Francesca Buontempo

Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia.

Constitutively active phosphoinositide 3-kinase (PI3K) 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 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G2/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose- and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway.

Cytotoxic activity of the casein kinase 2 inhibitor CX-4945 against T-cell acute lymphoblastic leukemia: targeting the unfolded protein response signaling.

Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945—a novel, highly specific, orally available, ATP-competitive inhibitor of CK2α. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1α and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2α/PI3K/Akt/mTOR signaling.

Activity of the selective IκB kinase inhibitor BMS-345541 against T-cell acute lymphoblastic leukemia

Several lines of evidence suggest that the IκB kinase (IKK)/nuclear factor-κB (NFκB) axis is required for viability of leukemic cells and is a predictor of relapse in T-cell acute lymphoblastic leukemia (T-ALL). Moreover, many anticancer agents induce NFκB nuclear translocation and activation of its target genes, which counteract cellular resistance to chemotherapeutic drugs. Therefore, the design and the study of IKK-specific drugs is crucial to inhibit tumor cell proliferation and to prevent cancer drug-resistance. Here, we report the anti-proliferative effects induced by BMS-345541 (a highly selective IKK inhibitor) in three Notch1-mutated T-ALL cell lines and in T-ALL primary cells from pediatric patients. BMS-345541 induced apoptosis and an accumulation of cells in the G2/M phase of the cell cycle via inhibition of IKK/NFκB signaling. We also report that T-ALL cells treated with BMS-345541 displayed nuclear translocation of FOXO3a and restoration of its functions, including control of p21Cip1 expression levels. We demonstrated that FOXO3a subcellular re-distribution is independent of AKT and ERK 1/2 signaling, speculating that in T-ALL the loss of FOXO3a tumor suppressor function could be due to deregulation of IKK, as has been previously demonstrated in other cancer types. It is well known that, differently from p53, FOXO3a mutations have not yet been found in human tumors, which makes therapeutics activating FOXO3a more appealing than others. For these features, BMS-345541 could be used alone or in combination with traditional therapies in the treatment of T-ALL.

Nuclear phosphoinositides and their roles in cell biology and disease.

Since the late 1980s, a growing body of evidence has documented that phosphoinositides and their metabolizing enzymes, which regulate a large variety of cellular functions both in the cytoplasm and at the plasma membrane, are present also within the nucleus, where they are involved in processes such as cell proliferation, differentiation, and survival. Remarkably, nuclear phosphoinositide metabolism operates independently from that present elsewhere in the cell. Although nuclear phosphoinositides generate second messengers such as diacylglycerol and inositol 1,4,5 trisphosphate, it is becoming increasingly clear that they may act by themselves to influence chromatin structure, gene expression, DNA repair, and mRNA export. The understanding of the biological roles played by phosphoinositides is supported by the recent acquisitions demonstrating the presence in the nuclear compartment of several proteins harboring phosphoinositide-binding domains. Some of these proteins have functional roles in RNA splicing/processing and chromatin assembly. Moreover, recent evidence shows that nuclear phospholipase Cβ1 (a key phosphoinositide metabolizing enzyme) could somehow be involved in the myelodysplastic syndrome, i.e. a hematopoietic disorder that frequently evolves into an acute leukemia. This review aims to highlight the most significant and updated findings about phosphoinositide metabolism in the nucleus under both physiological and pathological conditions.

Autophagy in acute leukemias: A double-edged sword with important therapeutic implications

