Induction of Apoptosis and Autophagy By Nampt Inhibition in Adult T-Cell Leukemia/Lymphoma and Leukemic Cell Lines

Abstract Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus type I (HTLV-1). Despite the recent advances in chemotherapy, allogeneic hematopoietic stem cell transplantation, and supportive care, the prognosis for patients with acute, lymphoma, or unfavorable chronic subtypes is one of the poorest among hematological malignancies. The identification of new molecular targets for ATL prevention and treatment is desired. SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, plays crucial roles in various physiological processes, including aging and apoptosis. We previously reported that ATL patients had significantly higher SIRT1 protein levels and novel small-molecule SIRT1 inhibitors are highly effective against ATL cells.1,2 Nicotinamide phosphoribosyltransferase (Nampt) also known as pre-B-cell colony-enhancing factor 1 or visfatin is a rate-limiting enzyme in NAD+ biosynthesis, and it regulates intracellular ATP levels in mammalian cells. Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma) is sensitive to low concentrations of FK866, Nampt inhibitor, as measured in cytotoxicity and clonogenic assays.3Here, we assessed how Nampt is regulated in ATL cells and leukemic cell lines. Results: We observed that ATL patients had significantly higher SIRT1 and Nampt protein levels than healthy controls. FK866 induced significant growth inhibition and apoptosis (Annexin V+ cells and TUNEL) in leukemia/lymphoma cell lines (HTLV-1-related cell lines: S1T, MT-2; Jurkat and HL60). FK866 showed potent activities with GI50values of 0.63, 3.7, 1.0, and 3,4 nM for S1T, MT-2, Jurkat, and HL60 cells, respectively. FK866 also activated caspase activity (caspase-3, 8, and 9) with DNA fragmentation. However, a caspase inhibitor did not inhibit this caspase-dependent cell death. Interestingly, FK866 increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation as well as autophagy. Autophagy detection was also performed using the CytoID Autophagy detection kit. Autophagy levels are increased in the presence of STF-62247 pre-treated with bafilomycin A1, a specific inhibitor of vacuolar proton ATPase, whose inhibition is known to block the fusion of autophagosomes with lysosomes for 2 h. Thus, FK866 simultaneously caused apoptosis and autophagy. Conclusion:These results suggest that Nampt inhibitor is highly effective against ATL cells in caspase-dependent or -independent manners with autophagy, and that its clinical application might improve the prognosis of patients with this fatal disease. 1. Kozako T, Aikawa A, Shoji T, et al. High expression of the longevity gene product SIRT1 and apoptosis induction by sirtinol in adult T-cell leukemia cells. Int J Cancer. 2012;131:2044-2055. 2. Kozako T, Suzuki T, Yoshimitsu M, et al. Novel small-molecule SIRT1 inhibitors induce cell death in adult T-cell leukaemia cells. Sci Rep. 2015;5:11345. 3. Nahimana A, Attinger A, Aubry D, et al. The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies. Blood. 2009;113:3276-3286. Disclosures Yoshimitsu: HUYA Bioscience International: Research Funding.

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Overexpression of SIRT1, a Longevity Gene-Encoded Protein, and Induction of Apoptosis by Its Inhibition In Adult T-Cell Leukemia Cells

Blood ◽

10.1182/blood.v116.21.2768.2768 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2768-2768

Author(s):

Tomohiro Kozako ◽

Makoto Yoshimitsu ◽

Yohann White ◽

Akiyoshi Aikawa ◽

Teruhisa Shoji ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Leukemic Cell ◽

Acute Type ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Leukemic Cell Lines ◽

