Method Article

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

DOI:

10.3791/58194

⸱

January 7th, 2019

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

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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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Protocol

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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.

1. Real-time qPCR Analysis

  1. Isolate total RNA from BaF3 cells growing in RPMI supplemented with 10% FBS and 10% WEHI spent cu....

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Results

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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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Discussion

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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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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Biological Materials
RPMICellgro (corning)15-040-CV
DMEMCellgro (corning)15-013-CV
IMDMCellgro (corning)15-016-CVR
RetroNectin Recombinant Human Fibronectin FragmentTakaraT100B
MethoCult GF M3434 (Methylcellulose for Mouse CFU)Stem Cell3434
MethoCult H4434 Classic (Methylcellulose for Human CFU)Stem Cell4434
4-HydroxytamoxifenSigmaH6278
Recombinant Murine SCFProspecCYT-275
Recombinant Murine Flt3-LigandProspecCYT-340
Recombinant Murine IL-6ProspecCYT-350
Recombinant Murine IL-7Peprotech217-17
DFCLKT Laboratories Inc.D3420
BCIChemzon ScientificNZ-06-195
ImatinibLC LaboratoryI-5508
CurcuminSigma458-37-7
NDGASigma500-38-9
Penn/StrepCellgro (corning)30-002-CI
FBSAtlanta biologicalS11150
Trypsin EDTA 1xCellgro (corning)25-052-CI
1x PBSCellgro (corning)21-040-CV
L-GlutamineCellgro (corning)25-005-CL5 mg/mL stock in water
PuromycinGibco (life technologies)A11138-03
HEPESSigmaH7006
Na2HPO4.7H2OSigmaS9390
Protamine sulfateSigmaP33695 mg/mL stock in water
Trypan Blue solution (0.4%)SigmaT8154
DMSOCellgro (corning)25-950-CQC
WST-1Roche11644807001
0.45 μM acro disc filterPALL2016-10
70 μm nylon cell starinerBecton Dickinson352350
FICOL (Histopaque 1083) (polysucrose)Simga1083
PBSCorning21040CV
LS ColumnsMiltenyi130-042-401
Protease Inhibitor CocktailRocheCO-RO
Phosphatase Inhibitor Cocktail 2SigmaP5762
Nitrocullulose MembraneBio-Rad1620115
SuperSignal West Dura Extended Duration Substrate ( chemiluminiscence substrate)Thermo Scientific34075
CD5eBioscience13-0051-82
CD11beBioscience13-0112-75
CD45R (B220)BD biosciences553092
CD45.1-FITCeBioscience11-0453-85
CD45.2-PEeBioscience12-0454-83
hCD45-FITCBD Biosciences555482
Anti-Biotin-FITCMiltenyi130-090-857 
Anti-7-4eBioscienceMA5-16539
Anti-Gr-1 (Ly-6G/c)eBioscience13-5931-82
Anti-Ter-119eBioscience13-5921-75
Ly-6 A/E (Sca1) PE Cy7BD 558612
CD117 APC BD 553356
BD Pharm LyseBD 555899
BD Cytofix/Cytoperm (Fixing and permeabilization solution)BD 554714
BD Perm/Wash  (permeabilization and wash solution for phospho flow)BD 554723
phospho p38Cell Signaling Technologies4511S
total p38Cell Signaling Technologies9212
Mouse IgG controlBD 554121
Alexa Flour 488 conjugated InvitrogenA-11034
Calcium ChlorideInvitrogenK278001
2x HBSInvitrogenK278002
EDTAAmbionAM9261
BSASigmaA7906
Blood Capillary TubesFisher22-260-950
Blood Collection TubeGiene Bio-One450480
Newborn Calf SerumAtlanta biologicalS11295
ErythropoieinAmgen5513-267-10
human SCFProspecCYT-255
Human IL-3ProspecCYT-210
G-SCFProspecCYT-220
GM-CSFProspecCYT-221
MyeloCult (media for LTCIC assay)Stem Cell Technologies5100
Hydrocortisone Sodium HemisuccinateStem Cell Technologies7904
MEM alphaGibco12561-056
1/2 cc Lo-Dose u-100 insulin syringe 28 G1/2Becton Dickinson329461
Mortor pestleCoor tek 60316 and 60317
Isoflorane (Isothesia TM)Butler Schien29405
SOCNew England BiolabsB90920s
AmpicillinSigmaA0166100 mg/mL stock in water
Bacto agar (agar)Difco214050
Terrific brothBecton Dickinson243820
AgaroseGenemateE-3119-500
Doxycycline chowTestDiet.com52662modified RMH1500, Autoclavable 5LK8  with 0.0625% Doxycycline 
TamoxifenSigmaT5648
Iodonitrotetrazolium chloride SigmaI10406
Kits
Dneasy Blood & tissue kitQiagen69506
GoTaq Green (taq polymerase with Green loadign dye)PromegaM1722
miRNeasy Mini Kit  (RNA isolation kit)Qiagen217084
DNA Free Dnase Kit (DNAse treatment for RT PCR)Ambion, Life TechnologiesAM1906
Superscript III First Strand Synthesis (reverse transcriptase for cDNA synthesis)Invitrogen18080051
SYBR Green (taq polymerase mix with green interchalating dye for qPCR)Bio-Rad1725270
CD117 MicroBead KitMiltenyi130-091-224
Human Long-Term Culture Initiating Cell AssayStemp Cell Technologies
Instruments
NAPCO series 8000 WJ CO2 incubatorThermo scientific
Swing bucket rotor cetrifuge 5810REppendorf
TC-10 automated cell counterBio-RAD
C-1000 Thermal cyclerBio-RAD
Mastercycler Real Plex 2Eppendorf
ChemiDoc Imaging System (imaging system for gels and western blots)Bio-RAD17001401
Hemavet (boold counter)Drew-Scientific
LSR II (FACS analyzer)BD 
Fortessa I (FACS analyzer)BD 
FACSAriaII (FACS Sorter)BD 
Magnet StandMiltenyi
Irradiator J.L. Shepherd and Associates, San Fernando CAMark I Model 68Asource Cs 137
Mice
ROSACreERT2Jackson Laboratory
Scl-tTA Dr. Claudia Huettner’s lab
BoyJ mouse core facility at CCHMC
C57Bl/6 Jackson Laboratory
NSGSmouse core facility at CCHMC
ROSACreERT2/c-Fosfl/fl Dusp1-/- Made in house
ROSACreERT2/c-Fosfl/flMade in house
Cells
BaF3Gift from George Daley, Harvard Medical School, Boston
WEHIGift from George Daley, Harvard Medical School, Boston
CML-CD34+ and Normal CD34+ cellsUniversity Hospital, University of Cincinnati

References

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  1. 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).
  2. Druker, B. J., et al. E....

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Tags

Oncogene Dependencec Fos Dusp1Tyrosine Kinase InhibitorsChronic Myeloid LeukemiaMinimal Residual DiseaseBone Marrow IsolationMagnetic Activated Cell SortingRetroviral TransductionColony Forming Unit AssayRT qPCR Western Blot

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