Targeting AKT Signaling in Pediatric Acute Lymphoblastic Leukemia with Sulforaphane

Abstract 1521

Refractory and relapse acute lymphoblastic leukemia (ALL) is one of the leading causes of cancer-related death in children. Novel agents are still in need as a frontline therapy in high risk patients and salvage agent in relapse patients. Leukemic cells acquire a survival advantage over normal blood cells by losing control of the cell cycle and activating pro-survival signals. There are emerging evidences showing that the PI3K/AKT/mTOR pathway is frequently activated in both T cell acute lymphoblastic leukemia (T-ALL) and precursor B cell acute lymphoblastic leukemia (pre-B ALL) (approximately 30% of ALL patients). Thus, drugs able to inhibit AKT signaling are potential candidates for ALL treatment. Sulforaphane (SF) is a dietary compound found in cruciferous vegetables with well-known chemopreventive and chemotherapeutic function in solid tumors by inducing apoptosis and inhibiting survival and cell division. However, the anti-leukemic capacity of SF in hematologic malignancies has not been extensively investigated. In this study, we found increased cleavage of PARP, caspase-3, caspase-8 and caspase-9 by immunoblots in pre-B and T-ALL cell lines treated with SF (5–10 μM) for 24 –48 hours. In addition, we detected increased expression of p21 and cyclin B that correlated with a G2/M block and DNA damage observed in SF treated cells by analysis of DNA content and H2AXγ expression. Noteworthy, SF-treated pre-B and T-ALL cell lines exhibit lower levels of total and phosphorylated AKT in addition to decreased levels of total and phosphorylated mTOR, which was independent of p53 and PTEN activities. Lower survival signals upon SF treatment correlated with a dose- and time-dependent cytotoxicity in pre-B ALL cell lines (Nalm-6, REH and RS-4) and T-ALL cell lines (Jurkat, RPMI, DND-41 and KOPTK1) compared to EBV-transformed lymphoblasts used as a control. Interestingly, SF showed a synergistic effect with vincristine in pre-B and T-ALL cell lines, suggesting a potential use of SF in adjunctive therapy. Aiming to support future clinical trials, we confirmed that SF is effective in lymphoblasts from pediatric patients suffering with ALL, both pre-B and T, in a time- and dose-dependent manner (95% confidence interval IC50: pre-B ALL = 9.9 – 12.13 μM, T-ALL = 8.3 –11.00 μM). To investigate whether SF is effective in vivo in a xenograft mouse model, we injected nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with Nalm-6 cells transduced with GFP-FFluc for bioluminescent imaging detection. SF treatment (2 mg i.p. daily) significantly reduced tumor burden compared to the control group (injected with vehicle). Collectively, our data show that SF has anti-leukemic properties in ALL, both in primary samples from patients and cell lines, by inducing cell death selectively in leukemic cells likely by inhibiting AKT-dependent survival signals.