Sirt1 Inhibitor ◽

Induced Apoptosis

Abstract Abstract 2768 Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm developing after a long-term infection with human T-cell leukemia virus (HTLV-1), in which NF-kB is also implicated as an exacerbation factor. Despite recent progress in both chemotherapy and supportive care for hematological malignancies, the prognosis of ATL is still poor; overall survival at 3 years is only 24%. New strategies for the therapy and prophylaxis of ATL (e.g., vaccines and novel molecular target agents) are still required. SIRT1, an NAD+-dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, due to its ability to deacetylate numerous substrates, such as histone and NF-kB. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines (S1T and MT-2). Sirtinol-induced apoptosis was mediated by activation of the caspase family, and inactivation of NF-kB, reducing IkBα phosphorylation. Interestingly, NAD+ augmented sirtinol-induced apoptosis following deacetylation of NF-kB via NAD+-dependent deacetylase. Thus, the SIRT1 inhibitor acted as a tumor suppressor, where NAD+ accelerated the SIRT1 inhibitor-induced apoptosis. These results suggest that SIRT1 is a crucial antiapoptotic molecule in ATL cells, and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL. Figure 1. SIRT1 inhibitor reduces viability of leukemic cell lines. Cell lines were incubated at 1×105 cells/mL in the presence of various concentrations of sirtinol for 72 h. Proliferation of cell lines in the absence or presence of the indicated concentrations of sirtinol was assessed by WST-8 assay. Data are means ± S.D. from 3 independent experiments. Figure 1. SIRT1 inhibitor reduces viability of leukemic cell lines. Cell lines were incubated at 1×105 cells/mL in the presence of various concentrations of sirtinol for 72 h. Proliferation of cell lines in the absence or presence of the indicated concentrations of sirtinol was assessed by WST-8 assay. Data are means ± S.D. from 3 independent experiments. Disclosures: No relevant conflicts of interest to declare.

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Sensitive Photodynamic Detection of Adult T-cell Leukemia/Lymphoma and Specific Leukemic Cell Death Induced by Photodynamic Therapy: Current Status in Hematopoietic Malignancies

Cancers ◽

10.3390/cancers12020335 ◽

2020 ◽

Vol 12 (2) ◽

pp. 335 ◽

Cited By ~ 2

Author(s):

Takashi Oka ◽

Ken-ichi Matsuoka ◽

Atae Utsunomiya

Keyword(s):

Cell Death ◽

Photodynamic Therapy ◽

T Cell ◽

Leukemic Cell ◽

Type I ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Cell Leukemia Virus Type ◽

Hematopoietic Malignancies ◽

Adult T Cell Leukemia

Adult T-cell leukemia/lymphoma (ATL), an aggressive type of T-cell malignancy, is caused by the human T-cell leukemia virus type I (HTLV-1) infections. The outcomes, following therapeutic interventions for ATL, have not been satisfactory. Photodynamic therapy (PDT) exerts selective cytotoxic activity against malignant cells, as it is considered a minimally invasive therapeutic procedure. In PDT, photosensitizing agent administration is followed by irradiation at an absorbance wavelength of the sensitizer in the presence of oxygen, with ultimate direct tumor cell death, microvasculature injury, and induced local inflammatory reaction. This review provides an overview of the present status and state-of-the-art ATL treatments. It also focuses on the photodynamic detection (PDD) of hematopoietic malignancies and the recent progress of 5-Aminolevulinic acid (ALA)-PDT/PDD, which can efficiently induce ATL leukemic cell-specific death with minor influence on normal lymphocytes. Further consideration of the ALA-PDT/PDD system along with the circulatory system regarding the clinical application in ATL and others will be discussed. ALA-PDT/PDD can be promising as a novel treatment modality that overcomes unmet medical needs with the optimization of PDT parameters to increase the effectiveness of the tumor-killing activity and enhance the innate and adaptive anti-tumor immune responses by the optimized immunogenic cell death.

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SIRT1, a Longevity Gene Encoded Protein, Regulates Apoptosis of Adult T-Cell Leukemia Cells and Its Inhibition by Sirtinol Induces Apoptosis.

Blood ◽

10.1182/blood.v114.22.3684.3684 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3684-3684

Author(s):

Tomohiro Kozako ◽

Teruhisa Shoji ◽

Akiyoshi Aikawa ◽

Satoru Hayashida ◽

Yukako Kuramoto ◽

...