Macroautophagy, usually referred to as autophagy, is a degradative pathway wherein cytoplasmatic components such as aggregated/misfolded proteins and organelles are engulfed within double-membrane vesicles (autophagosomes) and then delivered to lysosomes for degradation. Autophagy plays an important role in the regulation of numerous physiological functions, including hematopoiesis, through elimination of aggregated/misfolded proteins, and damaged/superfluous organelles. The catabolic products of autophagy (amino acids, fatty acids, nucleotides) are released into the cytosol from autophagolysosomes and recycled into bio-energetic pathways. Therefore, autophagy allows cells to survive starvation and other unfavorable conditions, including hypoxia, heat shock, and microbial pathogens. Nevertheless, depending upon the cell context and functional status, autophagy can also serve as a death mechanism. The cohort of proteins that constitute the autophagy machinery function in a complex, multistep biochemical pathway which has been partially identified over the past decade. Dysregulation of autophagy may contribute to the development of several disorders, including acute leukemias. In this kind of hematologic malignancies, autophagy can either act as a chemo-resistance mechanism or have tumor suppressive functions, depending on the context. Therefore, strategies exploiting autophagy, either for activating or inhibiting it, could find a broad application for innovative treatment of acute leukemias and could significantly contribute to improved clinical outcomes. These aspects are discussed here after a brief introduction to the autophagic molecular machinery and its roles in hematopoiesis.

Targeting signaling pathways in T-cell acute lymphoblastic leukemia initiating cells.

Leukemia initiating cells (LICs) represent a reservoir that is believed to drive relapse and resistance to chemotherapy in blood malignant disorders. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder of immature hematopoietic precursors committed to the T-cell lineage. T-ALL comprises about 15% of pediatric and 25% of adult ALL cases and is prone to early relapse. Although the prognosis of T-ALL has improved especially in children due to the use of new intensified treatment protocols, the outcome of relapsed T-ALL cases is still poor. Putative LICs have been identified also in T-ALL. LICs are mostly quiescent and for this reason highly resistant to chemotherapy. Therefore, they evade treatment and give rise to disease relapse. At present great interest surrounds the development of targeted therapies against signaling networks aberrantly activated in LICs and important for their survival and drug-resistance. Both the Notch1 pathway and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) network are involved in T-ALL LIC survival and drug-resistance and could be targeted by small molecules. Thus, Notch1 and PI3K/Akt/mTOR inhibitors are currently being developed for clinical use either as single agents or in combination with conventional chemotherapy for T-ALL patient treatment. In this review, we summarize the existing knowledge of the relevance of Notch1 and PI3K/Akt/mTOR signaling in T-ALL LICs and we examine the rationale for targeting these key signal transduction networks by means of selective pharmacological inhibitors.

Antioxidants in the prevention of UVB-induced keratynocyte apoptosis.

Skin cells can respond to UVB-induced damage by counteracting it through antioxidant activation and DNA repair mechanisms or, when damage is massive by undergoing programmed cell death. Antioxidant factors, and, in particular, food compounds, have attracted much interest because of their potential use in new protective strategies for degenerative skin disorders. Melatonin, creatine and hydroxytyrosol show a variety of pharmacological and clinical benefits including anti-oxidant and anti-inflammatory activities. Here, the potential protective actions of antioxidant compounds against UVB-induced apoptosis were investigated in human keratinocytes. The cells were pre-treated with antioxidants before UVB exposure and their effect evaluated by means of ultrastructural and molecular analyses. After UVB radiation typical morphological apoptotic features and in situ DNA fragmentation after TUNEL reaction, appeared. A significant numerical decrease of apoptotic patterns could be observed when antioxidants were administrated before cell death induction. Moreover, both the intrinsic and extrinsic apoptotic pathways appeared activated after UVB radiation, and their down-regulation has been shown when antioxidants were added to cells before death induction. In conclusion, these compounds are able to prevent apoptotic cell death in human keratinocytes exposed to UVB, suggesting, for these molecules, an important role in preventing skin damage.

Synergistic cytotoxic effects of bortezomib and CK2 inhibitor CX-4945 in acute lymphoblastic leukemia: turning off the prosurvival er chaperone BIP/GRP78 and turning on the pro-apoptotic NF-κB.

