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#1由 sufang 在 一, 09/07/2015 – 09:31 發表。
PMID 24768818 Structural Mechanisms Determining InhibitionStructural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors
Peter Canning1, Li Tan2,3, Kiki Chu4, Sam W. Lee4, Nathanael S. Gray2,3 and Alex N. Bullock1
1 – Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK 2 – Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02115, USA 3 – Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA 4 – Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA Correspondence to Nathanael S. Gray and Alex N. Bullock: N. S. Gray is to be contacted at: Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 250 Longwood Avenue, SGM 628, Boston, MA 02115, USA. A. N. Bullock. nathanael_gray@dfci.harvard.edu; alex.bullock@sgc.ox.ac.uk http://dx.doi.org/10.1016/j.jmb.2014.04.014 Edited by M. Guss Abstract The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1.
Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9 nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket.
Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.
Introduction
The discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among the receptor tyrosine kinases (RTKs) in being activated by interaction with the extracellular matrix [1,2]. Binding to triple-helical collagen is mediated by the receptor extracellular domains that include an N-terminal discoidin (DS) domain, a DS-like domain and a short juxtamem- brane (JM) region [3–5]. A single transmembrane helix links to the cytoplasmic domain, where a larger JM region precedes the catalytic C-terminal kinase domain. Both DDRs form constitutive dimers making them unusual among RTKs, which typically dimerize only upon activation [6–8]. DDRs regulate extracelular matrix remodeling, as well as cell adhesion, proliferation and migration [9]. DDR1 is expressed mainly in epithelial cells where it plays an important role in mammary gland development [10], whereas mesenchymal expression of DDR2 promotes bone growth, as suggested by dwarfism in DDR2 knock- out mice [11]. |
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#2由 sufang 在 六, 06/20/2015 – 17:09 發表。
2015/06/20 a copy of Q and ResponseReviewer#1:
Q 3    Is the language, specifically the grammar, of sufficient quality? The paper is quite readable, but would benefit from some English corrections and thorough proofing. For example: (Methods, p5, lines 176‐177) “Photographs of cell spheroids were taken by a microscope” (Response: Photographs of cell spheroids were taken and branch formation was quantitated by methods described in Andeson K et al pmid: 21195708)
Q 25    Other comments on the materials and methods.? Statistical analyses of the microarray datasets and the clinical staging are described, but tests used to analyze the other data are not mentioned. (Response: 補上Fig 1C shDDR1 qRT-PCR, Fig 3C–D 藥物抑制, Fig 4C branch formation)
Q 28    Other comments on the results.
The data are largely of good quality. However, there are some issues, as follows:
1. There are no controls for Fig 1A and Fig S1 (Response: 補一張western於Fig S1, 並強調normal 或是 dysplastic 或是OSCC culture condition不同, 並且DDR1會因density不同表現量差異) 2. Biological/morphological data with DDR1 knockdown should be shown (Response: 考慮補microscope data在Fig 1B下面)
3. The E‐cadherin blot (Fig 2C) does not show a convincing reduction when DDR1 is knocked down. At the very least, this needs to be quantified. Similarly, the migration assay (Fig 4C) should be quantified. (Response: 玉蓮幫忙補quantitation data) 4. Experiments shown in Figs 3 and 4 use the pharmacological inhibitors imatinib and dasatinib. These are not specific for DDR1, yet no mention is made of inhibiting other targets, and effects on Abl or Src do not seem to have been considered. (Response: 補回上一個version那一段話) 5. In Fig 5A, treatment of cells with collagen is reported to increase DDR1 expression. Although the protein levels in HaCaT cells seem to increase, data for the other two cell lines are not convincing (densitometry would help) and it is unclear whether the changes seen in the QPCR experiment are significant (looks like around 20% to 50% increase). (Response:玉蓮幫忙補 Fig 5A quantitation data/ 查清楚Fig 5A lower panel是否有統計意義) 6. On p8, line 303, the authors mention that adjacent normal tissues were used. This is a concern, given the effects of field cancerization. (Response: WHT: 加回tissue microarry data直接比較 normal and OSCC) Q 31 Are the conclusions justified? Most of the conclusions are justified, with the exception of those relying on data in the Results that are unclear (see section above). (Response:
Q 34 Human tissues are used in Figure S3, but no IRB approval is mentioned in the Methods section. (Response: These tissue microarrays were purchased from a US company, Biomax Inc. (http://www.biomax.us/index.php). All patient information have been de-linked thus are fully anonymized. According to our Institutional ethics regulation, these samples do not require IRB approval.)
Reviewer#2:
Q 3    Is the language, specifically the grammar, of sufficient quality? Reviewer 2 | 28 May 2015 | 01:12 #1
The paper is well written, however there are some minor typographic and grammatical errors that should be corrected. Examples: ‐ Current findings of the study or commonly known facts are often written in the past tense. ‐ “expressions” should be replaced with “expression levels”, e.g. lines 305, 315, 338, 540, 607, etc ‐ Line 71: weakening ‐ line 245: Depending ‐ Line 255: associated ‐ Line 282: lesser extent etc (Response: thank you for your corrections, we have corrected all the errors accordingly) Q 6    Is the study presented in a consistent and succinct form? No, Materials and Methods section is incomplete.
Additional materials and methods found in figure legends instead. Passages that belong into the Introduction section are found in the Results section. Passages that belong into the Results section are found in the Discussion section. The headings for each paragraph are not informative and should be rephrased. The descriptions should focus on what has been observed rather than speculative over‐ interpretations. Overall, a very confusing presentation of the data. The information is all over the place and the reader has to search for the relevant information. The aims of the study have not been clearly formulated. (Response: complete additional materials and methods in figure legend into Materials and Methods
Move “introduction” passages in “Results” into “Intrduction”
Move “results” passages in “Discussion” into “Results”
Rephrase “headings” to be more informative.
