c-fos 和 dusp1 在癌基因依赖性中的作用评价方法
Summary
在这里, 我们描述了 c-fos 和 dusp1 作为白血病药物靶标的遗传和化学验证协议, 在体外和体内使用遗传和人性化的小鼠模型。该方法可应用于遗传验证和治疗发展的任何目标。
Abstract
酪氨酸激酶抑制剂 (tki) 在治疗慢性髓系白血病 (cml) 方面的示范预示着癌症治疗的一个新时代。然而, 少数细胞对 tki 治疗没有反应, 导致残留疾病最小;即使是最强大的 tki 也无法消灭这些细胞。这些 mrd 细胞作为对治疗产生抵抗力的水库。为什么 tki 治疗对 mrd 细胞无效尚不清楚。生长因子信号转导与 tki 治疗过程中支持 mrd 细胞存活有关, 但缺乏机械理解。最近的研究表明, 由于 mrd 细胞中收敛的致癌和生长因子信号, c-fos 和 dusp1 表达升高, 可以介导 tki 的抗性。c-fos 和 dusp1 的遗传和化学抑制使 cml 对 tki 非常敏感, 并在遗传和人性化的小鼠模型中治愈 cml。我们使用来自 tki 敏感和耐药细胞的多个微阵列确定了这些目标基因。在这里, 我们提供了在体外和体内使用小鼠模型进行目标验证的方法。这些方法可以很容易地应用于任何目标的遗传验证和治疗发展。
Introduction
bcr-abl1 融合癌基因的本构酪氨酸激酶活性引起 cml, 为小分子抑制剂靶向激酶活性提供了依据。tki 在治疗 cml 患者方面的成功彻底改变了靶向治疗的概念1,2。随后, 抗激酶治疗作为精确的医学被开发了为其他几个恶性肿瘤, 包括实体肿瘤。到目前为止, 已有30多种激酶抑制剂被美国 fda 批准用于治疗各种恶性肿瘤。虽然 tki 治疗对抑制疾病非常有效, 但并不治疗。此外, 在治疗过程中, 少量癌细胞持续存在: mrd3、4、5。即使是表现出完全缓解的患者, 也会留下 mrd, 如果不持续抑制, 最终会导致复发。因此, 需要根除 mrd 细胞, 以实现持久或治疗性的反应。cml 代表了一个有价值的范式, 用于定义精确医学的概念、肿瘤发生的机制、合理的靶向治疗、疾病进展和耐药性。然而, 即使在今天, 导致 tki 诱导的癌细胞死亡的机制还没有完全了解, 也没有完全了解为什么 mrd 细胞 (由白血病干细胞组成) 对 tki4,6具有内在的耐药性。然而, “癌基因依赖” 突变激酶蛋白的现象与 tki 的有效性有关, tki 对靶向癌基因的急性抑制会导致致癌休克, 导致细胞出现大规模的原凋亡反应或静止上下文相关的方式6,7,8,9。然而, 癌基因依赖的机械基础是缺乏的。最近的研究表明, 生长因子信号减少了癌基因的依赖性, 从而导致抵抗 tki 治疗10,11,12。因此, 为了深入了解癌基因依赖的机制, 我们从 bcr-abl1 成瘾和非成瘾细胞 (随生长因子生长) 进行了全基因组表达谱分析, 揭示了 c-fos 和 dusp1 是犯罪基因依赖的关键介质。癌基因成瘾13。c-fos 和 dusp1 的遗传缺失对 bcr-abl1 表达细胞是合成致死的, 实验中使用的小鼠没有发生白血病。此外, 小分子抑制剂对 c-fos 和 dusp1 的抑制作用, 治愈了 bcr-abl1 诱导的小鼠 cml。结果表明, c-fos 和 dusp1 的表达水平定义了癌细胞的凋亡阈值, 因此较低的水平会产生药物敏感性,而较高的水平会导致对治疗的抵抗力13。
为了确定导致癌基因依赖性的基因, 我们在生长因子存在的情况下进行了几次全基因组表达分析实验, 并使用了鼠肉和 cml 患者衍生细胞 (k562)。这些数据与 cml 患者数据集在伊马替尼治疗前后从 cd34+造血干细胞中获得的数据进行了并行分析。这一分析揭示了三个基因 (转录因子 [c-fos]、双特异性磷酸酶 1 [dusp1] 和 rna 结合蛋白 [zfp36]) 通常在 tki 耐药细胞中被凝聚。为了验证这些基因在赋予耐药方面的意义, 我们进行了体外和体内的逐步分析。通过实时 qpcr (rt-qpcr) 和耐药细胞中的西方印迹证实了这些基因的表达水平。此外, cdna 过度表达和击倒的 shrna 发夹的 c-fos, dusp1 和 zfp36 揭示了升高的 c-fos 和 dusp1 表达是足够和必要的, 以赋予 tki 的抵抗力。因此, 我们仅使用 c-fos 和 dusp1 的小鼠模型进行了体内验证。为了对 c-fos 和 dusp1 进行基因验证, 我们创造了 rosacreert 诱导的 c-fosf函(条件淘汰赛)14 , 并与 dusp1-p/(直接淘汰赛)15交叉, 使 rosacreert2-c-fosfl双转基因 小鼠。在一个菌落形成单元 (cfu)中, 对表达 bcr-abl1的骨髓衍生的 c-kit + 细胞 (来自 c-fos fl/fl–/—和 c-fos fusp1-\-) 进行了体外分析, 活体骨髓移植在小鼠体内, 单独或共同检测白血病发展中对 c-fos 和 dusp1 的要求。同样, 用 bcr-abl1 表示, 用 bcr-abl1表达, 用 dfc (二氟姜黄素) 16 和 dusp1 对 c-fos 的化学抑制, 用 bci (苯并二己胺)-2 , 3-二氢-1h-1) 17 在体外和体内进行了测试。来自野生类型 (wt) 鼠标的骨髓衍生的 c-kit+细胞。为了确认白血病干细胞中对 c-fos 和 dusp1 的要求, 我们利用 cml 小鼠模型, 通过多西环素 (tet-转动剂在小鼠干细胞白血病 (scl) 基因 3 ‘ 基因下表达) 在其干细胞中特异性诱导 bcr-abl1章程)18,19。我们在体内移植试验中使用了这些小鼠的骨髓林-sca+c-kit+ (lsk) 细胞。此外, 我们还建立了 phopsho-p38 水平和 il-6 的表达, 分别作为药物动态生物标志物,分别用于 dusp1 和 c-fos 的抑制。最后, 为了扩大这项研究对人类的相关性, 患者衍生的 c-Kit+细胞 (相当于小鼠的 c-kit+细胞) 接受了长期的体外培养细胞检测 (ltcic) 和体内人性化的小鼠模型。cml20,21。免疫缺陷小鼠移植 cml cd34 + 细胞, 其次进行药物治疗和人白血病细胞存活分析。