The proteasome inhibitor bortezomib is a new targeted treatment option for refractory or relapsed acute lymphoblastic leukemia (ALL) patients. However, a limited efficacy of bortezomib alone has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. Recently, it has been documented that UPR disruption could be considered a selective anti-leukemia therapy. CX-4945, a potent casein kinase (CK) 2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. In this study, we analyzed in T- and B-ALL preclinical settings, the molecular mechanisms of synergistic apoptotic effects observed after bortezomib/CX-4945 combined treatment. We demonstrated that, adding CX-4945 after bortezomib treatment, prevented leukemic cells from engaging a functional UPR in order to buffer the bortezomib-mediated proteotoxic stress in ER lumen. We documented that the combined treatment decreased pro-survival ER chaperon BIP/Grp78 expression, via reduction of chaperoning activity of Hsp90. Bortezomib/CX-4945 treatment inhibited NF-κB signaling in T-ALL cell lines and primary cells from T-ALL patients, but, intriguingly, in B-ALL cells the drug combination activated NF-κB p65 pro-apoptotic functions. In fact in B-cells, the combined treatment induced p65-HDAC1 association with consequent repression of the anti-apoptotic target genes, Bcl-xL and XIAP. Exposure to NEMO (IKKγ)-binding domain inhibitor peptide reduced the cytotoxic effects of bortezomib/CX-4945 treatment. Overall, our findings demonstrated that CK2 inhibition could be useful in combination with bortezomib as a novel therapeutic strategy in both T- and B-ALL.

Tyrosol prevents apoptosis in irradiated keratinocytes

BACKGROUND Phenolic compounds, the biggest group of natural antioxidants, have attracted much attention due to their known and wide-ranging biological activities, as well as to their health effects. In particular, regardless their antioxidant activity, they play a key role in the control of several inflammation-associated processes as well as in improving antioxidant defense system. In an our previous work we have demonstrated the ability of Hydroxytyrosol, an ortho-diphenolic compound, essential component of oleuropein, in preventing apoptotic cell death induced by UVB radiation in HaCaT cell lines in vitro. In olive oil, besides Hydroxytyrosol, there are appreciable amounts of Tyrosol and its secoiridoid derivatives. OBJECTIVE It has been well established that Tyrosol has a significantly lower antioxidant activity than Hydroxytyrosol, but despite this, recent studies suggest that Tyrosol exerts a powerful protective effect against oxidative injuries in cell systems and that it is able to improve the intracellular antioxidant defenses. MATERIALS AND METHODS Here, Tyrosol effect has been evaluated in HaCaT cells exposed to UVB radiation by means of morphological and molecular analyses. RESULTS Our study revealed the polyphenol ability in reducing apoptotic markers and in protecting HaCaT cells from damage. CONCLUSION These findings suggest an important role of Tyrosol in protecting cells from apoptotic cell death and encourage the use of this phytochemical as biological ingredient in topical preparations as possible tool to prevent skin damage.

Targeting phosphatidylinositol 3-kinase signaling in acute myelogenous leukemia.

Introduction: Despite continuous advances in our knowledge of the biology of acute myelogenous leukemia (AML), the prognosis of AML patients treated with standard chemotherapy is still poor, especially in the elderly. Therefore, there is a need for novel targeted and less toxic therapies, particularly for patients who develop resistance to traditional chemotherapeutic drugs. Constitutively active phosphatidylinositol 3-kinase (PI3K) signaling characterizes many types of tumors, including AML, where it negatively influences response to therapeutic treatments. Areas covered: The literature data showed that small inhibitor molecules targeting PI3K signaling induced cell cycle arrest, apoptosis and decreased drug-resistance in AML cells. PI3K inhibitors were also capable of targeting leukemic initiating cells (LICs), the most relevant target for leukemia eradication, whereas they tended to spare healthy hematopoietic stem cells. Expert opinion: Data emerging from pre-clinical settings suggest that the PI3K pathway is critically involved in regulating proliferation, survival and drug-resistance of AML cells. Therefore, we propose that novel drugs targeting this signaling pathway may offer a novel and less toxic treatment option for AML patients, most likely in combination with a lower dosage of traditional chemotherapeutic agents or other innovative therapeutic agents.