Targeting Mutant p53 in Pediatric Acute Lymphoblastic Leukemia

Mutations of the tumor suppressor gene TP53 have been described to be associated with aggressive disease and inferior prognosis in different types of cancer, including hematological malignancies. In acute lymphoblastic leukemia (ALL), TP53 alterations are infrequently found at diagnosis but have recently been described in about 12% of patients at relapse. This suggests an association with therapy resistance in high risk/relapsed ALL and patients with TP53 mutated ALL have in fact an inferior outcome. Small molecule compounds targeting mutated TP53 such as APR-246, initially described as PRIMA-1^MET (p53-dependent reactivation and induction of massive apoptosis) leading to apoptosis induction have shown activity in several types of malignancies with mutated TP53. In ALL, however, mutant TP53has so far not been addressed as a target for therapeutic intervention.

In this study, we investigated a large cohort of patient-derived pediatric B cell precursor (BCP)-ALL primograft samples to identify cases with mutated TP53. Further, we analyzed the effects of APR-246 and evaluated its activity on BCP-ALL cell lines and primografts with mutated (mut) or wild type (wt) TP53.

Altogether, 62 BCP-ALL primograft samples established from patients at diagnosis (n=53) or relapse (n=9) by transplantation of primary ALL cells onto NOD/SCID mice were screened for TP53 mutations by denaturating high-performance liquid chromatography (dHPLC) followed by Sanger sequencing of exons 4 to 10 to confirm detected mutations. We identified 4 cases with TP53mut, 3 obtained from diagnosis (5.6%) and one at relapse (11.1%), corresponding to frequencies described in clinical studies. Mutated cases were further analyzed by fluorescence in situ hybridization (FISH), revealing a 17p deletion in one TP53mut sample. Similarly, we analyzed 6 BCP-ALL cell lines and identified 2 TP53mut and 4 TP53wt lines.

Exposure of BCP-ALL primograft (TP53mut n=4, TP53wt n=4) and cell line (TP53mut n=2, TP53wt n=4) samples to the DNA damaging agent doxorubicin showed, as expected, resistance of TP53mut leukemia cells for cell death induction, reflected by significantly higher half maximal inhibitory concentrations (IC50; TP53mut 49 and 143 ng/ml, TP53wt mean 12 ng/ml) and lower induction of cell death (TP53mut 16 to 23%, TP53wt 10 to 60%) in TP53mut ALL, corroborating the tumor-suppressive function of p53 in ALL.

We then investigated the sensitivity of BCP-ALL cell lines for cell death induction by APR-246 (kindly provided by Aprea, Stockholm, Sweden). We observed high sensitivity for APR-246 in TP53mut (IC50: 5 µM for both cell lines) as compared to TP53wt ALL (mean IC50: 58 µM). DNA fragmentation and Annexin-V/propidium-iodide (PI) positivity revealed apoptosis as mechanism of APR-246 mediated cell death. Reactive oxygen species (ROS) have recently been described to mediate APR-246 induced cell death in multiple myeloma cells. Therefore, we investigated ROS levels by detection of oxidation-specific fluorescence of dichlorodihydrofluorescein diacetate (DCFDA) in ALL cells. Interestingly, ROS quenching by N-acetyl cysteine abolished induction of cell death in TP53mut but not TP53wt ALL cells indicating ROS as a mediator of APR-246 induced cell death in TP53mut ALL. Furthermore, we addressed p53 activation in response to APR-246 by assessing phosphorylation of p53 (p53pSer15) using phosphoflow cytometry. Most interestingly, APR-246 led to 6-fold increased p53pSer15 levels in TP53mut compared to no activation in TP53wt leukemia cells, indicating restoration of p53 function upon APR-246 treatment in BCP-ALL.

Based on these findings, we addressed the effectivity of APR-246 on primary, patient-derived primografts and compared sensitivities for cell death induction in TP53mut (n=4) and TP53wt (n=4) samples. Importantly, the pattern of responsiveness of TP53mut ALL was also identified in TP53mut patient-derived ALL samples with induction of significantly higher cell death rates in TP53mut ALL (TP53mut 48%, TP53wt 18%, 5 µM APR-246, 24 h).

Taken together, we showed that TP53mut BCP-ALL can be targeted by APR-246 leading to re-activation of p53, induction of ROS dependent apoptosis and effective leukemia cell killing. Thus, targeting and re-activation of mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL.