The Mechanism And Combination Therapy Of GLI And AKT Inhibitors That Induce Apoptosis And Autophagy In Human Leukemia T Cells

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder of developing T cells in the thymus and accounts for 10%～15% of pediatric and 25% of adult ALL cases. Despite novel combination chemotherapy regiments, prognoses were still extremely poor. Among these patients, only 70～80% of children and as few as 40% of adults reach long-term remission.Hedgehog (Hh) signaling plays a critical role in embryogenesis and adult tissue homeostasis. The Hh ligands which include Sonic hedgehog (SHH), Desert hedgehog (DHH) and Indian hedgehog (IHH), bind to the receptor Patched (PTCH). This results in the activation of a second transmembrane protein Smoothened (SMO). Once activated, SMO results in stabilization and nuclear accumulation of GLI family members GLI family zinc finger 1 (GLI1), GLI family zinc finger 2 (GLI2) and GLI family zinc finger 3 (GLI3), defined as canonical Hh signaling. There is ample evidence to suggest that aberrantly activated Hh signaling is associated with the development of a variety of human tumors. The GLI antagonists GANT58 and GANT61 are more potent in inducing growth arrest and apoptosis compared to SMO inhibitor cyclopamine in a number of cancer cells. This has been found to be the case in myeloid leukemia cells, colon carcinoma cells, rhabdomyosarcoma cells, ovine squamouscell carcinoma cells and chronic leukemia cells. Despite these studies, information surrounding the role of Hh signaling and use of GLI or SMO inhibitors in T-ALL cell lines has been little investigated.In addition, recent findings have highlighted constitutively active phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling as a common feature of T-ALL. Genetic or post translational inactivation of the phosphatase and tensin homolog (PTEN) is common in T-ALL, leading to constitutively active PI3K/AKT/mTOR pathway. The signaling plays a central role in regulating cell growth, survival and autophagy of tumor cells. Perifosine is a novel alkylphospholipid analog and prevents AKT membrane localization and phosphorylation, resulting in inhibiting AKT. The new AKT inhibitor exhibits antitumor activity in various preclinical models and is currently undergoing phase Ⅰ, Ⅱ and Ⅲ clinical evaluation in many cancers such as chronic lymphocytic leukemia, renal cell carcinoma, ovarian cancer, multiple myeloma and colorectal cancer. However, the molecular mechanism underlying the antitumor activity of perifosine has not been fully elucidated.In this study, we used GLI antagonists and AKT inhibitor to investigate the role of Hh and AKT signaling, the interaction with two pathway, the potential cytotoxic effects and mechanism of the combination of different drugs in T-ALL cells.Part I Inhibition of hedgehog signaling by GANT58 induces apoptosis and shows synergistic antitumor activity with AKT inhibitor in acute T cell leukemia cellsThe hedgehog (Hh) and AKT signaling pathways have a crucial role in cell proliferation and survival, and the deregulation of these pathways can lead to tumorigenesis. Here we cultured four different T-ALL cell lines:CCRF-CEM, Jurkat, Human CEM clonal cell lines C7-14 and C1-15 and chose different inhibitors to investigate the expression and function of these pathways in T-ALL.1. Profiling of Hh pathway members revealed common expression of key Hh signaling effectors in all T-ALL cells. Using SYBR Green based real-time PCR, all four tested T-ALL cell lines expressed different levels of the Hh receptors PTCH1 and SMO, GLI1 transcription factors as well as a negative regulator of GLI proteins, Suppressor of Fused (SUFU). By western blot, we found two different variants of GL11 proteins expressed in T-ALL cell lines. GLI1△N was expressed in all T-ALL cell lines but at very low levels in CCRF-CEM, CEM7-14 and CEM1-15 cell lines. GLI1FL was strongly expressed in CCRF-CEM, CEM7-14 and CEM1-15 cells but was not detected in Jurkat cells.2. Cyclopamine confers only a weak cytotoxic