Additional file 3: of EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

Effects of CHS-828 and chemotherapeutics on protein translation. A) Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of CHS-828. Caspase 3/7 activity was quantified (using 5 μM of Camptothecin for 4 h as a positive control of apoptosis) and relative ATP levels were determined and then normalized to the number of viable cells. The levels of total AMPK, p-AMPK, total EIF2A and p-EIF2A, total 4EBP1, p-4EBP1 were evaluated by WB. Histogram shows the densitometric analysis of p-AMPK and p-EIF2A (* indicates p-value <0.05). Mean and SD of a biological triplicate. B) Jurkat cells were treated with the indicated concentration of drugs for 48 h and cell viability was measured by Cell Titer Glo. Data are represented as mean and SD of three independent experiments. C) Click-it chemistry based on the incorporation of an aminoacid analog (AHA) was used to monitor protein synthesis. Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866, Rapamycin (RAPA), Doxorubicin (DOXO), Cisplatin (CIS) and Dexamethasone (DEXA). The histogram quantifies the % of AHA positive cells (active protein-synthesizing cells) in the viable cell population. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 20000 events/sample. D) Jurkat cells were treated as in C and the level of p-EIF2A and p-4EBP1 was evaluated. Histogram shows the densitometric analysis of p-EIF2A (* indicates p-value <0.05). Mean and SD of a biological triplicate. E) Primary B-CLL cells were treated for 48 h with or without 30 nM FK866 in the presence or absence of 1 mM NA. Histogram shows the densitometric analysis of p-AMPK/AMPK. (PDF 691 kb

Activated T cells undergo massive NAD⁺ depletion upon Nampt inhibition.

<p>A, 3×10⁶ PBLs/well were stimulated (or not, unstim.) with 5 µg/ml PHA, 1 µg/ml con A, or 50 ng/ml PMA and 0.5 µM ionomycin in the presence or absence of the indicated FK866 concentrations. 48 h later, cells were lysed in 0.6 M PCA and NAD⁺ content was measured in neutralized extracts. NAD⁺ levels were normalized to those detected in the absence of FK866. B, Unstimulated or PHA-stimulated PBLs were treated with 33 nM FK866 for 48 h before NAD⁺ content was determined. Absolute NAD⁺ levels are presented. *: p<0.05. C, PBLs were cultured for 48 h with PHA with or without FK866 (33 nM) addition. Subsequently, pyridine dinucleotides levels were measured in acid (NAD⁺ and NAPD⁺) or alkaline (NADH and NADPH) cell extracts. Dinucleotide levels were normalized to those detected without FK866. D, PBLs were incubated with PHA and 33 nM FK866 for the indicated times. Thereafter cells were harvested and NAD⁺ and ATP levels were determined in cell extracts whereas cell viability was assessed by PI-staining and flow cytometry. Results were normalized to the values of FK866-untreated cells. E, Resting or PHA-stimulated PBLs were treated (or not) with 33 nM FK866 in the presence or absence of 1 mM NAD⁺. After five-days viability was assessed determining PI⁻ cells by flow cytometry. Results are means ± SD of five (A) or three (B–E) experiments.</p

Evidence for an involvement of Sirt6 in IFN-γ synthesis.

<p>A, PBLs were cultured for 24 h with or without PHA. Thereafter, Sirt6 levels were detected by Q-PCR. mRNA levels in PHA-stimulated cells were compared to those in unstimulated PBLs. B, C, Jurkat cells were transduced with PRS, (PRS) GFP-sh, or (PRS) S6 sh2, subsequently, Sirt6 mRNA levels or Sirt6 protein levels were determined by Q-PCR (B) and immunoblotting (C). D–F, Jurkat cells transduced with PRS or S6 sh2 were stimulated for 12 h with 5 µg/ml PHA, 50 ng/ml PMA, and 0.5 µM ionomycin. Thereafter, supernatants were harvested and TNF-α (D), IFN-γ (E), and IL-4 (F) levels were determined by ELISA. G, H9 cells transduced with GFP-sh or S6 sh2 were stimulated for 12 h with 5 µg/ml PHA, 50 ng/ml PMA, and 0.5 µM ionomycin. Thereafter, intracellular IFN-γ was detected by intracellular staining. Mean fluorescence intensity for IFN-γ expression is indicated for each histogram. H, 3×10⁶ splenocytes from wild type or <i>Sirt6 KO</i> mice/well were seeded in 24 well plates and stimulated for 24 h with 1 µg/ml Con A. Thereafter, supernatants were harvested and IFN-γ levels were determined by ELISA. *: p<0.05. Results are means ± SD of three experiments (A, B, D–F). Panel C and G are representative of three separate experiments.</p