Focuses on what has been observed rather than speculative over-interprtations.
Aims of the study have not been clearly formatulated)
Q 7 Are there any objective errors in the methodology? If so, please specify. Yes
1. It is unclear why the cell culture medium was changed to a different medium one day before RNA extraction for RTK profiling. Since the culture conditions for the individual cell lines have not been described, it is not possible to evaluate if a change in culture medium could have unexpected effects on gene expression. (Response: culture media for individual cell lines are provided in revised Materials and Methods) 2. It is also not clear why a cDNA pool of multiple cancer cell lines (not specified from which tissues) was used as the control and as a baseline to determine DDR1 overexpression. This should have been done using cDNA from normal oral keratinocyte cell lines.(Response: the aim of PTK profiling is to identify OSCC-specific PTKs, compared to other malignancues, thus common, pooled cDNAs from common malgnancies was used) 3. The sequence of shRNA clones used for DDR1 knockdown has not been described. Furthermore, they have not been tested for off‐target effects. It is therefore impossible to know if the knockdown is specific for DDR1 or if other off‐target effects have contributed to the experimental outcome.(Response: sequences of shDDR1 have now been included; explain adjusted score > 1 in The RNA Consortium) 4. The DDR1 expression analysis has only been performed on OSCC cell lines but not on control normal keratinocytes. It is therefore not clear if DDR1 is overexpressed and to what degree.(Response: try to emphasize not comparable between normal and keratinocytes because cell density and culture component might affact) 5. The used drugs to inhibit DDR1 signalling are not specific for DDR1 but also effect on other RTKs (see literature). Therefore, the conclusions from the experiments need to be reevaluated and more carefully phrased.(Response: list of imatinib-sensitive and dasatinib-sensitive tyrosine kinases) 6. The correlation between DDR1 expression and oral tumor stage should have been statistically analysed using ANOVA and not by multiple t‐tests.(Response: ANOVA has been used to evaluate the correlation between DDR1 expression and pathological staging as described in Methods, Results and Legend of Fig 5C) Q 8    Are there any objective errors in the results? If so, please specify. Yes
The experiments have been performed to a good standard and the figures generally are of good quality. However, some methodological errors could confound the results of the study and the interpretation of the data is occasionally incorrect or too speculative (see comments in individual sections). (Response: please see individual responses in the following) Q 17 Does the title clearly and precisely reflect the findings of the manuscript, as described in the author guidelines? No
The title only refers to the overexpression aspect of DDR1 but does not refer to the other findings of the study or, more importantly, does not send a key message, for example what the role of DDR1 is. (Response: too bad, we don’t have a clear answer yet) Q 21    Other comments on the introduction.
The introduction starts well but is too short and misses some information which can occasionally be found in other sections of the manuscript. The following aspects should be included or moved into the introduction from other sections of the manuscript: ‐ The roles of TGFB1 and collagen in oral submucous fibrosis or oral cancer should be made clearer (more carefully phrased). The cited studies merely show correlations and do not prove a causal effect of TGFB1 on collagen expression. (cite Khan Plos one 那一篇 23284772; ) ‐ A summary of DDR1 isoforms including Protein domains and their known functions (see lines 201‐209 in Results section) ‐ The used cell lines have been published and a reference to their basic charcteristics could be made. ‐ Lines 271‐279 of results section belongs into Introduction. ‐ The concluding paragraph is incomplete, too speculative and somehow disconnected. The purpose of the study could be made clearer. – The following statements need to be referenced: ‐ Lines 59‐60 (Response: OMG)
Q 23 Are the materials and methods sufficiently described to allow others to reproduce the findings? No
1. Not all used cell lines have been described in the M&M section. For example, A431 is missing. Furthermore, the basic characteristics of the used cell lines as well as the individual culture conditions could be briefly summarised rather than just referencing previous papers. It does not help with the interpretation of the results if the material is not described properly. For exampl,e in lines 364‐365 of the Discussion section, the authors mention that three of the analysed cell lines have very different characteristics but they do not elaborate on this and just refer to unpublished results. It is intriguing, for example, that the OC3 cell line shows a mesenchymal cell morphology (non‐keratinocyte) which is interesting considering that this cell line has also very different molecular characteristics. (Response: 2. The PTK profiling could benefit from more details, for example how qPCR data was analysed (for example this information can be found in the figure legend of Table1!) and why the baseline for comparison was a pooled cDNA sample from a range of different cancer cell lines and not from normal keratinocytes (e.g. HOK which have been used for other experiments in this study). The qPCR primers and their properties should be published as supplementary material in order to allow evaluation if these comply with accepted standards for qPCR primer design. For example, using the delta‐delta Ct method with SYBR Green chemistry is questionable unless the primers have been rigorously tested and validated. (Response: patent-related concern) 3. The target sequences for the shRNA constructs need to be made available and there needs to be a comment on specificty and off‐target effects. Could go in Supplement. (Response: TRC sequences will be provided) 4. The substances in which the PTK Inhibitors were dissolved should be mentioned. (Response: vehecial controls??) Q 24    Are the statistical methods used valid? No
The statistical analysis of DDR1 expression with different tumor stages should have been peformed using ANOVA and not multiple t‐tests. (Response: ANOVA has been used to evaluate the correlation between DDR1 expression and pathological staging as described in Methods, Results and Legend of Fig 5C) Q 26    Are the results presented appropriately? No (偉大的reviewer 共17題)
1. It is not clear what the criteria were for stating that the mentioned 5 PTKs were overexpressed. The cDNA control from a pool of various cancer cell lines (what tissues?) is not a good baseline since any of these PTKs might be overexpressed in some of these cancers but not in others. Biological significance can be better evaluated by comparing with normal keratinocytes (see point 2.) (Response: 不不不, 是故意要比cancer cell lines的啊, 要找出OSCC specific or PTKs preferentially expressed in 2.As already mentioned, the western blots showing DDR1 expression should include a normal keratinocyte control to allow the conclusion of DDR1 overexpression (Fig 1 & S1 & S2). a low and high expressing control could ideally be included. (Response: 3. The title “Overexpression of DDR1 contributes to cell growth of oral cancer cells” is misleading and should be changed to “Knockdown of DDR1 induces growth arrest and apoptosis of oral cancer cells”. The argument for oncogene addicition of the studied OSCC cell lines is speculative since knockdown of DDR1 has not been performed in normal oral keratinocytes. For example, if these cells also undergo apoptosis, it can be concluded that DDR1 is simply an important protein for survival of oral keratinocytes. To investigate oncogenic activity, DDR1 should be overexpressed in normal oral keratinocytes. (Response: constitutively active vs collagen-stimulated active) 4. Line 240: “autophosphorylated” ??? or do the authors mean “constitutively phosphorylated”? (Response: 5. The results from the analysis of potential downstream targets of DDR1 signalling (Fig 2c) should be rpoperly explained and discussed. Potential mechanism? (Response: 6. The study concluded that the effect of PTK inhibitory drugs on OSCC cell survival was dosage‐dependent. However,only very high doses have a differential effect in OSCC lines compared to controls. Are these doses physiologically relevant or feasible for therapy? (Response: 7. The authors claim that “among the four OSCC cell lines, OC3 had the lowest amount of DDR1 and was less responsive to inhibitor treatments, suggesting that drug sensitivity is related to the DDR1 expression level.”However, OC3 is sensitive to dasatinib (Fig 3D), and considering the known unspecificity of both PTK inhibitors, this conclusion is not valid. The different behaviour of OC3 cells could be explained by different cellular characteristics of this cell line such as acquirement of different cell fate. This hypothesis is supported by the authors claims that the OC3 cell line has a mesenchymal cell morphology and is also supported by the different molecular data shown in Figure 2c. (Response: 8. Line 263: refer to Figure 3a,b ? (Response: 9. Line 267: Imatinib and dasatinib are not specific “DDR1 Inhibitors” (Response: 10. “DDR1 is involved in collective invasion of OSCC TW2.6 cells.” It should be noted in this paragraph that the other cell lines were also tested but did not exhibit cell cohesiveness. this is only mentioned in the Discussion section but a more comprehensive description would help the reader in understanding why only one cell line has been analysed. (Response: 11. OEC‐M1 cell cohsion data mentioned in text but not shown in Figure 4. (Response: 12. Line 284: replace “actomyosin activity” with “myosin expression”. Is presence of myosin between cells generally accepted as an indicator of loss of cell cohesion? (Response: 13. Lines 289‐290: A cadherin immunostaining experiment would clarify this point. Why has it not been performed? 14. Results and Fig. 5a Collagen stimulation in oral keratinocytes results in minimal increase in DDR1 Expression. is this statistically significant? Why not? (Response: 15. Line 306+307: How can it be explained that only Col4a6 is co‐overexpressed with DDR1 when all collagens seem to stimulate DDR1 expression/activity in normal cells whereas DDR1 activity is independent of collagen stimulation in OSCC cells? What does that mean biologically? (Response: 16. Are the DDR1 expression values shown in Figures 5c and 5d from the microarray or from validated qPCR data? Fig 5c: Data needs to be statisticall analysed using ANOVA. (Response: ANOVA has been used to evaluate the correlation between DDR1 expression and pathological staging as described in Methods, Results and Legend of Fig 5C) 17. Unfortunately, the most exciting part of the study, the immunohistochemical analysis of collagen and DDR1 expression in oral tumours, has been buried as supplementary data (Supplementary figure S3) and has been insufficiently described in the Material and Methods section and the Results section. The images in Figure S3 are displayed at too high magnification yet too small to recognise details and therefore need reworking. Both the epithelial and mesenchymal (stromal) cell compartments should be visible and the images should have the same oientation. The origin of the analysed tissues should be described as well as any other information (e.g. patient data?). It needs to be made clear that increased expression levels of DDR1 positively correlate with tumor grade (if correct!). The source of collagen antibodies needs to be mentioned. I would suggest to include the immunohistochemical analysis in the main manuscript and move other, less important aspects into the supplement (if required). (Response: Q 28 Other comments on the results. 
Notwithstanding the concerns mentioned above, the experiments have been performed well. (被您批評得一無是處了嗚嗚) However, the lack of cohesion is evident. The study would benefit from focused experiments that tie all the experiments together into an interesting story. Focus should be on biological or clinical relevance and conclusions should be less speculative. (Response: Q 29 Does the Discussion address the research questions posed in the Introduction?
