Alice Cani

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G0/G1 phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/β and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34+/CD4−/CD7−), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.

Targeting the PI3K/Akt/mTOR signaling pathway in B-precursor acute lymphoblastic leukemia and its therapeutic potential

B-precursor acute lymphoblastic leukemia (B-pre ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. The prognosis of B-pre ALL has improved in pediatric patients, but the outcome is much less successful in adults. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K), Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) network is a feature of B-pre ALL, where it strongly influences cell growth and survival. RAD001, a selective mTORC1 inhibitor, has been shown to be cytotoxic against many types of cancer including hematological malignancies. To investigate whether mTORC1 could represent a target in the therapy of B-pre ALL, we treated cell lines and adult patient primary cells with RAD001. We documented that RAD001 decreased cell viability, induced cell cycle arrest in G0/G1 phase and caused apoptosis in B-pre ALL cell lines. Autophagy was also induced, which was important for the RAD001 cytotoxic effect, as downregulation of Beclin-1 reduced drug cytotoxicity. RAD001 strongly synergized with the novel allosteric Akt inhibitor MK-2206 in both cell lines and patient samples. Similar results were obtained with the combination CCI-779 plus GSK 690693. These findings point out that mTORC1 inhibitors, either as a single agent or in combination with Akt inhibitors, could represent a potential therapeutic innovative strategy in B-pre ALL.

Epstein-Barr virus-specific antibody response in cerebrospinal fluid and serum of patients with multiple sclerosis.

Cerebrospinal fluid and serum levels and intrathecal synthesis of anti-Epstein—Barr virus (EBV) IgG were measured by enzyme-linked immunosorbent assay in 80 relapsing—remitting multiple sclerosis patients grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. Eighty patients with other inflammatory neurological disorders (OIND) and 80 patients with non-inflammatory neurological disorders (NIND) served as neurological controls. Cerebrospinal fluid concentrations were higher in OIND than in multiple sclerosis (p < 0.0001) and NIND (p < 0.01) for anti-viral-capsid-antigen (anti-VCA) IgG, in multiple sclerosis than in NIND (p < 0.01) and in OIND than in NIND (p < 0.05) for anti-EBV nuclear antigen-1 (EBNA-1) IgG. Serum levels were more elevated in OIND than in multiple sclerosis (p < 0.05) and in MRI inactive than in MRI active multiple sclerosis (p < 0.0001) for anti-VCA IgG, and in multiple sclerosis than in OIND and NIND (p < 0.01) for anti-EBNA-1 IgG. Serum titres of anti-VCA and anti-EBNA-1 IgG were also positively (p < 0.05) and inversely (p < 0.001) correlated, respectively, with the Expanded Disability Status Scale. An intrathecal IgG production of anti-VCA and anti-EBNA-1 IgG, as indicated by Antibody Index, was present only in a limited number of multiple sclerosis patients and controls (range from 1.3 to 6.3%). These findings do not support a direct pathogenetic role of EBV-targeted humoral immune response in multiple sclerosis.

miR-199a-3p Modulates MTOR and PAK4 Pathways and Inhibits Tumor Growth in a Hepatocellular Carcinoma Transgenic Mouse Model

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Prognosis is poor, and therapeutic options are limited. MicroRNAs (miRNAs) have emerged as potential therapeutic molecules against cancer. Here, we investigated the therapeutic efficacy of miR-199a-3p, an miRNA highly expressed in normal liver and downregulated in virtually all HCCs. The therapeutic value of miR-199a-3p mimic molecules was assayed in the TG221 mouse, a transgenic model highly predisposed to the development of liver cancer. Administration of miR-199a-3p mimics in the TG221 transgenic mouse showing liver cancer led to a significant reduction of number and size of tumor nodules compared to control animals. In vivo delivery confirmed protein downregulation of the miR-199a-3p direct targets, mechanistic target of rapamycin (MTOR) and p21 activated kinase 4 (PAK4), ultimately leading to the repression of FOXM1. Remarkably, the anti-tumor activity of miR-199a-3p mimics was comparable to that obtained with sorafenib. These results suggested that miR-199a-3p may be considered a promising HCC therapeutic option.