effect on T-ALL cells and GANT58 confers a more potent cytotoxic effect than Cyclopamine. T-ALL cells were insensitive to specific Smoothened (SMO) inhibition following the use of low concentrations (1μM or 3μM) of the SMO antagonist cyclopamine. In contrast, treatment with the novel GLI antagonist GANT58 reduced expression of the target gene Patched 1 as well as GLI family zinc finger 1 (GLI1) and preferentially decreased the viability of T-ALL cells. The order of sensitivity to GANT58 for these cell lines is CEM 1-15> CEM7-14> CCRF-CEM> Jurkat, which is consistent with the expression level of GLI1. The decrease in cell viability was accompanied by the increase of apoptosis and cell cycle arrest.3. Perifosine down-regulates Hh/GLI pathway in T-ALL cell lines and Inhibition of MEK/ERK signaling pathway augments the effect of perifosine down-regulating GLI1 in T-ALL cell lines. Perifosine down-regulated GLI1 protein by dephosphorylation of AKT and GSK3β dose-dependently. Perifosine treatment also induced the increase of phosphorylation of ERK1/2 and that pretreatment with PD98059, a MEK/ERK pathway inhibitor, enhanced this down-regulation by 20%-30%.4. AKT inhibitor acts in synergy with GANT58 on the cytotoxic effect of T-ALL cells. By applying the Chou-Talalay method, low concentration of GANT58 induced T-ALL cell death in a synergism fashion with perifosine or GSK690693 when used simultaneously. These findings indicate that the combined use of GANT58 and AKT inhibitor could help treat a broad range of malignant tumors in conjunction with existing cancer treatments.Part II Perifosine induces protective autophagy by AKT/GSK3β and MEK/ERK pathways and acts synergistic antitumor activity with chloroquine in acute T cell leukemia cellsAutophagy is a cellular degradative process within lysosomes to recycle and turnover cellular constituents for maintaining cellular homeostasis. Many studies have shown that deregulation of autophagy is implicated in certain human diseases including tumor growth. The role of autophagy in tumor cells is complicated. It can facilitate cellular survival but may also contribute to cell death in response to cancer treatment as reported. The opposing consequences may depend on many circumstances such as tumor types, the stage of tumor development and the nature and extent of treatment. The effect of perifosine on autophagy has not yet been determined in T-ALL. In this study, we focus on whether perifosine has an impact on autophagy through PI3K/AKT or MAPK pathways in T-ALL and the role of autophagy in perifosine-induced cell death.1. Perifosine reduces cell viability and induces apoptosis of T-ALL cell lines. Perifosine inhibited cell growth of CCRF-CEM, CEM7-14, CEM1-15 and Jurkat cells with 50% inhibition (IC50) at 24h of 15.02,13.08,18.06 and 21.35μM, respectively. The sensitivities of the four cell lines to perifosine were different:C7-14> CCRF-CEM> C1-15> Jurkat. Caspase 3 was detected to be activated in a dose-dependent manner, indicating the cytotoxicity was related to apoptosis.2. Perifosine treatment induces autophagy in T-ALL cell lines. Perifosine induced the switch of LC3-Ⅰ to LC3-Ⅱ in a dose dependent manner in CEM7-14 and CEM1-15 cells, while both LC3-Ⅰ and LC3-Ⅱ decreased in a dose dependent fashion in CCRF-CEM and Jurkat cells after the treatment of perifosine.3. The inhibition of autophagy by lysosomotropic inhibitor chloroquine (CQ) increased the sensitivity of T-ALL cells to perifosine treatment. Blocking autophagy by CQ enhanced perifosine-inducing cell death in these cells by 10%-30%.4. Perifosine inhibited AKT/GSK3β and activated MEK/ERK pathway in T-ALL cells; Perifosine induces autophagy that is dependent on activation of MEK/ERK signaling pathway. Perifosine inhibited p-AKT and p-GSK3β while induced ERK1/2 phosphorylation in a dose-dependent fashion, accompanied with the changes in caspase 3 and LC3. Pre-treatment of MEK/ERK inhibitor PD98059 blocked autophagy induction and significantly augmented perifosine-inducing apoptosis in T-ALL cells by 40%.