p53 Suppression Mediates Blasts Survival, Proliferation and CD34 Maintenance in Pediatric Acute Myeloid Leukemia

Aberrations in the DNA damage responses contributes to the uncontrolled proliferation and therapy resistance in many cancers. Acute myeloid leukemia (AML) presents enhanced DNA damage as compared to MDS characterized by induced levels γ-2HAX and phosphorylated ATM that correlated with AML blast percentages (Boehrer et al. Oncogene 2009). The low incidence of p53 mutations (7% in adult AML, The Cancer Genome Atlas Research Network, N Engl J Med 2013) in AML indicates downstream suppression of DNA damage control system proteins, as was proposed previously by overexpression of MDM2 and BCL-2 (Wojcik et al. Neoplasma 2005). However, the role of p53 and downstream effectors regulating the DNA damage pathway in CD34+ AML blasts is still relatively unclear.

Surprisingly, using RPPA analysis, we found a common significant increase in p53 phosphorylation at serine 15 in pediatric AML as compared to CD34+ NBM (AML n=31 and NBM n=10, Mann-Whitney U, P = 0.003). Therefore, we challenged to target p53 using a shRNA approach. In the sh-p53 model, primary pediatric AML samples transduced with sh-p53 showed a significant increase in AML proliferation over time as compared to scrambled control (paired-samples t-test, P = 0.005). The overall AML cell viability was significantly improved in sh-p53 AML cultures (paired-samples t-test P = 0.001). DNA cell cycle analysis revealed that sh-p53 AML cultures contained significant increased percentages of cells in S and G2/M phases of the cell cycle as compared to their scrambled controls (paired samples t-test, P = 0.015). The CD34+ leukemic population was maintained in p53 knockdown cultures and thereby outcompeted scrambled control cultures. Knockdown of p53 in primary pediatric AML samples resulted in a higher proliferation rate, repressed differentiation and prolonged survival of AML blasts, especially in MLL-rearranged AML. Interestingly, we previously showed that MLL-rearranged AML samples express significantly higher BCL-2 peptide phosphorylation as compared to NBM (Kampen et al. Leukemia 2014). Growth factors are extrinsic cues that can regulate BCL-2 expression (Pidgeon et al. Br J Cancer 2001). Growth factors like VEGF, have been shown to be involved in leukemia pathogenesis in the bone marrow where leukemic stem cells are preserved in tightly controlled niches (Kampen et al. Cell Mol Life Sci 2013). Accordingly, we found that FGF and VEGFC stimulation of CD34+ AML blasts both significantly accelerated BCL-2 mRNA expression by 28-fold and 2-fold, indicating that indeed the niche is important for inhibiting pro-apoptotic p53-dependent signals further downstream the pathway, which protects AML CD34+ blasts (Student’s t-test, both P < 0.001), where p53 and p21 mRNA expression remained unchanged. The availability of VEGFR-2 and FGFR1 previously highlighted their therapeutic targeting potential in AML (Kampen et al. Leukemia 2006, Kentsis et al, Nature Med 2012). This study presents the effect of p53 suppression on AML blast proliferation, survival and maintenance in pediatric AML and indicates the importance of exploring extrinsic factors that contribute to DNA damage control system suppression in vivo for therapeutic interventions.