. Self-expanding stents, however, are often considered to be less preferred for coronary vessels as compared to balloon-expandable stents. One reason for this is that balloon-expandable stents can be precisely sized to a particular vessel diameter and shape since the ductile metal that is used can be plastically deformed to a desired size and shape. In contrast, self-expanding stents are designed with a particular expansible range. Thus, after being implanted, self-expanding stents continue to exert pressure against the vessel wall.
Commonly, delivery systems for self-expanding medical devices have a handle arrangement that remains outside of the patient's body during the deployment procedure. One portion of the handle is typically connected to an inner catheter upon which the self-expanding medical device is mounted, and another portion of the handle is typically connected to an outer sheath which restrains the self-expanding medical device in the compressed state. When the distal end of the delivery system is positioned within the patient's body at the intended treatment site, the physician actuates the handle by moving the two portions relative to each other so that the outer sheath is withdrawn from the self-expanding medical device and inner catheter. As a result, the self-expanding medical device expands outward away from the inner catheter. The handle may then be pulled by the physician to withdraw the inner catheter and outer sheath from the patient's body, while leaving the self-expanding medical device implanted in the body.
Precise placement of self-expanding medical devices is a concern in most medical procedures. However, precise placement can be more difficult with certain delivery systems due to their design, shape and other factors. Precise placement of self-expanding medical devices is generally a function of the relative movement and placement between the delivery system handle and the patient's body, and the relative movement between the portions of the handle connected to the inner catheter and outer sheath during deployment. A lack of control over any part of this system can result in inaccurate placement of a self-expanding medical device, and thus, less than desirable treatment of the medical condition being treated.
Accordingly, the inventor believes it would be desirable to provide a new delivery system for self-expanding medical devices.


The present invention relates to a new and distinct variety of rose plant of the hybrid tea class which was originated by me by crossing the variety Baccara, U.S. Plant Pat. No. 1,367 with pollen of the variety South Seas, U.S. Plant Pat. No. 2,184.
The primary objective of this breeding was to produce a new rose variety of the hybrid tea class with the color similar to that of Baccara, primarily a greenhouse cut flower variety, but with larger blooms and better formed blooms. This objective was substantially achieved along with other desirable improvements as evidenced by the following unique combination of characteristics which are outstanding in this new variety and which distinguish it from its parents as well as from all other varieties of which I am aware:
1. A very vigorous, upright, well branched plant.
2. Most flowers on the flowering stems are borne singly with few side buds.
3. Fewer petals than its parent, Baccara.
4. Flower color in overall appearance is near Signal Red with a pronounced yellow area at point of attachment.
5. Very little fragrance.
6. Nearly round leaflets, similar in shape to those of Baccara.
Asexual reproduction of this new cultivar by budding as performed at Wasco, Calif., shows that the foregoing characteristics and distinctions come true to form and are established and transmitted through succeeding propagations.


1. Field of the Invention
The present invention relates generally to the field of locking rings and more particularly to the field of locking rings for electrical energy measuring devices.
2. Background Art
Electrical energy measuring devices, both single and poly-phase, are used to indicate the actual electrical power consumption by a particular customer and are well known. One of the most common of such measuring devices is the electro-mechanical induction-type watt-hour meter which is used to measure actual electrical power consumed by most residential customers. The meter is electrically connected in the electrical power supply wiring to the customer's electrical load wiring network and is located at some convenient, accessible location on the customer's premises for minimum disruption of the customers activity. Periodically the meter is read and the customer is billed or charged for the actual consumption of electrical power.
Because the meter is located on the customer premises for convenient access by a meter reader, it is also accessible by others and thereby subject to possible theft or tampering. Because, for ease and speed of installation, the meter is normally operably mounted by plugging into a mounting base, it is also easy to remove. While theft of a meter by a determined individual probably cannot be economically avoided, some tampering and vandalism may be avoided by a suitable securing locking ring. Such locking ring also serves to operably connect the meter with the mounting base for maintaining proper electrical contact between the electrical bayonet connections of the meter and the mounting base.
In the past such meter locking rings have been formed of a gapped substantially circular metal retainer having a concave securing recess which extended about substantially the entire periphery of the engaged circular flanges of the mounting base and the meter. The metal locking retainer was sufficiently elastically deformable to enable concentric installation and then maintain the adjacent flanges in continuous peripheral sealing contact to exclude dirt and moisture from the mounting base. A soft metal end securing seal clamp is then used to close and seal the gapped ends of the installed retainer ring. Should the end clamp be removed, the impressed soft metal seal is broken and tampering is immediately apparent to the person reading the meter. In the event of an indication of tampering, a false meter reading is immediately suspected and a potential unauthorized power consumption problem identified for proper resolution.
The prior art metal securing clamps are expensive, are subject to corrosion and requires the use of a special tool to emboss or form the tattle-tale tampering seal in the soft metal of the end seal clamp.