在这个项目中, 我们开发了使用遗传和化学工具进行目标识别和验证的方法, 使用不同的临床前模型。这些方法可以成功地应用于验证其他目标开发化学模式的治疗发展。
Protocol
Representative Results
Discussion
对于大部分癌细胞, 对 tki 的治疗反应是通过阻断肿瘤所依赖的酪氨酸激酶-onco蛋白信号来介导的。然而, 对少数促成 mrd 的癌细胞如何摆脱癌基因依赖和治疗的了解相对较少4。最近的研究表明, 生长因子信号介导白血病和实体器官肿瘤的耐药性。这表明, 各种分子机制可能是内在阻力10、11、12 的基础。为了了?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢 g. q. dley 为 mscv-bcr-abres-yefp 结构提供了 baf3 和 wehi 细胞和 t. reya。作者感谢 m. carroll 提供了 cml 爆炸危机的病人样本。这项研究得到了 nci (1ro1ca155091)、白血病研究基金会和 v 基金会以及 nhlbi (R21HL114074-01) 向硕士提供的赠款的支持。
Materials
| Biological Materials | |||
| RPMI | Cellgro (corning) | 15-040-CV | |
| DMEM | Cellgro (corning) | 15-013-CV | |
| IMDM | Cellgro (corning) | 15-016-CVR | |
| RetroNectin Recombinant Human Fibronectin Fragment | Takara | T100B | |
| MethoCult GF M3434 (Methylcellulose for Mouse CFU) | Stem Cell | 3434 | |
| MethoCult H4434 Classic (Methylcellulose for Human CFU) | Stem Cell | 4434 | |
| 4-Hydroxytamoxifen | Sigma | H6278 | |
| Recombinant Murine SCF | Prospec | CYT-275 | |
| Recombinant Murine Flt3-Ligand | Prospec | CYT-340 | |
| Recombinant Murine IL-6 | Prospec | CYT-350 | |
| Recombinant Murine IL-7 | Peprotech | 217-17 | |
| DFC | LKT Laboratories Inc. | D3420 | |
| BCI | Chemzon Scientific | NZ-06-195 | |
| Imatinib | LC Laboratory | I-5508 | |
| Curcumin | Sigma | 458-37-7 | |
| NDGA | Sigma | 500-38-9 | |
| Penn/Strep | Cellgro (corning) | 30-002-CI | |
| FBS | Atlanta biological | S11150 | |
| Trypsin EDTA 1X | Cellgro (corning) | 25-052-CI | |
| 1XPBS | Cellgro (corning) | 21-040-CV | |
| L-Glutamine | Cellgro (corning) | 25-005-CL | 5mg/ml stock in water |
| Puromycin | Gibco (life technologies) | A11138-03 | |
| HEPES | Sigma | H7006 | |
| Na2HPO4.7H2O | Sigma | S9390 | |
| Protamine sulfate | Sigma | P3369 | 5mg/ml stock in water |
| Trypan Blue solution (0.4%) | Sigma | T8154 | |
| DMSO | Cellgro (corning) | 25-950-CQC | |
| WST-1 | Roche | 11644807001 | |
| 0.45uM acro disc filter | PALL | 2016-10 | |
| 70um nylon cell stariner | Becton Dickinson | 352350 | |
