Multiple distinct molecular mechanisms influence sensitivity and resistance to MDM2 inhibitors in adult acute myelogenous leukemia by Jianting Long, Brian Parkin, Peter Ouillette, Dale Bixby, Kerby Shedden, Harry Erba, Shaomeng Wang, and Sami N. Malek Blood Volume 116(1):71-80 July 8, 2010 ©2010 by American Society of Hematology

A substantial subset of primary AML displays primary resistance to MDM2 inhibitors. A substantial subset of primary AML displays primary resistance to MDM2 inhibitors. A total of 109 (MI219) and 60 (MI-63) AML samples were enriched to more than 90% blast purity through negative selection and incubated for 40 hours with various concentrations of either MI-219 or MI-63. Samples were prepared for annexin V and PI staining and analyzed by flow cytometry, and the residual live and nonapoptotic cell fraction was calculated for each concentration by comparison with the untreated control aliquots. (A) MI-219 assay results. Red represents p53 sequence mutants; green, cases with absent p53 mRNA; and black, wild-type p53 status. (B) MI-63 assay results. Red represents p53 sequence mutants; green, cases with absent p53 mRNA; and black, wild-type p53 status. Jianting Long et al. Blood 2010;116:71-80 ©2010 by American Society of Hematology

Sensitivities of 19 AML cell lines to MI219. Sensitivities of 19 AML cell lines to MI219. Nineteen AML cell lines (Table 2) were incubated for 40 hours 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 nonapoptotic cell fraction was calculated for each concentration by comparison with the untreated control incubation. Red represents p53 sequence mutants; and black, wild-type p53 status. Jianting Long et al. Blood 2010;116:71-80 ©2010 by American Society of Hematology

Results of p53 immunoblotting in sensitive and resistant primary AML blasts after MI219 or Nutlin3 treatment or external irradiation. Results of p53 immunoblotting in sensitive and resistant primary AML blasts after MI219 or Nutlin3 treatment or external irradiation. AML blasts were purified through negative selection and either left untreated or treated for 8 hours with MI219 (5μM), Nutlin 3 (5μM), or one-time external irradiation (5 Gy). After 8 hours, cells were lysed and protein fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each gel was also loaded with an aliquot of a MOLM13 AML 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-p53 and anti-actin antibody. Films for both p53 and actin were developed together. IC50 values for MI219 are indicated in brackets. Jianting Long et al. Blood 2010;116:71-80 ©2010 by American Society of Hematology

The mRNA levels of MDM2 and MDMX do not correlate with MI219 IC50 values. The mRNA levels of MDM2 and MDMX do not correlate with MI219 IC50 values. Normalized expression levels of MDM2 and MDMX mRNA were measured in cDNA made from RNA from FACS-sorted AML blasts. Displayed are δ Ct values (Ct mean MDM2 or MDMx − Ct mean PGK1) grouped by MI219 IC50 values as indicated. Red diamonds represent AML blasts with mutated p53. Jianting Long et al. Blood 2010;116:71-80 ©2010 by American Society of Hematology

p53 mutations or absent p53 mRNA expression in AML is associated with frequent copy-neutral LOH (acquired uniparental disomy) at 17p/p53 locus. p53 mutations or absent p53 mRNA expression in AML is associated with frequent copy-neutral LOH (acquired uniparental disomy) at 17p/p53 locus. Files generated through use of the Affymetrix program Genotyping Console for all patients were imported into the LOH tool, Version 2, using our software tool Pre-LOH Unification Tool, and all individual positions of LOH between buccal DNA and paired tumor DNA were graphed as a blue tick mark across the length of the chromosomes. Copy number estimates for all SNP positions for all patients were generated through dChipSNP, as described, and displayed across the length of the chromosomes. Copy losses are displayed with blue colors, copy gains with red colors. (A-B) Heatmap display of chromosomal copy number changes at 17p based on SNP 6.0 array profiling. Blue represents copy loss; and red, copy gain. (A) Buccal DNA. (B) AML blast DNA. (C) LOH analysis at 17p comparing paired blast and buccal DNA. Red numbering indicates copy-neutral LOH (acquired uniparental disomy); and black, LOH with copy loss. (D) p53 exon 5 to 9 mutation analysis results. (E) Normalized p53 mRNA expression in AML blasts grouped by MI219 IC50 values as indicated. Red diamonds represent AML blasts with mutated p53. Jianting Long et al. Blood 2010;116:71-80 ©2010 by American Society of Hematology

AML cases with mutated Flt3 (Flt3-ITD) are very sensitive to MI219. AML cases with mutated Flt3 (Flt3-ITD) are very sensitive to MI219. MI219 IC50 values categorized by (1) p53 mutation status, (2) presence of Flt3-ITD, and (3) all others. Differences in the mean IC50 value between Flt3-ITD+ and all other cases are significant (P = .02). Jianting Long et al. Blood 2010;116:71-80 ©2010 by American Society of Hematology