uHTS identification of HIF-2a Inhibitors in a luminesence assay
Most solid tumors and their metastases experience regions of hypoxia, which promotes both tumor progression and resistance to therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1alpha and HIF-2alpha. HIF-1alpha and HIF-2alpha are non-redundant and regulate both overlapping and unique downstream target genes. HIF-1alpha has been associated with poor patient more ..
BioActive Compounds: 2624
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, La Jolla, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 DA033980-01A1
Assay Provider: Mei Yee Koh, Ph.D., University of Texas M.D. Anderson Cancer Center
Most solid tumors and their metastases experience regions of hypoxia, which promotes both tumor progression and resistance to therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1alpha and HIF-2alpha. HIF-1alpha and HIF-2alpha are non-redundant and regulate both overlapping and unique downstream target genes. HIF-1alpha has been associated with poor patient survival in multiple tumor types and is currently being pursued as a target for inhibition for cancer therapy 1, 2. HIF-2alpha has also been associated with poor patient prognosis in specific tumor types such as renal cell carcinoma (RCC), neuroblastoma, glioblastoma (GBM) and non-small cell lung cancer (NSCLC) 3-6. The tumor type specific function of HIF-2alpha has been linked with the ability of HIF-2alpha (but not HIF-1alpha) to co-operate with a number of oncoproteins such as c-Myc, EGFR and K-Ras 5, 7, 8, and also to activate genes involved in proliferation, invasion and in the maintenance of cancer stem cells (CSCs) 9, 10. Hence, although the validity of HIF-1alpha as a target in multiple tumor types cannot be denied, emerging data suggest that in certain settings, the specific inhibition of HIF-2alpha versus HIF-1alpha would be warranted. Additionally, the newly established role of HIF-2alpha in the maintenance of neoplastic but not in normal stem cells, suggests that HIF-2alpha inhibition may provide a novel strategy for the targeting of CSCs 11, 12, 13. Thus our hypothesis is that the specific inhibition of HIF-2alpha will be a useful strategy for treating HIF-2alpha driven tumors and for targeting the CSC population. The overall goal of the study is to identify small molecule specific inhibitors of HIF-2alpha that will lead to the development of new anti-cancer therapies.
1. Gordan J D, Bertout J A, Hu C J, Diehl J A,Simon M C. HIF-2alpha promotes hypoxic cell proliferation by enhancing c-myc transcriptional activity. Cancer Cell 2007; 11: 335-47.
2. Scrideli C A, Carlotti C G, Jr., Mata J F, Neder L, Machado H R, Oba-Sinjo S M et al. Prognostic significance of co-overexpression of the EGFR/IGFBP-2/HIF-2A genes in astrocytomas. J Neurooncol 2007; 83: 233-9.
3. Kim W Y, Perera S, Zhou B, Carretero J, Yeh J J, Heathcote S A et al. HIF2alpha cooperates with RAS to promote lung tumorigenesis in mice. J Clin Invest 2009; 119: 2160-70.
4. Holmquist-Mengelbier L, Fredlund E, Lofstedt T, Noguera R, Navarro S, Nilsson H et al. Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype. Cancer Cell 2006; 10: 413-23.
5. Franovic A, Holterman C E, Payette J,Lee S. Human cancers converge at the HIF-2alpha oncogenic axis. Proc Natl Acad Sci U S A 2009; 106: 21306-11.
6. Giatromanolaki A, Koukourakis M I, Sivridis E, Turley H, Talks K, Pezzella F et al. Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival. Br J Cancer 2001; 85: 881-90.
7. Li Z, Bao S, Wu Q, Wang H, Eyler C, Sathornsumetee S et al. Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell 2009; 15: 501-13.
8. Seidel S, Garvalov B K, Wirta V, von Stechow L, Schanzer A, Meletis K et al. A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha. Brain 2010; 133: 983-95.
9. Pietras A, Hansford L M, Johnsson A S, Bridges E, Sjolund J, Gisselsson D et al. HIF-2alpha maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells. Proc Natl Acad Sci U S A 2009; 106: 16805-10.
10. Qing G,Simon M C. Hypoxia inducible factor-2alpha: a critical mediator of aggressive tumor phenotypes. Curr Opin Genet Dev 2009; 19: 60-6.
11. Carroll V A,Ashcroft M. Role of hypoxia-inducible factor (HIF)-1alpha versus HIF-2alpha in the regulation of HIF target genes in response to hypoxia, insulin-like growth factor-I, or loss of von Hippel-Lindau function: implications for targeting the HIF pathway. Cancer Res 2006; 66: 6264-70.
12. Dutta D, Ray S, Vivian J L,Paul S. Activation of the VEGFR1 chromatin domain: an angiogenic signal-ETS1/HIF-2alpha regulatory axis. J Biol Chem 2008; 283: 25404-13.
13. Saito T, Fukai A, Mabuchi A, Ikeda T, Yano F, Ohba S et al. Transcriptional regulation of endochondral ossification by HIF-2alpha during skeletal growth and osteoarthritis development. Nat Med 16: 678-86.
786-0 renal cell carcinoma cells (Assay Provider)
DMEM 4.5g/l glu w/glu w/o phenol red (CellGro 17-205-CV)
Fetal Bovine Serum (Hyclone SH30396.03)
Penicillin Streptomycin solution (Invitrogen 15140122)
L-glutamine (100X ) (Invitrogen 25030081)
G418 (50 mg/mL) (Omega Scientific GN-04)
T225 Tissue Culture flasks
DPBS without calcium and magnesium (1X)
TrypLE (Invitrogen 12563)
I. Compound Addition:
1- Transfer test compounds to columns 5-48 and DMSO to columns 1-4 using the Labcyte ECHO 555. Transfer volume of test compound and DMSO is 7.5 nL of 10 mM stock, making 18.75 uM compound concentration at 0.18% DMSO final.
II. Reagent Addition
2- Dispense 4 uL/well of assay media to col 1-2.
3- Dispense 4 uL/well of cells at 2.5x10^5 cells/ml to col 3-48.
4- Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.
5 - Put Kalypsis metal lids on plates, incubate plates at 37 degrees C with 5% C02 overnight.
6- Dispense 4 uL/well of Steady-Glo using Kalypsis dispenser to all columns.
7- Spin down plates without lids on Vspin at 2000 rpm for 1 min
8- Incubate plates at room temperature for 10 mins
III. Reading plates:
9-Read on luminesence protocol on viewlux
Compounds that demonstrated a normalized or corrected % activity of <=40% compared to the controls are defined as inhibitors of the reaction.
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)