Comprehensive Biomarker and Genomic Analysis Identifies p53 Status as the Major Determinant of Response to MDM2 Inhibitors in Chronic Lymphocytic Leukemia.

To understand determinants of sensitivity or resistance to MDM2 inhibitor therapy in chronic lymphocytic leukemia (CLL), we comprehensively analyzed highly pure CD19+ cells derived from 106 CLL patients for response to the MDM2 inhibitors MI-63 and Nutlin3 ex vivo and correlated these responses with clinically important biomarkers, including hierarchical FISH, IgV_H-mutations and ZAP70 expression. P53 exons 5–9 were sequenced for all cases, and p53 induction pre- and post-inhibitor treatment was determined by immunoblotting for all 106 samples. Furthermore, we employed high-density 50kSNP-arrays to analyze changes of copy number and allele status, including that of p53 on chromosome 17p, in 171 CLL patients and correlated this finding with p53 mutations and p53 expression. Patient-derived samples were incubated for 36 hours with both inhibitors in parallel and apoptotic (annexin-V) and necrotic cell death (PI) was assayed for all incubations using flow cytometry. IC50 values were calculated using XL-fit. Nineteen CLL cases demonstrated either p53 mutations or aberrant expression and all were relatively resistant to MDM2 inhibitor treatment; eighty-seven cases had functional p53 and all but 3 cases demonstrated sensitivity to MDM2 inhibitor treatment. Very few cases with functional p53 demonstrated IC50 values for both inhibitors that were within the range of p53 mutant cases. Del17p patient samples were resistant to MDM2 inhibitor-mediated cell kill, while all other CLL biomarker-based subgroups appeared equally sensitive. Univariate time to first therapy (TTFT) estimates demonstrated a statistically significant (p=0.02 using the log-rank test) shorter TTFT for the group of 19 CLL cases with aberrant p53 function as opposed to the group of 87 cases with functional p53. Importantly, TTFT analysis for the 87 patients with functional p53 found that patients with low IC50 values (high sensitivity to MDM2 inhibitors) had more aggressive CLL (shorter TTFT), identifying for the first time a susceptible target group for these inhibitors that demonstrates aggressive disease and wt p53. In conclusion, the results of these studies conclusively demonstrate that p53 status is the major determinant of response to MDM2 inhibitors in CLL. Additional defects in the p53 regulatory cascade do not appear operational in this leukemia. This comprehensive dataset provides supportive rationale for the design of early phase human trials using these compounds and supports ex vivo measurements of p53 induction pre- and post-MDM2 inhibitor treatment as part of future trial schemas.