Time and Dose Dependent Synergism Between Conventional Cytostatics and the p53-Modulating Small Molecule PRIMA-Meth in p53 Mutated Leukemic Cells

Background: P53 deletions and mutations can be found in 5 to 15% of patients with acute myeloid leukemia and in about 50% of all human cancers. Mutations in the p15 gene are associated with resistance to cancer chemotherapy. Furthermore, p53 is involved in the induction of apoptosis in response to many cellular stress factors. P53 mutations in AML are associated to poor prognosis and a decreased sensitivity to chemotherapeutic drugs. PRIMA-meth (APR-246) belongs to a new generation of the compounds proved to restore the function of p53. It has been shown that PRIMA-1 meth works synergistically with some cancer chemotherapeutic agents through mutant p53-dependent pathway.

Aim: To identify a novel effective regimen for AML patients by incorporating PRIMA-meth. The study was performed in vitro in order to evaluate a regimen that can exert the most pronounced synergistic effect when combining PRIMA-meth with conventional chemotherapeutic drugs.

Materials and Methods: After optimizing the doses of the drugs and the exposure time, cells were incubated with daunorubicin (0.01μM) cytarabine (0.5μM) and fludarabine (20μM) alone and in combination with PRIMA-meth (5, 10 and15μM). Three different timing schedules of the exposure were tested; one with 24 hours pretreatment with PRIMA-meth, one with 24 hours pretreatment with the cytostatic drugs and one with simultaneuous incubation of PRIMA-meth in combination with the cytostatics. After 96 hours of incubation, the cell viability was analyzed by a bioluminescence assay measuring ATP levels. Each experiment was done in triplicate.

Results: Pre-incubation for 24 hours in PRIMA-meth exerted synergistic effects at 15μM in combination with all three tested drugs (table 1). This effect was superior to the combination effect seen when cells were pre-incubated with the cytostatic drugs where synergism was seen only with fludarabine. In the 24 hour pretreatment regimens, more potent synergism was found with fludarabine in combination with the higher PRIMA-meth concentrations (10μM and 15μM) irrespectively which drug was added first. Generally, the higher the prima-meth concentration, the better the response to combination therapy. With simultaneous exposure, synergism was found only with fludarabine in combination with PRIMA-meth at the highest concentration.

View large

N.B: Values represent ratio values between actual cell survival percentage and expected cell survival percentage (synergistic effect< or =0,8; additive effect >0,8<1,2).

View Large

In conclusion, PRIMA-meth can exert synergistic effects in combination with conventional chemotherapeutic drugs in p53 mutated AML cells. Synergism could be seen with fludarabine irrespectively of the timing of the exposure whereas synergism with daunorubicin and cytarabine was seen only when preincubating cells with PRIMA-meth. Synergisms were seen when using PRIMA-meth at higher concentrations. This suggests that similar combinations are promising and should be used for combination treatments in clinical trials.