The tumor suppressor p53 plays a critical role in protecting cells from malignant transformation by inducing target gene transcription, cell cycle arrest, and apoptosis. MDM2 is the primary regulator of p53, targeting p53 for proteosomal degradation. The small molecule antagonist RG7112, which activates p53 by antagonism of the p53-MDM2 interaction, is currently in clinical trials. Clinical data from the Phase 1 program in leukemias has previously been presented (Andreeff et al., ASH 2010). Here we present pharmacodynamic biomarker data from the ongoing leukemia clinical trial.
Patients with relapsed/refractory leukemia were treated with escalating doses of study drug in two separate strata: A- AML, ALL, CML-BC; and B - CLL and S-CLL. Peripheral blood was collected for biomarkers pre-treatment and at multiple time points during the first 48 hours and on day 10. Bone marrow was collected pre-treatment, and on day 10 and 28. Specimens of blood and marrow were subjected to RBC lysis and MACS sorting (CD19, 33 or 34 Ab). After purification, the leukemic cells were divided for preparation of RNA, DNA and protein, or fresh frozen. The validated p53 transcriptional target and secreted protein, MIC-1 (macrophage inhibitory cytokine-1), was analyzed as a pharmacodynamic marker of p53 activation. Flow cytometric analysis was performed for assessment of apoptosis (CMxRos, AnnexinV) and differentiation, (CD34, CD19). Protein immunoblots for MDM2 and p53, and real-time PCR, after reverse transcription, for quantitation of 24 customized p53 targets by TaqMan low density arrays (TLDA) (Applied Biosystems) were undertaken. AmpliChip analysis of p53 mutational status was also performed. The AmpliChip p53 test detects single nucleotide substitutions and deletions in the entire coding region and splice sites of p53 (exons 2–11) by comparative analysis of probe hybridization patterns between sample and wild-type reference DNA.
To date, 68 patients (42 AML, 5 ALL, 1 CML-BC, 19 CLL/sCLL, 1 T-PLL) have been treated with RG7112, with doses ranging from 20 – 2430 (1215 mg BID) mg/m2/day x 10 days. P53 mutational status has been completed in 29 Arm A and 15 Arm B patients. Preliminary results reveal that 4/29 AML patients had mutations (3 MISSENSE, 1 SPLICE). 6 of 15 patients with CLL/SLL had mutations, including a patient with a 2bp deletion in exon 6, confirmed by deep sequencing, who has stable disease on therapy for 2 years. An increase in serum MIC-1 was seen at RG7112 concentrations of >2 μg/mL and was highly correlated (R2=0.4312) with exposure to RG7112 (AUC24h) at steady state. Consistent changes in p53 target genes were observed for PUMA, p21 and FDXR (ferrodoxin reductase) as early as 6 hours after the first dose; levels were found to be significantly (p<0.05) increased 6 hours after the last dose (day 10). Increases in PUMA and p21 in AML/ALL patient samples were greater in those patients whose blast percentages decreased during Cycle 1 (n=19). Linear regression analysis revealed a dose-dependent increase of p21 (r=0.65, p=0.01). In CLL patients, the same 3 genes (PUMA, p21 and FDXR) were significantly induced during treatment with RG7112. Apoptosis induction was documented by decreased mitochondrial membrane potential (CMxRos) and increased AnnexinV binding and immunoblotting confirmed PUMA and p53 induction at the protein level in selected cases.
We report evidence of concentration-related pharmacodynamic biomarker activity on the p53 pathway in blood and marrow specimens from patients with leukemia receiving the MDM2 antagonist, RG7112. The trial is ongoing with additional dose escalation. Further clinical and biomarker analyses are in progress and will be reported. We provide proof of mechanism for MDM2 antagonism by demonstrating p53 stabilization, activation of p53 targets and the p53 pathway in leukemic cells from patients treated with RG7112.
Andreeff:Roche: Research Funding. Younes:Genentech: Honoraria, Research Funding. Wu:Roche: Employment. Jukofsky:Roche: Employment. Vassilev:Roche: Employment. Zhi:Roche: Employment. Geho:Roche: Employment. Nichols:Hoffmann-LaRoche: Employment.
Asterisk with author names denotes non-ASH members.