PARP inhibitors and sirtinol attenuate FK866-induced T cell demise.

<p>A, PBLs were cultured for 24 h with or without PHA. Thereafter, Nampt and PARP1 levels were detected by Q-PCR. mRNA levels in PHA-stimulated cells were compared to those in unstimulated PBLs. B, Resting or PHA-stimulated PBLs were incubated with or without 33 nM FK866 in the presence or absence of 300 µM NU1025, 10 µM PJ34, or 300 µM 3-AB. 48 h later NAD⁺ levels were assessed (presented as % of values in FK866-untreated PBLs). *, p<0.05. C, PHA-stimulated PBLs were incubated for five days with or without 33 nM FK866 in the presence or absence of 300 µM NU1025, 10 µM PJ34, or 300 µM 3-AB. Thereafter, viability was assessed. D, 5×10⁵ Jurkat cells were treated for two days with 500 pM FK866 in the presence or absence of 300 µM NU1025, 5 µM PJ34, or 300 µM 3-AB. Subsequently, NAD⁺ content was determined and expressed as % of values in FK866-untreated Jurkat. E, 3×10⁴ Jurkat cells/well were incubated in 96-well plates with or without 300 pM FK866 in the presence or absence of the indicated concentrations of NU1025, PJ34, or 3-AB. Viability was determined 96 h later by PI cell staining and flow cytometry. F, PBLs were incubated for five days with PHA, with or without 33 nM FK866, in the presence or absence of 30 µM sirtinol. Viability was subsequently assessed by PI staining and flow cytometry. C, E, F, each treatment was tested in triplicate wells. Results are presented as means ± SD of three experiments.</p

Nam and Na prevent NAD⁺ shortage and cell death induced by FK866 in human T lymphocytes.

<p>A, PHA-stimulated PBLs were treated (or not) with 33 nM FK866 in the presence or absence of 10 mM Nam or of 10 µM Na. After 48 h, NAD⁺ content was determined (expressed as percentage of NAD⁺ content in FK866-untreated cells). B, PBLs were cultured for 24 h with or without PHA, 1 µg/ml Con A, or 50 ng/ml PMA and 0.5 µM ionomycin. Thereafter, Naprt1 mRNA levels were detected by Q-PCR. mRNA levels in mitogen-stimulated PBLs were compared to those in unstimulated PBLs. C, D, PHA-stimulated PBLs were incubated for five days with or without 10 mM Nam or 10 µM Na in the presence or absence of the indicated FK866 concentrations. Thereafter, cells were imaged by light microscopy (C), and cell viability was determined (D). E, PHA-stimulated PBLs were incubated for five days with or without 33 nM Fk866 in the presence of the indicated concentrations of Nam, Na, or tryptophan (Trp). Viability was subsequently determined. F, PBLs were stimulated with or without PHA in the presence or absence of 1 mM Nam or Na. Thymidine incorporation was measured after 48 h by a 16-h pulse with 0.5 µCi/well [³H]thymidine. D-F each treatment was tested in triplicate wells. Results are means ± SD of three (A, B, F) or four (D, E) experiments. In panel C, one representative experiment out of three is shown.</p

A putative model of Nampt's role in activated T lymphocytes.

