Blood, Vol. 116, Issue 1, 71-80, July 8, 2010

Multiple distinct molecular mechanisms influence sensitivity and resistance to MDM2 inhibitors in adult acute myelogenous leukemia
Blood Long et al. 116: 71

Supplemental materials for: Long et al

Files in this Data Supplement:

• Table S1. Detailed clinical and genomic patient characteristics (XLS, 54.5 KB)
  
  
• Table S2. Comparative IC50 values for MI219 in unpurified and purified (blasts) AML marrow samples (XLS, 17 KB)
  
  
• Figure S1. Non-blast AML derived marrow cells are less sensitive to MI219-mediated apoptosis than AML blasts (JPG, 264 KB) -
  Ten AML-derived Ficoll-enriched bone marrow samples (black) were incubated for 40h with various concentrations of MI-219. Samples were prepared for annexin-V and PI staining and analyzed by flow cytometry, and the residual live and non-apoptotic cell fraction was calculated for each concentration by comparison with the untreated control aliquots. Blue: MI-219 assay results for highly purified blasts. Black: MI-219 assay results for unpurified paired marrow samples.
  
  
  
  
  
  
• Figure S2. Reversible epigenetic changes due not appear to cause low p53 protein levels in MI219 resistant AML blasts (JPG, 356 KB) -
  Purified AML blasts obtained through negative selection as described (see methods) were incubated in aliquots with either trichostatin A (TSA), 5-azacytidine, both drugs combined or solvent for 48 hours followed by treatment with MI219 at various concentrations (0, 2.5, 5, and 10 µM, respectively) as indicated. Blasts were subsequently prepared for FACS-based annexin V/PI apoptosis measurements (A) or p53 immunoblotting (B).
  
  
  
  
  
  
• Figure S3. Results of MDM2 and MDMX immunoblotting in sensitive and resistant primary AML blasts after MI219 or Nutlin3 treatment or external irradiation (JPG, 453 KB) -
  AML blasts were purified through negative selection and either left untreated or treated for 8 hours with MI219 (10 µM), Nutlin 3 (10 µM) or onetime external irradiation (5Gy). After 8 hours, cells were lysed and protein fractionated by SDS-PAGE. Each gel was also loaded with an aliquot of a LNCaP cell line lysate as an internal standard (loaded as 1.25, 2.5, and 5 µg of MI219-treated lysate or 5 µg of untreated lysate UT, respectively). Protein was transferred to membrane and prepared for immunoblotting with an anti-MDM2 or anti-MDMX and anti-actin antibody. IC50 values for MI219 are indicated in brackets.
  
  
  
  
  
  
• Figure S4. Results of p21 immunoblotting in sensitive and resistant primary AML blasts after MI219 or Nutlin3 treatment or external irradiation (JPG, 533 KB) -
  AML blasts were purified through negative selection and either left untreated or treated for 8 hours with MI219 (10 µM), Nutlin 3 (10 µM) or onetime external irradiation (5Gy). After 8 hours, cells were lysed and protein fractionated by SDS-PAGE. Each gel was also loaded with an aliquot of a LNCaP cell line lysate as an internal standard (loaded as 1.25, 2.5, and 5 µg of MI219 treated lysate or 5 µg of untreated lysate UT, respectively). Protein was transferred to membrane and prepared for immunoblotting with an anti-p21 and anti-actin antibody. IC50 values for MI219 are indicated in brackets.