No
The aims of the study have not been clearly formulated, however the Discussion makes a good attempt. (Response: Q 30 Does the Discussion interpret the results in light of previous knowledge? No
The Discussion is very short and lacks substance. It is mainly a summary of the Results and Introduction sections. Comparison with DDR1 function in other contexts is sparse. Biological/clinical relevance or mechanistic insights have not been discussed. The links to oral cancer are insufficiently discussed. (Response: Q 31    Are the conclusions justified? No
See examples above. The conclusions are frequently either too far‐fetched and speculative or absent. Relevant experiments could be performed to fill the gaps in knowledge. Overall, there are still too many gaps in this study to conclude that DDR1 could be a therapeutic target in oral cancer. (Response: Q 32    Other comments on the discussion or the conclusions. The Discussion session is very short and lacks substance. It is written as a repetition of the results and does not put the findings into the wider context of the literature. Sometimes it contains new results (e.g. DDR1 gene sequencing) that belong in the Results section. Potential molecular mechanisms have not been discussed. (Response:
Q 40    Please add here any further comments on this manuscript.
Major revision of the manuscript is suggested. (Response: O-Mi-To-Fo)
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#3由 sufang 在 五, 06/19/2015 – 15:53 發表。
2015/02/18 懺悔記2015/02/18 (除夕日) 一晃眼又果三個月了… 只做實驗 不發表paper 是不行的, 是不負責任的的科學家. 慚愧懺悔…
Antibodies and chemicals. Antibodies used in this study were purchased from various companies: p-AKT T308 (#9275), active caspase 3 (#9664), CCND1 (#2926), CDK4 (#2906), CDK6 (#3136), CDKN1A (#2946), CDKN1B (#2552), CDKN2A (#4824), CDKN2B (#4822), E-cadherin (#3195), ERK1/2 (#9107), p-ERK1/2 (#4376), p-MLC (Ser19) (#3671), SHC1 (#2432), SHP2 (#3397), p-SHP2 (#3751) (Cell Signaling, Danvers, MA); AXL (sc-1096), CCNB1 (sc-752), CDK1 (sc-54), CDK2 (sc-163), CDKN1C (sc-1040), DDR1 (sc-532), FGFR3 (sc-123), PARP-1 (sc-8007) (Santa Cruz Biotechnology, Santa Cruz, CA); AKT (#05-796), p-AKT S473 (#05-669), BCL2 (#05-729), CCNE2 (#04-223), α-tubulin (#05-829), p-SHC (#07-209), p-tyrosine 4G10 (#16-316) (Millipore, Billerica, MA); N-cadherin (#610920), STAT3 (#610189) (BD Biosciences, San Jose, CA); β-actin (A5441), EGFR (E3138) (Sigma-Aldrich, St. Louis, MO); collagen I (ab34710) (Abcam, Cambridge, UK); collagen IV (M0785) (DAKO, Glostrup, Denmark); p-STAT3 (#2236-1) (Epitomics, Burlingame, CA); GAPDH (GTX100118) (GeneTex, Hsinchu, Taiwan). Imatinib mesylate salt was provided by Novartis (Basel, Switzerland) and dasatinib was purchased from Selleckchem (Houston, TX).
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Two and half μg total RNAs was subjected to reverse transcription in a 10 μl SuperScript III reaction mixtures according to the manufacturer’s instructions (Invitrogen). Each real-time PCR reaction was composed of 2 μl diluted cDNA, 3 μl primer mix (0.66 μM) and 5 μl Power SYBR Green Master. The reaction was conducted and detected by Step One Plus Real-Time PCR system (Applied Biosystems).
Spheroid invasion assays. A431 cells (5,000 cells/well) and TW2.6 cells (1,500 cells/well) were seeded in 1% agar-coated U-bottom 96 well plates and incubated for 48 h to allow aggregation into multiple individual spheroids. To observe spheroids invasion in 3D collagen matrix, 2.4 mg/ml bovine type I collagen was first neutralized with 10× PBS (1/10 V) and 0.1 N NaOH (1/10 V) to reach pH 7.5. About 15-25 spheroids were suspended in serum free medium and mixed with neutralized collagen at 1:1 ratio (V/V). Cell–matrix mixture was placed in 24 well plates and incubated for 30 min at 37°C followed by adding culture media containing 10 μM imatinib or 0.1 μM dasatinib on top of the cell–matrix mixtures for 2 – 9 d. Photographs of cell spheroids were taken by a microscope.
Figure 4. DDR1 is involved in collective cell migration of A431 and TW2.6 cells. (A–B) Confocal microscopy cell images of phosphorylated myosin light chain (p-MLC) staining in A431 (A) and TW2.6 (B) cells. Multi-cell cohesiveness, as evident by decreased p-MLC activity at cell-cell contacts, was observed in the control cells (shLuc and vehicle control, VC). By contrast, cell cohesiveness was disrupted in cells treated with DDR1 shRNA or inhibitors (10 μM imatinib and 0.1 μM dasatinib, respectively), indicating that DDR1 is involved in cell-cell cohesion. (C–D) Three-dimensional reconstruction of A431 (C) and TW2.6 (D) cell spheroids embedded in collagen I gel invasion assays. Fifteen to twenty-five spheroids suspended in serum free medium mixed with neutralized collagen were placed in 24-well plates and incubated for 30 min at 37°C. Cell culture media with or without DDR1 inhibitors were added carefully on top of the cell–matrix mixtures and cultured for additional 2–9 d. In the vehicle control cells, arrowheads indicate collective cell invasion. Photographs of cell spheroids were taken by a microscope.
2015/04/27 來投吧! 加油~
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#4由 sufang 在 六, 09/20/2014 – 14:28 發表。
Frontiers in oncologyAuthor Guidelines: http://www.frontiersin.org/oncology/authorguidelines
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#5由 sufang 在 三, 08/06/2014 – 10:31 發表。
DDR1 Inhibitors相關1 Bantscheff, M., D. Eberhard, Y. Abraham, S. Bastuck, M. Boesche, S. Hobson, T. Mathieson, J. Perrin, M. Raida, C. Rau, V. Reader, G. Sweetman, A. Bauer, T. Bouwmeester, C. Hopf, U. Kruse, G. Neubauer, N. Ramsden, J. Rick, B. Kuster, and G. Drewes. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. (2007). Nat Biotechnol. 25: 1035-44. pdf 3805. DDR1 inhibitor (cited by J Proteomics pdf 3217 ref #22) We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.