Abstract B46: Activity of the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic leukemia and its therapeutic potential to prevent AKT reactivation

mTOR is a highly conserved and widely expressed serine/threonine kinase, that is a member of the phosphatidylinositol-3 kinase–like kinase (PIKK) family. mTOR plays a pivotal role in the PI3K/Akt/mTOR signaling pathway, which senses growth factor and serves as a central regulator of fundamental cellular processes such cell growth/apoptosis, autophagy, translation, and metabolism. Hyperactivation of this pathway through loss of negative regulators, such as PTEN, or mutational activation of receptor tyrosine kinases of phosphoinositide 3-kinase (PI3K) is a frequent occurrence in leukemia patients, where it negatively influences response to therapeutic treatments. In B-precursor acute lymphoblastic leukemia (B-pre ALL) many research efforts are currently devoted to the development of targeted therapies to limit side effects of chemotherapy and to increase treatment efficacy for poor prognosis patients, i.e. poor outcome following relapse. Thus, targeting the PI3K/AKT /mTOR pathway is an attractive therapeutic strategy. Numerous inhibitors targeting these kinases are currently undergoing clinical evaluation in hematological malignancies including ALL, mostly in combination with conventional chemotherapy. Prolonged suppression of mTORC1 also results in disruption of a negative feedback loop and often results in re/hyper-phosphorylation of Akt through activation of IRS1 and PI3K, which may counteract rapalogs activity. A new class of ATP-competitive mTOR inhibitors, such as Torin-2, has revealed that these inhibitors potently targets mTORC1 and mTORC2. Together, these features have generated hope that this new generation of inhibitors will exhibit broader clinical efficacy when compared to the rapalogs. We therefore hypothesized that dual inhibition of mTORC1 and mTORC2 by Torin-2 would provide a superior outcome in ALL as compared to inhibition of mTORC1 alone with RAD001 in B-pre-ALL. We tested the capability of Torin-2 to prevent AKT reactivation after mTORC1 and mTORC2 inhibition. Furthermore we explored if dual targeting of mTORC1 and AKT may achieve results similar to those obtained with Torin-2 alone. Drugs cytotoxic activity were analyzed in a panel of B-pre-ALL cells by MTT Assay and Western Blotting at different time points. Cell cycle, apoptosis and autophagy were analyzed by flow cytometry, Western Blotting and fluorescent staining. In all the B-pre-ALL cell lines Torin-2 showed a powerful cytotoxic activity, inhibiting the growth of each cell line in a dose-dependent manner, with an IC50 in the nanomolar range as assessed by MTT assays. The major proteins along the PI3K/AKT/mTOR signaling pathway where heavily dephosphorylated after 2 hrs of drug exposure. This inhibition lasted up to at least 48 hrs at variance to RAD001, that already after 24 hrs was unable to prevent AKT reactivation. However the association of RAD001 with MK-2206, an allosteric AKT inhibitor, prevented AKT reactivation and reached a significant cytotoxicity. These data suggest an interesting cytotoxicity of Torin-2 in B-pre-ALL acting on both mTORC1 and mTORC2 as assessed by their specific substrate inhibition. Feedback activation of PI3K/AKT was suppressed by Torin-2 alone, whereas RAD001 required the addition of MK-2206 to achieve the same efficacy. These two pharmacological strategies targeting PI3K/Akt/mTOR at different points of the signaling pathway cascade might represent a new therapeutic option in B-pre-ALL patients also preventing Akt reactivation. Citation Format: Carolina Simioni, Alice Cani, Alberto M. Martelli, Giorgio Zauli, Silvano Capitani, Luca M. Neri. Activity of the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic leukemia and its therapeutic potential to prevent AKT reactivation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B46.

