In Vitro and Ex Vivo Studies On Cell Lines and Primary Human Leukemia Cells of the Effects of APR-246 Alone and in Combination with Conventional Chemotherapeutic Drugs.

Mutations of TP53 are associated with an extremely poor prognosis in hematopoietic malignancies and is found in 10 to 15% of patients with AML. APR-246 is a methylated form of the small molecule PRIMA-1. It primarily targets mutated p53 protein, but has cytotoxic and apoptosis inducing effects on primary leukemia cells from AML and CLL patients irrespectively of p53 status even though cells with mutated p53 have been shown to be more sensitive. In May 2009, a first-in-man trial has been initiated evaluating the effect of APR-246 in hematologic malignancies.

Leukemic blast cells from 32 untreated patients with AML were isolated and analyzed. Sixteeen selected patients had normal karyotype and 16 selected patients had complex karyotype. All patient DNA were sequenced at exon 5-8 of TP53 gene. Cells were exposed to APR-246 (2.5μM and 5μM), Ara-C (0.5μM), daunorubicin (0,05μM) and fludarabin (1μM) alone and in combinations. Different timing schedules of the exposure were also used. Cell viability was assayed by bioluminescence measuring ATP. Expression of p53, Bax, Bcl-2 and active caspase –3 and induction of reactive oxygen species (DCF-fluorescence) was analyzed by flow cytometry. We also analyzed two leukemia cell lines, KBM3 and NB4, with and without p53mutation respectively, in order to study the combination effect with chemotherapeutic drugs and the role of preincubation with either of the drug. The Combination Index (CI) was calculated according to the additive model where a CI of less than 0.8 indicates synergy (Möllgård et al CCP, 2008). We also performed global gene expression analysis with Affymetrix platform 1.0 after 18h of APR-246 treatment at 15μM and 25μM in vitro.

APR-246 induced dose dependent cytotoxic effects in primary AML samples with a IC50 value of 5.0 μM after 4 days of incubation. A statistically significant increase in the expression of active caspase-3 after 48h treatment with APR-246 (p<0.001) and a tendency to p53 up-regulation (p=0.056) could be detected in patient cells with both normal and complex karyotype. Patients that up-regulated p53 after APR-246 exposure were more sensitive to APR-246 (p<0.05) and had a significantly lower level of Bax (p<0.05) before APR-246 treatment. The seven patients with TP53 mutation had significant lower sensitivity to daunorubicin and fludarabin (p<0.01) in vitro but not to APR-246, indicating a possible treatment alternative in multidrug resistant cells. In primary AML cells, the combination of APR-246 and daunorubicin was the most effective drug combination and gave strong synergy at simultaneous incubation. The TP53 mutated KBM3 cell line was significantly more sensitive to APR-246 (p<0.01) than wt TP53 NB4 cell line with IC50 of 15μM and 45μM respectively. Simultaneous exposure of APR-246 and conventional chemothperapeutic drugs showed synergy in drug combinations tested in both the mutated and the wild type cell line. Pre-incubation with APR-246 gave significantly stronger synergism with all tested drugs combined with 15μM APR-246 (CI for DNR 0.76, for Ara-C 0.65 and for fludarabin 0.60). Pre-incubation with conventional chemotherapeutic drugs showed significantly less synergy. Flow cytometry demonstrated a 2-fold increase in reactive oxygene species (ROS) after 24h and a 15-fold increase after 48h of APR-246 treatment in the TP53 mutated cell line. Global gene expression analysis showed that 50 % of the genes that were upregulated more than a 2.0 fold by APR-246 were associated to the redox status of the cells. Genes that were significantly upregulated were SLC7A11 and oxygenase 1, indicating that APR-246 may exert some of its cytotxic effects by incuding oxidative stress.

We conclude that APR-246 exhibits a concentration and time dependent cytotoxic effects in wt and p53 mutated cell lines and primary AML cells ex vivo. APR-246 shows strong synergism together with conventional chemotherapeutic drugs, especially with pre-incubation with APR-246. The anti-leukemic effects are associated with an increase in ROS. A first-in-man trial with APR-246 in haematological malignancies has been initiated and an update of the trial will be given at the meeting.

Paul:Aprea AB: Consultancy, Research Funding. Lehmann:Aprea AB: Consultancy, Membership on an entity's Board of Directors or advisory committees.