2 Rix, U., O. Hantschel, G. Durnberger, L. L. Remsing Rix, M. Planyavsky, N. V. Fernbach, I. Kaupe, K. L. Bennett, P. Valent, J. Colinge, T. Kocher, and G. Superti-Furga. Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets. (2007). Blood. 110: 4055-63. pdf 2687 The BCR-ABL tyrosine kinase inhibitor imatinib represents the current frontline therapy in chronic myeloid leukemia. Because many patients develop imatinib resistance, 2 second-generation drugs, nilotinib and dasatinib, displaying increased potency against BCR-ABL were developed. To predict potential side effects and novel medical uses, we generated comprehensive drug-protein interaction profiles by chemical proteomics for all 3 drugs. Our studies yielded 4 major findings: (1) The interaction profiles of the 3 drugs displayed strong differences and only a small overlap covering the ABL kinases. (2) Dasatinib bound in excess of 30 Tyr and Ser/Thr kinases, including major regulators of the immune system, suggesting that dasatinib might have a particular impact on immune function. (3) Despite the high specificity of nilotinib, the receptor tyrosine kinase DDR1 was identified and validated as an additional major target. (4) The oxidoreductase NQO2 was bound and inhibited by imatinib and nilotinib at physiologically relevant drug concentrations, representing the first nonkinase target of these drugs.
3 Day, E., B. Waters, K. Spiegel, T. Alnadaf, P. W. Manley, E. Buchdunger, C. Walker, and G. Jarai. Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation by imatinib, nilotinib and dasatinib. (2008). Eur J Pharmacol. 599: 44-53. pdf 2679 Imatinib, nilotinib and dasatinib are protein kinase inhibitors which target the tyrosine kinase activity of the Breakpoint Cluster Region-Abelson kinase (BCR-ABL) and are used to treat chronic myelogenous leukemia. Recently, using a chemical proteomics approach another tyrosine kinase, the collagen receptor Discoidin Domain Receptor1 (DDR1) has also been identified as a potential target of these compounds. To further investigate the interaction of imatinib, nilotinib and dasatinib with DDR1 kinase we cloned and expressed human DDR1 and developed biochemical and cellular functional assays to assess their activity against DDR1 and the related receptor tyrosine kinase Discoidin Domain Receptor2 (DDR2). Our studies demonstrate that all 3 compounds are potent inhibitors of the kinase activity of both DDR1 and DDR2. In order to investigate the question of selectivity among DDR1, DDR2 and other tyrosine kinases we have aligned DDR1 and DDR2 protein sequences to other closely related members of the receptor tyrosine kinase family such as Muscle Specific Kinase (MUSK), insulin receptor (INSR), Abelson kinase (c-ABL), and the stem cell factor receptor (c-KIT) and have built homology models for the DDR1 and DDR2 kinase domains. In spite of high similarity among these kinases we show that there are differences within the ATP-phosphate binding loop (P-loop), which could be exploited to obtain kinase selective compounds. Furthermore, the potent DDR1 and DDR2 inhibitory activity of imatinib, nilotinib and dasatinib may have therapeutic implications in a number of inflammatory, fibrotic and neoplastic diseases.
4 Grimminger, F., R. T. Schermuly, and H. A. Ghofrani. Targeting non-malignant disorders with tyrosine kinase inhibitors. (2010). Nature reviews. Drug discovery. 9: 956-70. (pdf 4134)
Receptor and non-receptor tyrosine kinases are involved in multiple proliferative signalling pathways. Imatinib, one of the first tyrosine kinase inhibitors (TKIs) to be approved, revolutionized the treatment of chronic myelogenous leukaemia, and other TKIs with different spectra of kinase inhibition are used to treat renal cell carcinoma, non-small-cell lung cancer and colon cancer. Studies also support the potential use of TKIs as anti-proliferative agents in non-malignant disorders such as cardiac hypertrophy, and in benign-proliferative disorders including pulmonary hypertension, lung fibrosis, rheumatoid disorders, atherosclerosis, in-stent restenosis and glomerulonephritis. In this Review, we provide an overview of the most recent developments–both experimental as well as clinical–regarding the therapeutic potential of TKIs in non-malignant disorders.
5 Rix, U., L. L. Remsing Rix, A. S. Terker, N. V. Fernbach, O. Hantschel, M. Planyavsky, F. P. Breitwieser, H. Herrmann, J. Colinge, K. L. Bennett, M. Augustin, J. H. Till, M. C. Heinrich, P. Valent, and G. Superti-Furga. A comprehensive target selectivity survey of the BCR-ABL kinase inhibitor INNO-406 by kinase profiling and chemical proteomics in chronic myeloid leukemia cells. (2010). Leukemia. 24: 44-50. pdf 2686, DDR1 inhibitor 2/2. Resistance to the BCR-ABL tyrosine kinase inhibitor imatinib poses a pressing challenge in treating chronic myeloid leukemia (CML). This resistance is often caused by point mutations in the ABL kinase domain or by overexpression of LYN. The second-generation BCR-ABL inhibitor INNO-406 is known to inhibit most BCR-ABL mutants and LYN efficiently. Knowledge of its full target spectrum would provide the molecular basis for potential side effects or suggest novel therapeutic applications and possible combination therapies. We have performed an unbiased chemical proteomics native target profile of INNO-406 in CML cells combined with functional assays using 272 recombinant kinases thereby identifying several new INNO-406 targets. These include the kinases ZAK, DDR1/2 and various ephrin receptors. The oxidoreductase NQO2, inhibited by both imatinib and nilotinib, is not a relevant target of INNO-406. Overall, INNO-406 has an improved activity over imatinib but a slightly broader target profile than both imatinib and nilotinib. In contrast to dasatinib and bosutinib, INNO-406 does not inhibit all SRC kinases and most TEC family kinases and is therefore expected to elicit fewer side effects. Altogether, these properties may make INNO-406 a valuable component in the drug arsenal against CML.