Three hits are superior than one: multiple Akt inhibition as a new therapeutic strategy in T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder of T lymphocytes characterized by a poor clinical outcome, especially for relapsed patients [1]. The PI3K/Akt/mTOR signaling pathway is crucial for cell growth and survival in many types of solid and blood tumors, including T-ALL, influencing the response to therapeutic treatments [2]. The PI3K/Akt/mTOR network is often hyperactivated in T-ALL and therefore could constitute a target of inhibitory strate- gies, such as those that use small molecules inhibitors (SMI). The combined administration of multiple drugs is an attractive attempt to overcome drug resistance and to improve clinical outcome [3]. We tested in a panel of T-ALL cell lines three drugs directed against Akt with totally different modes of action: GSK690693, ATP- competitive, MK-2206, allosteric, and Perifosine, alkylphospholipid-Akt inhibitor. We showed that multiple Akt inhibition with this drug combination in T-ALL cell lines was cytotoxic and displayed a synergistic effect which was also related to the timing and the sequence of every drug administration. In fact, our findings showed that 6h of Perifosine pre-treatment followed by the combined administration of MK-2206 and GSK690693 for 30 min was necessary for the complete switch off of the activated protein. This combination caused a potent cell cycle arrest in G0/G1 phase and induced apoptosis and autophagy with more efficacy than single or double drug administration. In conclusion, our data demonstrated that this pharmacological strategy could represent a new promising treatment for patients affected by T-ALL with hyperacti- vated PI3K/Akt/mTOR signaling pathway.

Cytotoxic activity of the novel dual PI3K/mTOR inhibitor NVP-BGT226 in both normoxic and hypoxic hepatocarcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common lethal human malig- nancies worldwide. One of the most prevalent causes for the high mortality rate in patients with HCC is the lack of effective treatment, especially for patients with advanced disease (1). For this reason, an effective and well-tolerated pharmaceutical profile for the treatment of advanced HCC is requested to introduce new, potential therapeutic approaches. We evaluated the cytotoxic effect of the orally bioavailable dual PI3K/mTOR inhibitor, NVP-BGT226, on a panel of HCC cell lines, since hyperac- tivated PI3K/Akt/mTOR signaling pathway could represent a biomolecular target for Small Inhibitor Molecules in this neoplasia (2). We analyzed the activity of the drug in both normoxia and hypoxia conditions, which play often a relevant role in the induc- tion of chemoresistance and angiogenesis. Indeed, it has been recently demonstrated that PI3K/Akt signaling pathway regulates VEGF and HIF-1α expression, and inhibitors targeting PI3K p110α decrease both VEGF expression and angiogenesis in an vitro HCC model. In normoxia NVP-BGT226 caused cell cycle arrest in the G0/G1 phase of the cell cycle and induced apoptosis and autophagy at low concentrations. Interestingly, the drug inactivated p-Akt and p-S6 at a concentration lower than 10 nM. In hypoxia NVP-BGT226 maintained its cytotoxic efficacy at the same concentration, as documented by MTT assays and Western blot analysis. Moreover, in hypoxia the drug showed inhibitory properties against angiogenesis by lowering the expression of the transcription factor HIF-1α and of VEGF. Our results indicate that NVP-BGT226 has a potent cytotoxic effect on HCC cell lines also in hypoxia condition, thus emerging as a potential candidate for cancer treatment in HCC targeted therapy.

Abstract A34: Therapeutic potential of the novel mTOR inhibitor Torin-2 to overcome AKT reactivation in B-precursor acute lymphoblastic leukemia (B-pre ALL).

