Effective Targeting Of The P53/MDM2 Axis In Preclinical Models Of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

While the overall cure rate for the most common pediatric cancer, acute lymphoblastic leukemia (ALL) now approaches 90%, infants (<12 months) diagnosed with ALL harboring translocations in the mixed-lineage leukemia oncogene (infant MLL-ALL) experience shorter remission duration and a significantly reduced likelihood of survival (∼50%). Therefore, new treatments that can be incorporated into conventional chemotherapy regimens to extend patient remission and improve survival are urgently required. Mutations in the p53 tumor suppressor are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be of therapeutic benefit. Nutlin cis-imidazole molecules selectively inhibit p53-MDM2 binding, resulting in activation of the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenograft models.

In vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. P53 protein levels and subcellular distribution were assessed by immunoblotting. Patient-derived xenografts were established from infant MLL-ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice, and their gene expression profiles generated using Illumina Human Ref-12 Expression BeadChips. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45⁺ cells in the PB. Mice with established disease received vehicle, RG7112 (100 mg/kg daily x 5 p.o.), a combination of vincristine (0.15 mg/kg once i.p.) dexamethasone (5 mg/kg daily x 5 i.p.) and L-asparaginase (1,000 IU/kg daily x 5 i.p.) (VXL), or RG7112 plus VXL. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups from treatment initiation. Therapeutic enhancement was considered to occur when the RG7112/VXL combination significantly extended mouse EFS compared with that of both of the RG7112 and VXL treated groups.

Unsupervised hierarchical clustering of gene expression profiles revealed that the MLL-ALL (n=9), BCP-ALL (n=7) and T-ALL (n=13) xenografts clustered according to leukemia subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA gene family, were significantly upregulated in MLL-ALL xenografts. The specificity of RG7112 was validated by cytotoxicity assays against leukemia cell lines of known p53 status; p53 wild-type cell lines (RS4;11, IC50 1.4 µM; NALM-6, IC50 3.0 µM) were markedly more sensitive than those with mutant p53 (CEM, IC50 >10 µM; JURKAT, IC50 >10 µM). The in vitro sensitivity of BCP-ALL (n=3) and infant MLL-ALL (n=4) xenografts was consistent with wild-type p53 status, with IC50s of 0.11 - 2.2 µM. Exposure of ALL xenograft cells to nutlin-3 (10 µM, 6h) caused marked p53 up-regulation and nuclear translocation. Since we had previously shown that RG7112 administered as a single agent for 14 days induced significant regressions [Complete Responses (CRs) or greater] in 7/7 infant MLL-ALL xenografts in vivo, we assessed its efficacy in a 5-day combination treatment with an induction-type regimen (VXL) against two infant MLL-ALL xenografts (MLL-5 and MLL-14). The RG7112/VXL combination caused a Partial Response in MLL-5 compared with Progressive Disease for both RG7112 and VXL. The efficacy of RG7112/VXL was even more pronounced against MLL-14, causing a Maintained CR compared with CRs for both RG7112 and VXL, which met the criteria for Therapeutic Enhancement (the median EFS of RG7112/VXL-treated mice, 65.0 days, was significantly greater, P< 0.0001, than that of RG7112, 22.2 days, and VXL, 28.5 days).

RG7112 induces significant regressions in a high proportion of infant MLL-ALL xenografts and enhances the efficacy of an induction-type regimen. The utility of targeting the p53-MDM2 axis in combination with established drugs for the clinical management of infant MLL-ALL warrants further investigation.

This study was supported by NCI NO1CM42216. The authors thank Roche Pharmaceuticals, Inc., for providing RG7112.

No relevant conflicts of interest to declare.