| FICOL (Histopaque 1083) (polysucrose) | Simga | 1083 | |
| PBS | Corning | 21040CV | |
| LS Columns | Miltenyi | 130-042-401 | |
| Protease Inhibitor Cocktail | Roche | CO-RO | |
| Phosphatase Inhibitor Cocktail 2 | Sigma | P5762 | |
| Nitrocullulose Membrane | Bio-Rad | 1620115 | |
| SuperSignal West Dura Extended Duration Substrate ( chemiluminiscence substrate) | Thermo Scientific | 34075 | |
| CD5 | eBioscience | 13-0051-82 | |
| CD11b | eBioscience | 13-0112-75 | |
| CD45R (B220) | BD biosciences | 553092 | |
| CD45.1-FITC | eBioscience | 11-0453-85 | |
| CD45.2-PE | eBioscience | 12-0454-83 | |
| hCD45-FITC | BD Biosciences | 555482 | |
| Anti-Biotin-FITC | Miltenyi | 130-090-857 | |
| Anti-7-4 | eBioscience | MA5-16539 | |
| Anti-Gr-1 (Ly-6G/c) | eBioscience | 13-5931-82 | |
| Anti-Ter-119 | eBioscience | 13-5921-75 | |
| Ly-6 A/E (Sca1) PE Cy7 | BD | 558612 | |
| CD117 APC | BD | 553356 | |
| BD Pharm Lyse | BD | 555899 | |
| BD Cytofix/Cytoperm (Fixing and permeabilization solution) | BD | 554714 | |
| BD Perm/Wash (permeabilization and wash solution for phospho flow) | BD | 554723 | |
| phospho p38 | Cell Signaling Technologies | 4511S | |
| total p38 | Cell Signaling Technologies | 9212 | |
| Mouse IgG control | BD | 554121 | |
| Alexa Flour 488 conjugated | Invitrogen | A-11034 | |
| Calcium Chloride | Invitrogen | K278001 | |
| 2X HBS | Invitrogen | K278002 | |
| EDTA | Ambion | AM9261 | |
| BSA | Sigma | A7906 | |
| Blood Capillary Tubes | Fisher | 22-260-950 | |
| Blood Collection Tube | Giene Bio-One | 450480 | |
| Newborn Calf Serum | Atlanta biological | S11295 | |
| Erythropoiein | Amgen | 5513-267-10 | |
| human SCF | Prospec | CYT-255 | |
| Human IL-3 | Prospec | CYT-210 | |
| G-SCF | Prospec | CYT-220 | |
| GM-CSF | Prospec | CYT-221 | |
| MyeloCult (media for LTCIC assay) | Stem Cell Technologies | 5100 | |
| Hydrocortisone Sodium Hemisuccinate | Stem Cell Technologies | 7904 | |
| MEM alpha | Gibco | 12561-056 | |
| 1/2cc Lo-Dose u-100 insulin syringe 28 G1/2 | Becton Dickinson | 329461 | |
| Mortor pestle | Coor tek | 60316 and 60317 | |
| Isoflorane (Isothesia TM) | Butler Schien | 29405 | |
| SOC | New England Biolabs | B90920s | |
| Ampicillin | Sigma | A0166 | 100mg/ml stock in water |
| Bacto agar (agar) | Difco | 214050 | |
| Terrific broth | Becton Dickinson | 243820 | |
