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Cancer Research

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

Published: January 7th, 2019

DOI:

10.3791/58194

1Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati Children's Hospital Medical Center

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Constitutive tyrosine kinase activity of BCR-ABL1 fusion oncogene causes CML, which provides a rationale to target the kinase activity by small molecule inhibitors. The success of TKIs in treating CML patients revolutionized the concept of targeted therapy1,2. Subsequently, anti-kinase therapy as precision medicine was developed for several other malignancies, including solid tumors. So far, more than thirty kinase inhibitors have been approved by the United State FDA for treating various malignancies. While TKI treatment is very effective in suppressing the disease, it is not curative. Besides, a small popula....

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All animal experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children's Hospital Medical Center (CCHMC). Human specimens (Normal BM and that from CML (p210-BCR-ABL+) leukemia) were obtained through Institutional Review Board-approved protocols (Institutional Review Board: Federalwide Assurance #00002988 Cincinnati Children's Hospital Medical Center) and donor-informed consent from CCHMC and the University of Cincinnati.

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Oncogene addiction has been implicated in the therapeutic efficacy of TKIs. However, the mechanisms driving the oncogene dependence are not understood. We performed multiple unbiased gene expression analyses to identify the genetic component involved in orchestrating the addiction. These analyses revealed the upregulation of three genes, c-Fos, Dusp1, and Zfp36, in cancer cells that are not dependent on oncogenic signaling for survival and, thus, are insensitive to TKI treatment. The down.......

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For the bulk of cancer cells, the therapeutic response to TKI is mediated by a blockade of tyrosine-kinase-oncoprotein signals to which the tumor is addicted. However, relatively little is known about how a minority of cancer cells contributing to MRD escape the oncogene dependence and therapy4. Recent studies revealed that growth factor signaling mediates drug resistance in both leukemia and solid organ tumors. This suggests that various molecular mechanisms might underlie intrinsic resistance

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The authors are thankful to G. Q. Daley for providing the BaF3 and WEHI cells and T. Reya for the MSCV-BCR-ABL-Ires-YFP constructs. The authors are thankful to M. Carroll for providing the patient samples from the CML blast crisis. This study was supported by grants to M.A. from the NCI (1RO1CA155091), the Leukemia Research Foundation and V Foundation, and from the NHLBI (R21HL114074-01).

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Name Company Catalog Number Comments
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

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