B-precursor acute lymphoblastic leukemia (B-pre ALL) is characterized by malignant proliferation and accumulation of early lymphoid precursor cells in the bone marrow, blood and lymphoid organs, due to acquired mutations in early B-cells. Around 20% of children and the majority of adults relapses after the treatment and the poor outcome following relapse did not significantly changed over the past twenty years. For this reason, many research efforts are currently devoted to the development of targeted therapies to limit side effects of chemotherapy and to increase treatment efficacy for poor prognosis patients. Constitutively active phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is a common feature of leukemia patients, where it negatively influences response to therapeutic treatments. Thus, targeting the PI3K/AKT /mTOR pathway is an attractive therapeutic strategy. Rapamycin, an allosteric inhibitor of mTORC1, acutely inhibits mTORC1 but not mTORC2 activity. Prolonged suppression of mTORC1 also results in disruption of a negative feedback loop and consequently results in hyperphosphorylation of Akt through activation of IRS1 and PI3K. mTOR inhibitors are currently in clinical trial for a range of hematological malignancies including ALL, mostly in combination with conventional chemotherapy. RAD001, a mTORC1 inhibitor, has been shown to affect cell cycle progression and survival with a potentially relevant therapeutic efficacy. However, prolonged inhibition of mTORC1 often leads to AKT re-phosphorylation at Ser473which may counteract RAD001 activity. A new class of ATP competitive mTOR inhibitors, such as Torin-2, have revealed that this new class of inhibitors potently targets mTORC1 and mTORC2. Together, these features have generated hope that the new generation of ATP-competitive mTOR inhibitors will exhibit broader clinical efficacy relative to the rapalogs. We therefore hypothesized that dual inhibition of mTORC1 and mTORC2 by Torin-2 would provide a superior outcome in ALL as compared to inhibition of mTORC1 alone with RAD001 in B-pre-ALL. We tested the capability of Torin-2 to prevent AKT reactivation after mTORC1 and mTORC2 inhibition. Furthermore we explored if dual targeting of mTORC1 and AKT may achieve results similar to those obtained with Torin-2 alone. We used a panel of B-pre-ALL to test the drugs by MTT Assay and Western Blotting after different time exposures of the cells. Cell cycle, apoptosis and autophagy were analyzed by flow cytometry, Western Blotting and fluorescent staining. In all the B-pre-ALL cell lines Torin-2 showed a powerful cytotoxic activity, inhibiting the growth of each cell line in a dose-dependent manner, with an IC50 in the nanomolar range as assessed by MTT assays. The major proteins along the PI3K/AKT/mTOR signaling pathway where heavily dephosphorylated after 2 hrs of drug exposure. This inhibition lasted up to at least 48 hrs at variance to RAD001, that already after 24 hrs was unable to prevent AKT reactivation. However the association of RAD001 with MK-2206, an allosteric AKT inhibitor, prevented AKT reactivation and reached a significant cytotoxicity. Our data suggest an interesting cytotoxicity of Torin-2 in B-pre-ALL acting on both mTORC1 and mTORC2 as assessed by their substrate inhibition. Torin-2 alone suppresses feedback activation of PI3K/AKT, whereas RAD001 requires the addition of MK-2206 to achieve the same efficacy. These two pharmacological options targeting PI3K/Akt/mTOR at different points of the signaling pathway cascade might represent a new therapeutic potential for treatment of B-pre-ALL patients. Citation Format: Alice Cani, Carolina Simioni, Alberto M. Martelli, Giorgio Zauli, Silvano Capitani, Luca M. Neri. Therapeutic potential of the novel mTOR inhibitor Torin-2 to overcome AKT reactivation in B-precursor acute lymphoblastic leukemia (B-pre ALL). [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A34.

Abstract 3750: The novel Akt inhibitor MK-2206, is cytotoxic in T-cell acute lymphoblastic leukemia: Therapeutic implications