| Agarose | Genemate | E-3119-500 | |
| Doxycycline chow | TestDiet.com | 52662 | modified RMH1500, Autoclavable 5LK8 with 0.0625% Doxycycline |
| Tamoxifen | Sigma | T5648 | |
| Iodonitrotetrazolium chloride | Sigma | I10406 | |
| Kits | |||
| Dneasy Blood & tissue kit | Qiagen | 69506 | |
| GoTaq Green (taq polymerase with Green loadign dye) | Promega | M1722 | |
| miRNeasy Mini Kit (RNA isolation kit) | Qiagen | 217084 | |
| DNA Free Dnase Kit (DNAse treatment for RT PCR) | Ambion, Life Technologies | AM1906 | |
| Superscript III First Strand Synthesis (reverse transcriptase for cDNA synthesis) | Invitrogen | 18080051 | |
| SYBR Green (taq polymerase mix with green interchalating dye for qPCR) | Bio-Rad | 1725270 | |
| CD117 MicroBead Kit | Miltenyi | 130-091-224 | |
| Human Long-Term Culture Initiating Cell Assay | Stemp Cell Technologies | ||
| Instruments | |||
| NAPCO series 8000 WJ CO2 incubator | Thermo scientific | ||
| Swing bucket rotor cetrifuge 5810R | Eppendorf | ||
| TC-10 automated cell counter | Bio-RAD | ||
| C-1000 Thermal cycler | Bio-RAD | ||
| Mastercycler Real Plex 2 | Eppendorf | ||
| ChemiDoc Imaging System (imaging system for gels and western blots) | Bio-RAD | 17001401 | |
| Hemavet ( boold counter) | Drew-Scientific | ||
| LSR II ( FACS analyzer) | BD | ||
| Fortessa I ( FACS analyzer) | BD | ||
| FACSAriaII ( FACS Sorter) | BD | ||
| Magnet Stand | Miltenyi | ||
| Irradiator | J.L. Shepherd and Associates, San Fernando CA | Mark I Model 68A | source Cs 137 |
| Mice | |||
| ROSACreERT2 | Jackson Laboratory | ||
| Scl-tTA | Dr. Claudia Huettner’s lab | ||
| BoyJ | mouse core facility at CCHMC | ||
| C57Bl/6 | Jackson Laboratory | ||
| NSGS | mouse core facility at CCHMC | ||
| ROSACreERT2/c-Fosfl/fl Dusp1-/- | Made in house | ||
| ROSACreERT2/c-Fosfl/fl | Made in house | ||
| Cells | |||
| BaF3 | Gift from George Daley, Harvard Medical School, Boston | ||
| WEHI | Gift from George Daley, Harvard Medical School, Boston | ||
| CML-CD34+ and Normal CD34+ cells | University Hospital, University of Cincinnati |
Referências
- Daley, G. Q., Van Etten, R. A., Baltimore, D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science. 247, 824-830 (1990).
- Druker, B. J., et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nature Medicine. 2, 561-566 (1996).
