Cecilia Grimaldi

Activity of the Novel Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 against T-Cell Acute Lymphoblastic Leukemia

Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival, and drug resistance. These observations lend compelling weight to the application of PI3K/Akt/mTOR inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. NVP-BEZ235 was cytotoxic to a panel of T-ALL cell lines as determined by MTT assays. NVP-BEZ235 treatment resulted in cell cycle arrest and apoptosis. Western blots showed a dose- and time-dependent dephosphorylation of Akt and mTORC1 downstream targets in response to NVP-BEZ235. Remarkably, NVP-BEZ235 targeted the side population of both T-ALL cell lines and patient lymphoblasts, which might correspond to leukemia-initiating cells, and synergized with chemotherapeutic agents (cyclophosphamide, cytarabine, dexamethasone) currently used for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic to T-ALL patient lymphoblasts displaying pathway activation, where the drug dephosphorylated eukaryotic initiation factor 4E-binding protein 1, at variance with rapamycin. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment of those T-ALLs that have aberrant upregulation of this signaling pathway for their proliferation and survival.

The emerging role of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in normal myelopoiesis and leukemogenesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway mediates diverse and important physiological cell functions which include proliferation, differentiation, survival, motility, autophagy, and metabolism. However, dysregulated PI3K/Akt/mTOR signaling has been documented in a wide range of neoplasias, including malignant hematological disorders. It is now emerging that this signaling network plays a key role during normal hematopoiesis, a tightly regulated process resulting in the formation of all blood lineages. Blood cell development encompasses a complex series of events which are mainly regulated by actions of cytokines, a family of extracellular ligands which stimulate many biological responses in a wide array of cell types. Hematopoiesis is strictly dependent on the correct function of the bone marrow microenvironment (BMM), as BMM cells secrete most of the cytokines. Several of these cytokines activate the PI3K/Akt/mTOR signaling network and regulate proliferation, survival, and differentiation events during hematopoiesis. Here, we review the evidence that links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of hematopoietic stem cells and the process of myelopoiesis, including lineage commitment. We then highlight the emerging role played by aberrant PI3K/Akt/mTOR signaling during leukemogenesis.

AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be downmodulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4+ T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34+/CD7−/CD4−) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL.

Erucylphosphohomocholine, the first intravenously applicable alkylphosphocholine, is cytotoxic to acute myelogenous leukemia cells through JNK- and PP2A-dependent mechanisms

Alkylphospholipids and alkylphosphocholines (APCs) are promising antitumor agents, which target the plasma membrane and affect multiple signal transduction networks. We investigated the therapeutic potential of erucylphosphohomocholine (ErPC3), the first intravenously applicable APC, in human acute myelogenous leukemia (AML) cells. ErPC3 was tested on AML cell lines, as well as AML primary cells. At short (6–12 h) incubation times, the drug blocked cells in G2/M phase of the cell cycle, whereas, at longer incubation times, it decreased survival and induced cell death by apoptosis. ErPC3 caused JNK 1/2 activation as well as ERK 1/2 dephosphorylation. Pharmacological inhibition of caspase-3 or a JNK 1/2 inhibitor peptide markedly reduced ErPC3 cytotoxicity. Protein phosphatase 2A downregulation by siRNA opposed ERK 1/2 dephosphorylation and blunted the cytotoxic effect of ErPC3. ErPC3 was cytotoxic to AML primary cells and reduced the clonogenic activity of CD34+ leukemic cells. ErPC3 induced a significant apoptosis in the compartment (CD34+ CD38Low/Neg CD123+) enriched in putative leukemia-initiating cells. This conclusion was supported by ErPC3 cytotoxicity on AML blasts showing high aldehyde dehydrogenase activity and on the side population of AML cell lines and blasts. These findings indicate that ErPC3 might be a promising therapeutic agent for the treatment of AML patients.

The Emerging Role of the Phosphatidylinositol 3-Kinase/ Akt/Mammalian Target of Rapamycin Signaling Network in Cancer Stem Cell Biology

The cancer stem cell theory entails the existence of a hierarchically organized, rare population of cells which are responsible for tumor initiation, self-renewal/maintenance, and mutation accumulation. The cancer stem cell proposition could explain the high frequency of cancer relapse and resistance to currently available therapies. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway regulates a wide array of physiological cell functions which include differentiation, proliferation, survival, metabolism, autophagy, and motility. Dysregulated PI3K/Akt/mTOR signaling has been documented in many types of neoplasias. It is now emerging that this signaling network plays a key role in cancer stem cell biology. Interestingly, cancer stem cells displayed preferential sensitivity to pathway inhibition when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling pathways between neoplastic stem cells and healthy stem cells could be identified. In this review, we present the evidence which links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of cancer stem cells, both in solid and hematological tumors. We then highlight how targeting PI3K/Akt/mTOR signaling with small molecules could improve cancer patient outcome.

PKR Activity Is Required for Acute Leukemic Cell Maintenance and Growth: A Role for PKR-Mediated Phosphatase Activity to Regulate GSK-3 Phosphorylation

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p‐T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant‐negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time‐dependent augmentation of AKT S473 and GSK‐3α S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK‐3α was not dependent on PI3K activity. PKR inhibition augmented levels of p‐S473 AKT and p‐S21/9 GSK‐3α/β in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK‐3α or β phosphorylation in the presence of the AKT inhibitor, A443654. Pre‐treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2α, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2α phosphorylation. The effects of PKR inhibition on AKT and GSK‐3 phosphorylation were found to be, in part, PP2A‐dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK‐3. J. Cell. Physiol. 221: 232–241, 2009.

The side population of T-cell acute lymphoblasticleukemia cells is sensitive to modulators of PI3K/AKT/ mTOR signaling

The side population (SP), which overexpresses ABC-family membrane transporters including ABCG2 (also referred to as Breast Cancer Resistance Protein or BRCP), is thought to be enriched in cancer stem cells (CSC). SP cells are characterized by the capacity of extruding the Hoechst 33342 dye. The difficulty in eradicating tumors might be due to the fact that conventional treatments target the bulk of the tumor cells leaving behind the CSCs. Since CSCs may be responsible for aggressive behaviour of certain tumors as well as their sustained growth, strategies that target these cells will have significant clinical implications. So far the SP of T-cell acute lymphoblastic leukemia (T-ALL) has not been identified. Here, we have investigated the possible existence of SP cells in T-ALL cell lines and patients. In addition, we have studied if SP cells were sensitive to modulators of the PI3K/Akt/mTOR pathway. SP cells were analyzed using a Cell Lab Quanta SC flow cytometer equipped with an UV lamp and a 488 solid state laser. The Hoechst 33342 dye was excited at 366 nm. SP cells were gated on the FL1/FL3 histogram, while ABCG2 staining was evaluated on the FL2 channel. Hoechst staining specificity was demonstrated by the use of ABC-family transporter blocking agents (verapamil, fumitremorgin C, Ko143). The analysis documented the existence of cells displaying SP features (2.7-7.5%). Flow cytometric analysis demonstrated that the T-ALL SP expresses high levels of ABCG2, whereas the expression of two other ABC-family member transporters, P-gp or MRP1, was not detected. SP cells disappeared in samples treated with BEZ-235 (a dual inhibitor of PI3K/mTOR) or rapamycin (an mTORC1 inhibitor). Our findings indicate that SP cells exist also in T-ALL and are sensitive to PI3K/Akt/mTOR inhibition. Taken together, our preclinical findings strongly suggest that modulators of PI3K/AKT/mTOR could be valuable compounds for treating T-ALL because of their capability of targeting a population enriched in CSCs.