Parts formed with several portions are known, for example profiled elements of a turbine engine comprising a main body in a composite material and a metal reinforcement. Upon using the profiled element in operation or during production phases, the reinforcements may be worn or damaged while the main body is protected. Thus, it may be advantageous, in maintenance and in production, to replace the reinforcement while retaining the original main body.
In order not to weaken the main body, it is preferable to reuse the attachment elements (for example a riveting, tapping hole, etc.) already provided on the main body. Such an operation however requires exact localization, on the new reinforcement, of the positions of these attachment elements. Therefore there exists a need for a device or a method for localizing a position on a part.


In media distribution over an IP network, such as video and TV distribution over the Internet, the bandwidth to a client device will vary depending on various circumstances. When accessing the distributed media content over a mobile data network or a Wi-Fi network the capacity is shared between client devices. Further, individual client devices might enter locations with weaker or stronger signal affecting the bandwidth received by the client.
Today, variation in bandwidth as conceived by the client device is typically handled by three mechanisms: congestion control mechanisms of TCP (transmission control protocol of the TCP/IP protocol stack), buffering, and adjusting the video bitrate (ABR). Basically, congestion control is handled by the TCP protocol stack which adjusts the retransmission rate of lost packets to adapt the client device to use a fair share of the available bandwidth in the network (or actually in the bottleneck of the transmission). In such system, the client device needs to buffer data since it is not certain the network can offer enough bandwidth required by the video stream. In order to maintain the viewer experience the client device needs to have video data to present, so buffering is needed to absorb variances in bitrate introduced by the network, specifically by the TCP congestion avoidance mechanisms and the jitter introduced by the network. As the capacity and jitter varies in the IP network, the receiving client device must pause the presentation of the current video on the screen to accumulate more video data in its buffer. The accumulation is one method, meaning that the delay will increase and not decrease for a specific session. Such adjustments will introduce delay and the video cannot be considered live distribution due to the added delay.
According to the European Broadcasting Union, EBU, which defines TV standards in Europe, live TV is defined as a broadcasting delay from the ingress to the client device display or screen lower than seven seconds. Delay caused by the adjustments above may however end up in several minutes of delay.
More specifically, one of the most common ways to distribute video over the Internet is to use HLS (HTTP Live Streaming) or MPEG-DASH where the video stream is divided into typically 10 second (2-10 sec) video files (segments) making the linear video stream a series of 10 second video files. Every stream is typically represented by several bitrates (different video qualities), each being segmented into equivalent segment files. The client device then requests these files using normal http technology. To ensure that the client device always has video data to present, at least 3 time wise consecutive files are buffered in the device. This means that buffering will impose at least 30 seconds of delay. At start-up the buffer is filled up to a certain level, typically 30 seconds, corresponding to three 10 second segments. If packets are lost, the transport protocol TCP used by the HTTP protocol requests the data again and if uncertain if it can recover the whole segment file, also reduces the bitrate on outgoing traffic by requesting the next corresponding segment file of a lower bitrate to avoid congestion.
Over IP networks the bandwidth of a distributed video, i.e. the encoded bitrate of the video, is adjusted to the bandwidth available in the network to the client device. The video is encoded in different predefined bitrates (i.e. the level of compression of the video is differentiated, which in turn provides different quality levels of the video). This is typically done by a transcoding system which takes in an encoded video stream and then “re-encodes” it into one or several video streams with different bitrates, qualities and formats for different devices. The network system decides which encoded bitrate is applicable for the specific moment and selects the most suitable video quality to transfer to the client. In existing solutions, ABR adjustments, i.e. adjustment of the video bitrate/quality, are done based on the fill level of the buffer of the client device. More particularly, the decision to change to another encoded bitrate is done by monitoring the buffer fill level in the client device. A decreasing fill level indicates that the bandwidth of the network is lower than needed and the system needs to select a video with lower quality. Shifting to a higher quality video stream is done by simply testing a higher video bit rate and watching if the buffer fill level decreases. If it decreases, the system needs to go back to a lower video bit rate. This means that there will be continuous changes in ABR levels during the operation with the exception if the system is running on the highest ABR level and the buffer is not becoming empty. To avoid glitches in the video when changing between bit streams of different bit rates, the change is performed between segments, i.e. one start using the corresponding new segment of the new bitrate stream.
Although this approach to perform ABR adjustments of video by monitoring the buffer fill level may be applicable for video on demand services, there is a need for an improved method to perform ABR adjustments for video in IP network systems since the buffering of multiple segments at the device and possibly also in edge caches, and also the continuous adjustments of the ABR level increases the buffer fill level at the device, thereby increasing the delay before the video is displayed in the device. The accumulated delay happens because imperfections of using TCP to determine the network bit rate (which continuously is changing) force the client device to buffer more video data to avoid the buffer to run empty which in turn disrupts the video presentation. Typically, today's OTT systems start with an initial delay of 30-60 seconds depending on encoding delay, segment sizes, distribution network and client player implementation. This accumulates over time and e.g., the HLS protocol allows a client to buffer up to 15 minutes.