- Mahon, F. X., et al. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncology. 11, 1029-1035 (2010).
- O’Hare, T., Zabriskie, M. S., Eiring, A. M., Deininger, M. W. Pushing the limits of targeted therapy in chronic myeloid leukaemia. Nature Reviews Cancer. 12, 513-526 (2012).
- Rousselot, P., et al. Imatinib mesylate discontinuation in patients with chronic myelogenous leukemia in complete molecular remission for more than 2 years. Blood. 109, 58-60 (2007).
- Krause, D. S., Van Etten, R. A. Tyrosine kinases as targets for cancer therapy. The New England Journal of Medicine. 353, 172-187 (2005).
- Sharma, S. V., Settleman, J. Exploiting the balance between life and death: targeted cancer therapy and “oncogenic shock”. Biochemical Pharmacology. 80, 666-673 (2010).
- Sharma, S. V., Settleman, J. Oncogene addiction: setting the stage for molecularly targeted cancer therapy. Genes & Development. 21, 3214-3231 (2007).
- Weinstein, I. B. Cancer. Addiction to oncogenes–the Achilles heal of cancer. Science. 297, 63-64 (2002).
- Corbin, A. S., et al. Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. The Journal of Clinical Investigation. 121, 396-409 (2011).
- Straussman, R., et al. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature. 487, 500-504 (2012).
- Wilson, T. R., et al. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature. 487, 505-509 (2012).
- Kesarwani, M., et al. Targeting c-FOS and DUSP1 abrogates intrinsic resistance to tyrosine-kinase inhibitor therapy in BCR-ABL-induced leukemia. Nature Medicine. 23, 472-482 (2017).
- Zhang, J., et al. c-fos regulates neuronal excitability and survival. Nature Genetics. 30, 416-420 (2002).
- Dorfman, K. Disruption of the erp/mkp-1 gene does not affect mouse development: normal MAP kinase activity in ERP/MKP-1-deficient fibroblasts. Oncogene. 13, 925-931 (1996).
- Padhye, S., et al. Fluorocurcumins as cyclooxygenase-2 inhibitor: molecular docking, pharmacokinetics and tissue distribution in mice. Pharmaceutical Research. 26, 2438-2445 (2009).
- Molina, G., et al. Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages. Nature Chemical Biology. 5, 680-687 (2009).
- Zhang, B., et al. Effective targeting of quiescent chronic myelogenous leukemia stem cells by histone deacetylase inhibitors in combination with imatinib mesylate. Cancer Cell. 17, 427-442 (2010).
- Koschmieder, S., et al. Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis. Blood. 105, 324-334 (2005).
- Abraham, S. A., et al. Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells. Nature. 534, 341-346 (2016).
- Li, L., et al. Activation of p53 by SIRT1 inhibition enhances elimination of CML leukemia stem cells in combination with imatinib. Cancer Cell. 21, 266-281 (2012).
- . Qiagen-miRNAeasy kit Available from: https://www.qiagen.com/us/shop/sample-technologies/rna/mirna/mirneasy-mini-kit/#resources (2018)
- . DNA-free DNA removal kit Available from: https://www.thermofisher.com/order/catalog/product/AM1906?gclid=EAIaIQobChMIg4D-_ZvC2wIVx5-zCh00Cg8fEAAYASAAEgIv6vD_BwE&s_kwcid=AL!3652!3!264318446624!b!!g!!&ef_id=V7SO5AAAAck3ba89:20180607185004:s (2018)
- . SuperScript™ III First-Strand Synthesis System Available from: https://www.thermofisher.com/order/catalog/product/18080051 (2018)
- . Human Long Term Culture Initiating Cell Itc-ic Assay Available from: https://www.stemcell.com/human-long-term-culture-initiating-cell-ltc-ic-assay.html (2018)
- Abarrategi, A., et al. Modeling the human bone marrow niche in mice: From host bone marrow engraftment to bioengineering approaches. Journal of Experimental Medicine. 215, 729-743 (2018).
- Kesarwani, M., Huber, E., Kincaid, Z., Azam, M. A method for screening and validation of resistant mutations against kinase inhibitors. Journal of Visualized Experiments. , (2014).