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising within the thymus from the clonal proliferation of T-cell precursors. T-ALL accounts for 15% of ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of intensive polychemotherapy schemes, the outcome of relapsed and chemoresistant T-ALL is still poor, especially in adults, with a 35-40% survival at 5 years. Therefore, efforts are being made to develop targeted therapies against deregulated signaling cascades that sustain T-ALL cell proliferation, survival, and drug-resistance. A signaling pathway that is frequently upregulated in T-ALL, is the PI3K/Akt/mTOR network. To explore whether or not Akt could represent a potential target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines (MOLT-4, CEM, drug-resistant CEM) and primary cells from T-ALL patients, characterized by pathway upregulation. MK-2206 decreased T-ALL cell line viability as documented by MTT assays. IC50 for MK-2206 ranged from 1.0 to 4.8 μM at 48 hours. The drug was effective against drug-resistant CEM cells, overexpressing 170-kDa P-glycoprotein. MK-2206 blocked leukemic cells in the G1 phase of the cell cycle and induced caspase-dependent apoptotic cell death, as documented by Annexin V/propidium iodide staining and western blot analysis. In CEM cells, MK-2206 induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3β and FOXO3A, in response to MK-2206. mTORC1 downstream targets were also efficiently dephosphorylated by MK-2206, including p70S6K and 4E-BP1. MK-2206 decreased mTORC2 activity, as indicated by the downregulation of Ser 2481 p-mTOR levels, a readout for mTORC2 activity In MOLT-4 and CEM cells, MK-2206 strongly synergized (combination index: 0.1-0.33) with doxorubicin. Moreover, MK-2206 dephosphorylated Akt and induced apoptosis in a T-ALL patient cell subset (CD34+/CD4−/CD7−) which is enriched in leukemia initiating cells. Our findings indicate that Akt inhibition, either alone or in combination with chemotherapeutic drugs, represents a potential therapeutic target in T-ALL cells that require upregulation of the PI3K/Akt/mTOR signaling pathway for their proliferation and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3750. doi:1538-7445.AM2012-3750

Abstract 3736: The mTOR inhibitor, RAD001, displays higher cytotoxicity in leukemias with hyperactivated PI3K/AKT/mTOR pathway

Acute myelogenous leukemia (AML) is a disease resulting from the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. B-cell Acute lymphoblastic leukemia (B-ALL) is a form of leukemia characterized by lymphoblast abnormal increase. B-ALL is the most common form of cancer in childhood with a peak of incidence at 2-5 years of age, and another peak in the elderly. The overall cure rate in children is about 80%, and about 38% to 60% of adults have long-term disease-free survival. Although the prognosis of AML and ALL has improved in the last decades, the outcome of relapsed and chemoresistant AML and ALL is still poor, especially in adults, with only a 35-40% survival at 5 years. Therefore, major efforts are being made to develop rationally targeted therapies against alterated signaling cascades that sustain AML and ALL cell proliferation, survival, and drug-resistance. A signaling pathway that is frequently upregulated in AML and ALL is the PI3K/Akt/mTOR network. To explore whether this pathway could represent a potential pharmacological target in AML and ALL, we evaluated the effects of the mTOR inhibitor, RAD001, on the HL60 AML cell line and on the SEM B-ALL cell line.RAD001decreased viability in AML and ALL cell lines, as demonstrated by MTT experiments. The values of IC50 at 24 and 48 hours in HL60 cell line was 8μM, while in SEM cell line, that displays an hyperactivation of the PI3K/Akt/mTOR pathway, the IC50 range was from 4,7 to 6,8 μM, thus showing an higher sensitivity of this cell line to this targeted therapy. As documented by Western Blotting, RAD001 showed a concentration-dependent induction of apoptosis with a relevant increment of PARP, Caspase 3, 8 and 9 cleavage in both cell lines. The ALL cell line SEM, with hyperactivated PI3K/Akt/mTOR pathway, displayed a much higher Caspase and PARP cleavage dependent apoptosis.PI3K/Akt/mTOR downstream targets were also partially dephosphorylated by RAD001, in particular GSK3β and 4E-BP1. RAD001 induced no significant changes in Akt dephosphorylation in HL60 and SEM cell lines, and this is in agreement with several reports showing no relevant changes in the phosphorylation of Akt. The cells always expressed unchanged total Akt. This indicates that PI3K/Akt/mTOR inhibition could be an attractive target to develop innovative therapeutic strategies directed towards AML and ALL leukemia cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3736. doi:1538-7445.AM2012-3736