Eva Moran

Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.

Neoadjuvant chemotherapy and vaginal radical trachelectomy for fertility-sparing treatment in women affected by cervical cancer (FIGO stage IB-IIA1).

OBJECTIVES The aim of the present report is to support the feasibility and the safety of a new fertility-sparing treatment in young women affected by bulky cervical cancer. METHODS Between February 2007 and October 2010, seven patients presenting large IB-IIA1 tumors (30-45 mm) were scheduled for conservative treatment. All patients underwent neoadjuvant chemotherapy (NACT) followed by laparoscopic pelvic lymphadenectomy and vaginal radical trachelectomy (VRT). RESULTS One patient presented hematological toxicity during NACT (grade 3). All patients showed complete disappearance of tumor (n=4/7) or partial response (a 50% or more decrease in total tumor size, n=3/7) to neoadjuvant treatment, and they were all treated with pelvic lymphadenectomy and VRT. Additional treatment (interstitial brachytherapy) was offered to only one woman because of a persistent parametrial tumoral lesion. After a mean follow up of 22 months (range 5-49), no relapse was observed. To date, only one woman in our study attempted to conceive and she is currently pregnant. CONCLUSIONS Neoadjuvant chemotherapy for fertility sparing treatment is an innovative approach which is potentially quite interesting for many young women affected by bulky cervical cancer. These women, i.e. those with tumors larger than 2 cm (2-5 cm), are traditionally not offered fertility sparing treatment, thus the preliminary data we report here might have a promising impact. Nevertheless, for these patients it may be suitable to use the more radical, and time-tested, conservative surgical approach to allow for a complete and conservative excision of the residual tumor after neoadjuvant treatment. Studies with a larger number of patients and adequate follow-up are required to validate this conservative approach and to define clearly the good indications for this treatment.

Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD+-independent HDACs are an established therapeutic target, the relevance of NAD+-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD+-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD+ levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD+-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.

Potent synergistic interaction between the Nampt inhibitor APO866 and the apoptosis activator TRAIL in human leukemia cells

OBJECTIVE The nicotinamide phosphoribosyltransferase (Nampt) inhibitor APO866 depletes intracellular nicotinamide adenine dinucleotide (NAD(+)) and shows promising anticancer activity in preclinical studies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to plasma membrane receptors DR4 and DR5 and induces apoptosis via caspase-8 and -10. Here we have explored the interaction between APO866 and TRAIL in leukemia cell lines and in primary B-cell chronic lymphocytic leukemia cells. MATERIALS AND METHODS Cells were treated with APO866, TRAIL, or their combination. Viability and mitochondrial transmembrane potential (ΔΨ(m)) were determined by cell staining with propidium iodide and tetramethylrhodamine ethyl ester, respectively, and flow cytometry. Nampt and γ-tubulin levels, as well as caspase-3 cleavage were detected by immunoblotting. DR4 and DR5 expression were assessed by immunostaining and flow cytometry. Caspases were inhibited with zVAD-FMK and zDEVD-FMK; autophagy with 3-methyladenine, LY294002, and wortmannin. Intracellular NAD(+) and adenosine triphosphate (ATP) were measured by cycling assays and high-performance liquid chromatography (HPLC), respectively. RESULTS APO866 induced NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage in leukemia cells, thereby leading to autophagic cell death. TRAIL induced caspase-dependent apoptosis. TRAIL addition to APO866 synergistically increased its activity in leukemia cells by enhancing NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage. No DR5 upregulation at the cell surface in response to APO866 was observed. Remarkably, in healthy leukocytes APO866 and TRAIL were poorly active and failed to show any cooperation. CONCLUSIONS Activation of the extrinsic apoptotic cascade with TRAIL selectively amplifies the sequelae of Nampt inhibition in leukemia cells, and appears as a promising strategy to enhance APO866 activity in hematological malignancies.

Grb7 Upregulation Is a Molecular Adaptation to HER2 Signaling Inhibition Due to Removal of Akt-Mediated Gene Repression

The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2+ breast cancer cell line SKBR3, and Grb7 in MCF7 cells. Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs.

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as α-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Proteasome inhibitors as immunosuppressants: biological rationale and clinical experience.

Accumulating evidence supports the potential of proteasome inhibitors as immunosuppressants. Proteasome inhibitors interfere with antigen processing and presentation, as well as with the signaling cascades involved in immune cell function and survival. Both myeloma and healthy plasma cells appear to be highly susceptible to proteasome inhibitors due to impaired proteasomal activity in both cell types. As a consequence, these agents can be used to reduce antibody production and thus prevent antibody-induced tissue damage. Several clinical studies have explored the potential of bortezomib, a peptide boronate proteasome inhibitor, for treating immune disorders, such as antibody-mediated organ rejection and graft-versus-host disease (GVHD), with encouraging results. Here, we discuss the biological rationale for the use of proteasome inhibitors as immunosuppressive agents and review the clinical experience with bortezomib in immune-mediated diseases.

APO866 activity in hematologic malignancies: a preclinical in vitro study

To the editor: Nahimana and coworkers have recently reported that the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor APO866 elicited massive cell death in primary leukemia cells and in numerous leukemia/lymphoma cell lines.[1][1] In particular, in 32 primary leukemias (including 12 B-cell

Ras-Induced Resistance to Lapatinib is Overcome by MEK Inhibition

Lapatinib, a dual HER2 and EGFR tyrosine kinase inhibitor is highly active in HER2+ breast cancer. However, its efficacy is limited by either primary or acquired resistance. Although mutations in ras genes are rarely found in breast cancer, H-ras overexpression is frequently observed. Moreover, genetic alterations that do not directly involve ras such as Brk amplification, ultimately result in increased ras signaling. Using SKBR3 cells, a HER2+ breast cancer cell line that is naturally devoid of mutations in PI3KCA, PTEN, BRAF, and ras we show that both H-ras overexpression and expression of an oncogenic ras allele (ras V12) reduce susceptibility to lapatinib in analogy to what observed with activating PI3KCA mutations and with a constitutively active form of Akt. Importantly, we found that resistance to lapatinib due to ras overexpression or to ras V12 is overcome by MEK inhibition with U0126, suggesting a key role for the MEK-Erk pathway in ras-induced resistance. Similar results were obtained in BT474 cells, another HER+ breast cancer cell line. Therefore, our data indicate that overexpressed/mutated ras may act as a biological modifier of the response to lapatinib. Combining MEK inhibitors with lapatinib may help overcome this form of resistance and increase the efficacy of lapatinib in these tumors.

Monoclonal Antibodies for Non-Hodgkin's Lymphoma: State of the Art and Perspectives

Monoclonal antibodies have been the most successful therapeutics ever brought to cancer treatment by immune technologies. The use of monoclonal antibodies in B-cell Non-Hodgkins lymphomas (NHL) represents the greatest example of these advances, as the introduction of the anti-CD20 antibody rituximab has had a dramatic impact on how we treat this group of diseases today. Despite this success, several questions about how to optimize the use of monoclonal antibodies in NHL remain open. The best administration schedules, as well as the optimal duration of rituximab treatment, have yet to be determined. A deeper knowledge of the mechanisms underlying resistance to rituximab is also necessary in order to improve the activity of this and of similar therapeutics. Finally, new antibodies and biological agents are entering the scene and their advantages over rituximab will have to be assessed. We will discuss these issues and present an overview of the most significant clinical studies with monoclonal antibodies for NHL treatment carried out to date.