6 Hammerman, P. S., M. L. Sos, A. H. Ramos, C. Xu, A. Dutt, W. Zhou, L. E. Brace, B. A. Woods, W. Lin, J. Zhang, X. Deng, S. M. Lim, S. Heynck, M. Peifer, J. R. Simard, M. S. Lawrence, R. C. Onofrio, H. B. Salvesen, D. Seidel, T. Zander, J. M. Heuckmann, A. Soltermann, H. Moch, M. Koker, F. Leenders, F. Gabler, S. Querings, S. Ansen, E. Brambilla, C. Brambilla, P. Lorimier, O. T. Brustugun, A. Helland, I. Petersen, J. H. Clement, H. Groen, W. Timens, H. Sietsma, E. Stoelben, J. Wolf, D. G. Beer, M. S. Tsao, M. Hanna, C. Hatton, M. J. Eck, P. A. Janne, B. E. Johnson, W. Winckler, H. Greulich, A. J. Bass, J. Cho, D. Rauh, N. S. Gray, K. K. Wong, E. B. Haura, R. K. Thomas, and M. Meyerson. Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer. (2011). Cancer discovery. 1: 78-89. pdf not yet While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials. SIGNIFICANCE: DDR2 mutations are present in 4% of lung SCCs, and DDR2 mutations are associated with sensitivity to dasatinib. These findings provide a rationale for designing clinical trials with the FDA-approved drug dasatinib in patients with lung SCCs.
7 Lemeer, S., A. Bluwstein, Z. Wu, J. Leberfinger, K. Muller, K. Kramer, and B. Kuster. Phosphotyrosine mediated protein interactions of the discoidin domain receptor 1. (2011). J Proteomics. 75: 3465-77. pdf 3217 in BB2012Mar. The receptor tyrosine kinase DDR1 has been implicated in multiple human cancers and fibrosis and is targeted by the leukemia drug Gleevec. This suggests that DDR1 might be a new therapeutic target. However, further insight into the DDR1 signaling pathway is required in order to support its further development. Here, we investigated DDR1 proximal signaling by the analysis of protein-protein interactions using proteomic approaches. All known interactors of DDR1 were identified and localized to specific phosphotyrosine residues on the receptor. In addition, we identified numerous signaling proteins as new putative phosphotyrosine mediated interactors including RasGAP, SHIP1, SHIP2, STATs, PI3K and the SRC family kinases. Most of the new proteins contain SH2 and PTB domains and for all interactors we could directly point the site of interaction to specific phosphotyrosine residues on the receptor. The identified proteins have roles in the early steps of the signaling cascade, propagating the signal from the DDR1 receptor into the cell. The map of phosphotyrosine mediated interactors of DDR1 created in this study will serve as a starting point for functional investigations which will enhance our knowledge on the role of the DDR1 receptor in health and disease.
8 Montero, J. C., S. Seoane, A. Ocana, and A. Pandiella. Inhibition of SRC family kinases and receptor tyrosine kinases by dasatinib: possible combinations in solid tumors. (2011). Clin Cancer Res. 17: 5546-52. Dasatinib is a small molecule tyrosine kinase inhibitor that targets a wide variety of tyrosine kinases implicated in the pathophysiology of several neoplasias. Among the most sensitive dasatinib targets are ABL, the SRC family kinases (SRC, LCK, HCK, FYN, YES, FGR, BLK, LYN, and FRK), and the receptor tyrosine kinases c-KIT, platelet-derived growth factor receptor (PDGFR) alpha and beta, discoidin domain receptor 1 (DDR1), c-FMS, and ephrin receptors. Dasatinib inhibits cell duplication, migration, and invasion, and it triggers apoptosis of tumoral cells. As a consequence, dasatinib reduces tumoral mass and decreases the metastatic dissemination of tumoral cells. Dasatinib also acts on the tumoral microenvironment, which is particularly important in the bone, where dasatinib inhibits osteoclastic activity and favors osteogenesis, exerting a bone-protecting effect. Several preclinical studies have shown that dasatinib potentiates the antitumoral action of various drugs used in the oncology clinic, paving the way for the initiation of clinical trials of dasatinib in combination with standard-of-care treatments for the therapy of various neoplasias. Trials using combinations of dasatinib with ErbB/HER receptor antagonists are being explored in breast, head and neck, and colorectal cancers. In hormone receptor-positive breast cancer, trials using combinations of dasatinib with antihormonal therapies are ongoing. Dasatinib combinations with chemotherapeutic agents are also under development in prostate cancer (dasatinib plus docetaxel), melanoma (dasatinib plus dacarbazine), and colorectal cancer (dasatinib plus oxaliplatin plus capecitabine). Here, we review the preclinical evidence that supports the use of dasatinib in combination for the treatment of solid tumors and describe various clinical trials developed following a preclinical rationale.