<p>Nampt activity is responsible for providing sufficient NAD⁺ supplies during T cell activation. NAD⁺, in turn, is required for ATP synthesis, metabolic reactions, and to replenish NADPH levels. In addition, NAD⁺ represents the substrate of NAD⁺-degrading enzymes such as PARP, CD38, and the sirtuins. Among these, Sirt6 appears to have a central role in IFN-γ and TNF-α production. Nampt inhibitors such as FK866 (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune disorders.</p

Nampt inhibition with FK866 prevents T lymphocyte proliferation and selectively kills activated T cells.

<p>A, PBLs were seeded in 96-well plates in the presence or absence (unstim.) of PHA and 33 nM FK866. Proliferation was assessed 96 h later by standard [³H]thymidine incorporation assay. B, PBLs were incubated in 96-well plates in the presence or absence of 5 µg/ml PHA, 1 µg/ml Con A, with or without the indicated concentrations of FK866. Five days later viability was detected by PI staining and flow cytometry. Spontanous cell death was 12.2% and 28.1% for PHA- and Con A-stimulated PBLs, respectively. C, D, PBLs were stimulated for 7 days with or without allogeneic mature DCs before FK866 at the indicated concentrations was added. After 5 days viability was assessed by PI staining and flow cytometric analysis using the lymphocyte gate (C). Spontaneous PBL death was 18.4%. D: phenotype of unstimulated or DC-stimulated PBLs. E, Immature or LPS stimulated DCs were cultured for 7 days with 33 nM FK866 before staining with FITC-conjugated Annexin-V and PI and flow cytometry. F, Resting or PHA-stimulated PBLs were treated with 33 nM FK866 for the indicated times and subsequently stained with FITC-conjugated Annexin-V and PI for flow cytometric analysis. Mean values ± SD of five (B) and three (A, C) different donors are presented. D–F One representative experiment out of three is shown.</p

Nampt inhibition with FK866 induces mitochondria depolarization and ATP depletion in activated T lymphocytes.

<p>A, PHA- stimulated PBLs were incubated with 33 nM FK866 and <i>ΔΨ</i>_m was determined at the indicated days of exposure. B, Resting of PHA-stimulated PBLs were cultured with 33 nM FK866 for five days. Thereafter PBLs with conserved <i>ΔΨ</i>_m-high were quantified by flow-cytometry. C, Bcl2-overexpressing Jurkat and the respective vector control cells were incubated with 10 nM FK866 for the indicated number of days. Thereafter, <i>ΔΨ</i>_m was determined. Inset, Western blot for Bcl2 and γ-tubulin expression. D, 5×10⁵ Bcl2-overexpressing Jurkat and the vector control cells were incubated with or without 10 nM FK866 for the indicated times before ATP was detected. ATP levels are presented as % of ATP in FK866-untreated cells. E, 3×10⁴ Bcl2-overexpressing and control Jurkat cells/well were incubated in 96-well plates with or without the indicated FK866 concentrations. Viability was determined by PI staining and flow cytometry 96 h later. F, PHA-stimulated PBLs were incubated in the presence or absence of 33 nM FK866 with or without the indicated concentrations of 3-MA. Viability was detected after five days. *, p<0.05. G, PBLs incubated in 96-well plates in the presence of 5 µg/ml PHA were treated for five days with the indicated FK866 concentrations in the presence or absence of 20 µM LY294002. Viability was subsequently determined by PI staining and flow cytometry. B, Results are presented as means ± SD of three experiments (B, D–G). Panels A and C are representative of three separate experiments.</p

FK866 ameliorates EAE.

<p>10 mg/kg body weight FK866 were administered to mice from day 12 after rMOG immunization for 10 days. A, NAD(H) and NADP(H) levels were measured in mononuclear cells isolated from spleen and lymph nodes of treated or untreated animals at day 16. Dinucleotide levels in FK866-treated mice were expressed as % of those detected in control animals. B, FK866 halts EAE severity compared with controls (p<0.05 from day 19 onward). Arrows indicate the days of FK866 administration. C, Luxol fast Blue staining of the spinal cord shows areas of demyelination in control mice compared with FK866-treated animals.</p

Clinical-pathologic features of EAE-affected mice treated with FK866.

<p>*p<0.05 (Mann-Whitney test).</p