Keyword(s):

T Cell ◽

Infected Cell ◽

Cell Lines ◽

Caspase 3 ◽

Leukemic Cell ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Sirt1 Inhibitor ◽

The Mean

Abstract Abstract 3684 Poster Board III-620 Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm with a poor prognosis developing after long-term infection with human T-cell leukemia virus-1 (HTLV-1). HTLV-1 Tax is closely related to leukemic cell proliferation through nuclear factor-kappa B (NF-ƒÈB) activation. Recent studies have demonstrated that histone deacetylase class I/II inhibitors induce growth arrest and apoptosis of HTLV-1-infected T-cells via blockade of NF-ƒÈB signaling. SIRT1, an NAD(+)-dependent class III histone deacetylase, is widely recognized for its link to caloric restriction and longevity. SIRT1 plays a crucial role in a variety of physiological processes including metabolism, neurogenesis, cell survival, apoptosis and aging due to its ability to deacetylate numerous substrates such as histone, p53 and NF-ƒÈB. Existing reports on the role of SIRT1 in oncogenesis are controversial, with some evidence of an oncogenic role due to its increased expression in prostate cancer, acute myeloid leukemia and colon cancer, possibly mediated by inactivation of proteins involved in tumor suppression and DNA damage repair. Contrasting evidence of reduced SIRT1 expression in breast and hepatocellular carcinomas may support a tumor suppressor role, especially if the tumor is related to a p53 mutation. Such conflicting reports raise intriguing questions regarding its role in oncogenesis, and even less is known about its role in ATL in particular. We therefore set out to assess the expression of SIRT1 and the effect of its inhibition in HTLV-1 infected cell lines and ATL cells from patients. We observed SIRT1 protein and mRNA expression in ATL patient cells, an HTLV-1-infected cell line (MT-2), an ATL cell line (S1T), as well as HTLV-1 unrelated cell lines, Jurkat and HL60, as controls. SIRT1 expression in ATL patients was significantly higher than asymptomatic HTLV-1-carriers and healthy donors. The SIRT1 inhibitor, sirtinol, inhibited growth of all cell lines tested, with greater selectivity for HTLV-1 related cell lines (Figure 1) and ATL patients. Sirtinol induced apoptosis by activation of caspase-3, 8, 9 (Figure 2) and reducing IkBa phosphorylation, but did not significantly increase p53 acetylation in HTLV-1 infected cell lines. SIRT1 activation by NAD+ augmented apoptosis induction by sirtinol in MT-2 cells. These findings suggest that SIRT1 may be involved in T-cell immortalization by HTLV-1 and may be a crucial anti-apoptotic molecule in ATL cells. SIRT1 inhibition could therefore be useful in treating ATL. Figure 1 Inhibitory effects of sirtinol, SIRT1 inhibitor, on cell viability of leukemic cell lines. Cell lines were treated with sirtinol (0, 0.1, 10, 25 and 50μM) for 24hr. Each bar represents the mean ±S.D. of 3 independent experiments. Figure 1. Inhibitory effects of sirtinol, SIRT1 inhibitor, on cell viability of leukemic cell lines. Cell lines were treated with sirtinol (0, 0.1, 10, 25 and 50μM) for 24hr. Each bar represents the mean ±S.D. of 3 independent experiments. Figure 2 The activities of caspase-3, 8 and 9 in S1T and MT-2. Cell lines were treated with sirtinol (50μM) for 6 hr. Each bar represents the mean ±S.D. of 3 independent experiments. Figure 2. The activities of caspase-3, 8 and 9 in S1T and MT-2. Cell lines were treated with sirtinol (50μM) for 6 hr. Each bar represents the mean ±S.D. of 3 independent experiments. Disclosures: No relevant conflicts of interest to declare.

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APO-1-induced apoptosis of leukemia cells from patients with adult T- cell leukemia

Blood ◽

10.1182/blood.v81.11.2972.2972 ◽

1993 ◽

Vol 81 (11) ◽

pp. 2972-2977 ◽

Cited By ~ 86

Author(s):

KM Debatin ◽

CK Goldman ◽

TA Waldmann ◽

PH Krammer

Keyword(s):

Cell Death ◽

T Cell ◽

Cell Lines ◽

Ex Vivo ◽

Apoptotic Cell ◽

Leukemic Cells ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Induction Of Apoptosis