9 Canning, P., L. Tan, K. Chu, S. W. Lee, N. S. Gray, and A. N. Bullock. Structural mechanisms determining inhibition of the collagen receptor DDR1 by selective and multi-targeted type II kinase inhibitors. (2014). J Mol Biol. 426: 2457-70. pdf 4135
J Mol Biol. 2014 Jun 26;426(13):2457-70. The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and alphaD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a beta-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer. |
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#6由 sufang 在 二, 08/05/2014 – 13:36 發表。
代Maruko移轉NAS上資料DDR1 Paper Candidates: 2014/08/05
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#7由 maruko 在 五, 08/08/2014 – 10:43 發表。
老闆我後來挑中的是:1.Mol Cell Biol http://mcb.asm.org/ IF=5.0
BIOCHEMISTRY & MOLECULAR BIOLOGY (55/291)
CELL BIOLOGY (50/185)
BIOCHEMISTRY & MOLECULAR BIOLOGY (65/291)
不知您覺得如何 若您沒特別意見 我就從中選一本
由於JBC中 刊登DDR1相關的paper較多 所以我應該會選他
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#8由 maruko 在 四, 04/24/2014 – 10:36 發表。
DDR1 MS Figures回應Figure 1. 會加入Table1的數據
Figure 5. 我是要說這柱狀圖的數據的n=4(來自two independent experiments, 每次實驗各兩重複)
Figure 6. a.2011年的DDR1-collective migration的paper的論述是說DDR1的功能是將細胞與細胞間p-MLC維持在低低的狀態 以利細胞間的cohesive 所以一但將DDR1拿掉 細胞與細胞間的p-MLC activity就會上升 另外作者有其他data佐證 他在3D culture的確都可以看到cancer cell collective migration現象 (而在3D culture下也可以染到p-MLC一圈的現象) 我會再找找是否有可以加強這張圖的data 會再寫過figure legends
b.我當時沒進行OC3 treated imatinib後的p-MLC染色
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#9由 EVKVLIN 在 五, 04/18/2014 – 12:14 發表。
Referee #1的所有concernHowever, the conclusions are not supported by the evidence. Results were over interpreted and the conclusion overreaching. Figures are not convincing.
Major points
1. Methods and reagents are poorly described. –DDR1 antibody used (catalog number, type and epitope) and its concentration in the immunohistochemistry were not described. (舊版material/method有抗體之cat#, DDR1 epitope?)
–Sequences of the shRNAs should have been provided.
–In Figures 1, 2 and 3, blots of DDR1 expression and phosphorylation should be shown in their entirety. (OK now)
–The proliferation assays of Figure 1 were not described in the methods. (OK now)
–The collagen stimulation and DDR1 phosphorylation methods should have been described in detail and not cited by a reference, which does not deal with DDR1 conditions. It is therefore difficult to evaluate the reliability of the authors’ conclusions. (OK now)
–Figure 2. Please show %stats and p values to prove significance of actual cell death vs. viable cells. Decreased cell growth is evident, but apoptosis is not clear in shDDR1 cells. (OK now)
–Figure 2 b: Student t-test was performed on duplicated data. However, Student t test data is performed when n {greater than or equal to} 3. Also is mentioned in the figure legend that shLuc control group is set to 100%. However, this is not indicated in the figure. (OK now)
–Based on results authors drawn a conclusion that a pro survival role of DDR1 is common to all OSCC cell lines tested. However, their conclusion cannot be justified with SCC-15 cell line. (OK now)
– Figure 3. This is a very difficult figure to understand, as described. These data are not convincing without a detailed protocol of the experimental conditions. Moreover, these results are unclear because there is no evidence that high level of DDR1 expression results in constitutive receptor activation. Indeed, T47D express very high levels of DDR1. TW2.6 does not appear to be constitutively active. The CM lane shows strong phosphorylation, whereas the (-) collagen serum starved lane shows no phosphorylation. These two lanes should be approximately equal in band size/intensity. Explain what is meant by “+ or +/- phosphorylation state.” It is mentioned that the DDR1 expression level for OC3 cell line is relatively low compared to the other cell lines used. However, in this figure compared to figures 1a and b this is not the case. Therefore the conclusions that DDR1 in OSCC cells is ligand-independent and is constitutively active are not supported by the evidence provided. (OK now)
–Figure 4. These data are not well described and therefore unconvincing. #For instance, phosphorylation of SHP2 in C9 or OC3 cell even in shLuc lanes cannot be seen. A faint band of p-SHP2 is found in shDDR1 lane of C9. This does not correlate to the claim that SHP2 is “consistently” lower in DDR1 knockdown cells. Also does not point out why SHP2 total levels increase when DDR1 is knocked-down, but phosphorylation is still decreased.
#p-STAT3 is also claimed to be increased with a decrease in p-SHP2. This is only clear in TW2.6 cell line. p-STAT3 is actually decreased in shDDR1 lane of OEC-M1 even though the p-SHP2 is decreased. OC3 doesn’t seem to have any p-SHP2, so a related p-STAT2 claim cannot be made there, as well as the same in C9. A strong p-STAT3 band is shown in shDDR1 C9, but a faint band is also shown in p-SHP2, whereas no band is detectable in the shLuc for p-SHP2. Results seem to be inconsistent with claims.
#Not convinced with lower p-SHC1 claim. Consider better/clearer western blot.
#Lower p-ERK1/2 is only clear in TW2.6, but not the other cell lines. Cannot make a significant claim to such a pathway.
#Noticeable E-Cad decreased levels only evident in OEC-M1. Consider a better/clearer western blot, or scanning analysis.