Abstract The 48-Kd cell-surface protein APO-1 is a new member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily. APO-1 is expressed on various cells, including activated T and B cells and some lymphoid and nonlymphoid cell lines. Triggering of APO-1 by the monoclonal antibody anti-APO-1 induces programmed cell death (apoptosis) in APO-1-expressing cells. APO-1 is also present on T-cell lines derived from patients with adult T-cell leukemia (ATL). Therefore, we investigated APO-1 expression and APO-1-mediated induction of apoptosis ex vivo in cells from patients with ATL. Fresh leukemic cells from nine patients with ATL were assayed for APO-1 expression by two-color immunofluorescence. The leukemic cells from all patients strongly expressed APO-1. Incubation of ATL cells with anti- APO-1 in vitro inhibited spontaneous and cytokine-mediated DNA synthesis. Furthermore, DNA isolated from cells treated with anti-APO-1 exhibited polynucleosomal DNA fragmentation (DNA ladder) characteristic for apoptotic cell death. The analysis of APO-1-mediated apoptosis may represent a new approach to the study of growth control in lymphoid malignancies. In addition, induction of apoptosis by administration of anti-APO-1 may represent a new therapeutic approach for aggressive T- cell malignancies such as ATL.

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Expression of different combinations of interleukins by human T cell leukemic cell lines that are clonally related.

Journal of Experimental Medicine ◽

10.1084/jem.169.5.1853 ◽

1989 ◽

Vol 169 (5) ◽

pp. 1853-1858 ◽

Cited By ~ 47

Author(s):

T Noma ◽

H Nakakubo ◽

M Sugita ◽

S Kumagai ◽

M Maeda ◽

...

Keyword(s):

T Cell ◽

Cell Clone ◽

Expression Patterns ◽

Leukemic Cell ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Leukemic Cell Lines ◽

Human T Cell ◽

T Cell Clone

We have analyzed expression patterns of 7 lymphokine mRNAs by Northern blot analyses in 19 different human T cell clones derived from patients with adult T cell leukemia. However, we were not able to reveal particular combinations of lymphokine production that allowed classification of human T cells. Especially, four clonally related leukemic lines that were established independently from the same patient with adult T cell leukemia expressed different combinations of lymphokine mRNAs, indicating that the expression of various lymphokines is not fixed but rather variable even among progenies of a single T cell clone.

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APO-1-induced apoptosis of leukemia cells from patients with adult T- cell leukemia

Blood ◽

10.1182/blood.v81.11.2972.bloodjournal81112972 ◽

1993 ◽

Vol 81 (11) ◽

pp. 2972-2977 ◽

Cited By ~ 1

Author(s):

KM Debatin ◽

CK Goldman ◽

TA Waldmann ◽

PH Krammer

Keyword(s):

Cell Death ◽

T Cell ◽

Cell Lines ◽

Ex Vivo ◽

Apoptotic Cell ◽

Leukemic Cells ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Induction Of Apoptosis

The 48-Kd cell-surface protein APO-1 is a new member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily. APO-1 is expressed on various cells, including activated T and B cells and some lymphoid and nonlymphoid cell lines. Triggering of APO-1 by the monoclonal antibody anti-APO-1 induces programmed cell death (apoptosis) in APO-1-expressing cells. APO-1 is also present on T-cell lines derived from patients with adult T-cell leukemia (ATL). Therefore, we investigated APO-1 expression and APO-1-mediated induction of apoptosis ex vivo in cells from patients with ATL. Fresh leukemic cells from nine patients with ATL were assayed for APO-1 expression by two-color immunofluorescence. The leukemic cells from all patients strongly expressed APO-1. Incubation of ATL cells with anti- APO-1 in vitro inhibited spontaneous and cytokine-mediated DNA synthesis. Furthermore, DNA isolated from cells treated with anti-APO-1 exhibited polynucleosomal DNA fragmentation (DNA ladder) characteristic for apoptotic cell death. The analysis of APO-1-mediated apoptosis may represent a new approach to the study of growth control in lymphoid malignancies. In addition, induction of apoptosis by administration of anti-APO-1 may represent a new therapeutic approach for aggressive T- cell malignancies such as ATL.

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