#CDK6 is not lowered in “majority” of cell lines. Only evident in OC3
–The conclusion that DDR1 is the highest kinase expressed in these cell lines has not been convincingly demonstrated; therefore the inhibitor studies are questionable. Moreover, Imatinib and Dasatinib are broad range spectrum drugs and will most likely affect other pathways of the cell that can lead to similar results. This does not prove DDR1 is solely responsible for decreased cell growth in a dose dependent manner.
#In Figure 5c, STAT3 phosphorylation data for TW2.6 goes against the conclusions.
#For OEC-M1 and TW2.6 no significant decrease in phosphorylation for the knockdowns is shown.
#The conclusion that “in addition to the kinase activity, other structural domains of DDR1 may also contribute to cell fate determination” has not data and mechanistic bases and thus seems to be a total speculation, especially using non-specific inhibitors. Thus, the data and conclusions of Figure 6 are questionable. –The immunohistochemical data are not convincing. The specificity and quality of the staining is unclear.
–In Figure 7c: TGFB1 and Collagen data in T/N in addition to DDR1 should have been shown.
–Page 12: “Thus, DDR1 is likely to be the final effecter of the TGFB1-collagen accumulation axis, which transmits and executes oncogenic processes in oral epithelial cells.” This claim is over-reaching as there is no experimental proof to back it up. –Supplement Figure 1. How are DDR1a and DDR1b differentiated in the western blot from the other isoforms when the antibody recognizes all isoforms? |
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#10由 EVKVLIN 在 一, 04/28/2014 – 08:49 發表。
Referee #2 的所有concernThe authors showed that expression of discoidin domain receptor-1 (DDR1) was elevated in tissue specimens from patients with oral squamous cell carcinoma (OSCC) and in OSCC cell lines. Depletion of DDR1 by shRNA induced activation of caspase-3 which indicated the increased apoptosis. Depletion of DDR1 also affected a series of cellular factors that are associated with cancer progression. The study shows a novel role of DDR1 in OSCC progression. However, there are some major concerns about this study.
Major concerns: 1. The authors presented too many unsorted data especially lots of western blots throughout the manuscript, but did not provide interpretations about those data, how they can support the main aims. For example: many blots showed expression of cell cycle regulators in OSCC cells transfected with control-shRNA or DDR1-shRNA, but the authors did not provide mention why DDR1 depletion induced expression of some cell cycle regulators but reduced expression of the similar cell cycle regulators. There are many overstatements and over-interpretation throughout the manuscript. 2. The authors did not provide direct evidence on that imatinib or dasatinib caused severe cell growth arrest was associated with its inhibition of DDR1. Unless they show that imatinib or dasatinib directly inhibit DDR1 phosphorylation kinetics. 3. The authors state that DDR1 overexpression is associated with pathogenesis of OSCC, but they did not introduce overexpression of DDR1 into OSCC cells. The overexpression experiments need to be performed to support their statement about the role of DDR1. 4. The conclusion on that (page 4-5) “overexpression of DDR1 is also a hallmark of oral cancer cells” is overstated. They only analyzed 40 patients by immunohistochemical analysis and observed that expression levels of DDR1 expression and collagens were elevated in cancer specimens compared with normal tissues. No functional study using cell line systems to examine whether overexpression of DDR1 may increase tumor cell growth and survival. 5. Materials and methods section, experimental procedures need to be described in great details. No description about how cell cycle analysis is performed. How cell growth in Figure 1 is determined. (OK now) 6. Figure 1. Figure 1b, It is not clear how cell growth is determined. Stringent methods using flow cytometry analysis to determine viable cells are required. 7. Figure 2a. It is not appropriate to use trypin-blue to measure the rate of apoptosis. Statistical calculations need to be indicated. (OK now) 8. Figure 3a. It is difficult to interpreted Figure 3a. The authors stated that Four OSCC cell lines were treated with collagen 1 for 18 hours to increase DDR1 expression. The authors did not explain how this experiment is performed. The authors did not use specific antibody against tyrosin-phosphorylated DDR1. It is not appropriate to use word “overexpressed DDR1” to describe that DDR1 expression is increased after treatment with collagen 1. It is difficult to understand the conclusion from the authors: “overexpressed DDR1 in OSCC cells was constitutively active, regardless of collagen stimulation”. The quality of bands is poor. Semi-quantification of bands of western blots in Figure 3 needs to be performed to show whether increase in DDR1 expression is significant in these cell lines. Figure 4b. I am surprised that DDR1 shRNA depletion reduced CDK1 expression, but increased CDK2, cyclin D1 expression which are the key cell cycle regulators to promote cell proliferation. This indicates that DDR1 depletion may have positive effect on cell growth. To clarify this issue, the authors need to perform Brdu-based proliferation assay using cells transfected with Sh-control RNA and ShRNA-DDR1. These data are discrepancy to the data showing that DDR1 depletion reduced cell growth and increased apoptosis in these cancer cell lines. Figure 5. It is not appropriate to state that Imatinib and dasatinib are DDR1 inhibitor. The only data which is relevant to the statement was that treatment of cells with Imatinib or dasatinib inhibited phosphor-tyr expression in DDR1-associated immunocomplex. It is not known whether these two kinase inhibitors indeed inhibited DDR1 phosphorylation or expression. The authors did not provide sufficient evidence to support their statement. The quality of Figure 5C is poor. The result from Figure 5C is inclusive. Why the authors did not perform same experiments using cells which have been treated for 24 or 48 hours to examine both P-Tyr and expression of DDR1. Figure 6a and b. The quality of upper panels of Figure 6a and b are poor. The statistical significance from three independent experiments should be indicated in Figure 6a and b. (OK now) #1由 EVKVLIN 在 四, 04/17/2014 – 09:17 發表。
CDDis的二位Reviewer之critiques
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