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• W2783452613 abstract "•Stabilization of NIK suppresses MLL-AF9-induced AML•NIK-induced leukemic suppression acts through NF-κB non-canonical signaling•NF-κB non-canonical signaling upregulates Dnmt3a and downregulates Mef2c•NF-κB non-canonical and canonical signaling have opposite roles in AML stem cells Canonical NF-κB signaling is constitutively activated in acute myeloid leukemia (AML) stem cells and is required for maintenance of the self-renewal of leukemia stem cells (LSCs). However, any potential role for NF-κB non-canonical signaling in AML has been largely overlooked. Here, we report that stabilization of NF-κB-inducing kinase (NIK) suppresses AML. Mechanistically, stabilization of NIK activates NF-κB non-canonical signaling and represses NF-κB canonical signaling. In addition, stabilization of NIK-induced activation of NF-κB non-canonical signaling upregulates Dnmt3a and downregulates Mef2c, which suppresses and promotes AML development, respectively. Importantly, by querying the connectivity MAP using up- and downregulated genes that are present exclusively in NIK-stabilized LSCs, we discovered that verteporfin has anti-AML effects, suggesting that repurposing verteporfin to target myeloid leukemia is worth testing clinically. Our data provide a scientific rationale for developing small molecules to stabilize NIK specifically in myeloid leukemias as an attractive therapeutic option. Canonical NF-κB signaling is constitutively activated in acute myeloid leukemia (AML) stem cells and is required for maintenance of the self-renewal of leukemia stem cells (LSCs). However, any potential role for NF-κB non-canonical signaling in AML has been largely overlooked. Here, we report that stabilization of NF-κB-inducing kinase (NIK) suppresses AML. Mechanistically, stabilization of NIK activates NF-κB non-canonical signaling and represses NF-κB canonical signaling. In addition, stabilization of NIK-induced activation of NF-κB non-canonical signaling upregulates Dnmt3a and downregulates Mef2c, which suppresses and promotes AML development, respectively. Importantly, by querying the connectivity MAP using up- and downregulated genes that are present exclusively in NIK-stabilized LSCs, we discovered that verteporfin has anti-AML effects, suggesting that repurposing verteporfin to target myeloid leukemia is worth testing clinically. Our data provide a scientific rationale for developing small molecules to stabilize NIK specifically in myeloid leukemias as an attractive therapeutic option. Acute myeloid leukemia (AML) is the most common acute leukemia in adults; it occurs increasingly with age, with devastating outcomes (Dombret and Gardin, 2016Dombret H. Gardin C. An update of current treatments for adult acute myeloid leukemia.Blood. 2016; 127: 53-61Crossref PubMed Scopus (349) Google Scholar). AML is initiated and maintained by a small minority of self-renewing leukemia stem cells (LSCs) (Kreso and Dick, 2014Kreso A. Dick J.E. Evolution of the cancer stem cell model.Cell Stem Cell. 2014; 14: 275-291Abstract Full Text Full Text PDF PubMed Scopus (1524) Google Scholar). Defining and targeting the key molecules specific to LSCs hold clinical promise for the eradication of AML (Pollyea and Jordan, 2017Pollyea D.A. Jordan C.T. Therapeutic targeting of acute myeloid leukemia stem cells.Blood. 2017; 129: 1627-1635Crossref PubMed Scopus (159) Google Scholar). Despite advances in understanding of the pathophysiology of AML, current treatment still largely relies on standard “7+3” chemotherapy and allogeneic stem cell transplantation (Dombret and Gardin, 2016Dombret H. Gardin C. An update of current treatments for adult acute myeloid leukemia.Blood. 2016; 127: 53-61Crossref PubMed Scopus (349) Google Scholar). Canonical nuclear factor κB (NF-κB) signaling is constitutively activated in AML stem cells required for maintenance of LSC self-renewal and positively correlates with resistance to therapy (Bosman et al., 2016Bosman M.C. Schuringa J.J. Vellenga E. Constitutive NF-κB activation in AML: causes and treatment strategies.Crit. Rev. Oncol. Hematol. 2016; 98: 35-44Crossref PubMed Scopus (46) Google Scholar, Gasparini et al., 2014Gasparini C. Celeghini C. Monasta L. Zauli G. NF-kappaB pathways in hematological malignancies.Cell. Mol. Life Sci. 2014; 71: 2083-2102Crossref PubMed Scopus (127) Google Scholar, Guzman et al., 2007Guzman M.L. Rossi R.M. Neelakantan S. Li X. Corbett C.A. Hassane D.C. Becker M.W. Bennett J.M. Sullivan E. Lachowicz J.L. et al.An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells.Blood. 2007; 110: 4427-4435Crossref PubMed Scopus (336) Google Scholar). Suppression of canonical signaling by ectopic expression of the so-called super-repressor form of IκBα attenuates AML development, validating targeting the pathway as potential therapy in AML in combination with current treatment (Cilloni et al., 2007Cilloni D. Martinelli G. Messa F. Baccarani M. Saglio G. Nuclear factor kB as a target for new drug development in myeloid malignancies.Haematologica. 2007; 92: 1224-1229Crossref PubMed Scopus (90) Google Scholar, Hsieh and Van Etten, 2014Hsieh M.Y. Van Etten R.A. IKK-dependent activation of NF-κB contributes to myeloid and lymphoid leukemogenesis by BCR-ABL1.Blood. 2014; 123: 2401-2411Crossref PubMed Scopus (35) Google Scholar). However, the existing studies have focused almost entirely on the canonical NF-κB pathway, and any potential role for NF-κB non-canonical signaling in AML has been largely overlooked. A limited number of papers suggest that expression of the non-canonical signaling components may be beneficial in AML; for example, the upregulation of non-canonical NF-κB components (NF-κB-inducing kinase [NIK] and p52) induces AML cell differentiation (Olsnes et al., 2009Olsnes A.M. Ersvaer E. Ryningen A. Paulsen K. Hampson P. Lord J.M. Gjertsen B.T. Kristoffersen E.K. Bruserud Ø. The protein kinase C agonist PEP005 increases NF-kappaB expression, induces differentiation and increases constitutive chemokine release by primary acute myeloid leukaemia cells.Br. J. Haematol. 2009; 145: 761-774Crossref PubMed Scopus (26) Google Scholar). In contrast, using a biphenotypic B-myelomonocytic leukemia cell line with lymphoblastic morphology, expression of RelB was shown to repress a tumor suppressor, death-associated protein kinase 1 (Shanmugam et al., 2012Shanmugam R. Gade P. Wilson-Weekes A. Sayar H. Suvannasankha A. Goswami C. Li L. Gupta S. Cardoso A.A. Baghdadi T.A. et al.A noncanonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia.Clin. Cancer Res. 2012; 18: 360-369Crossref PubMed Scopus (31) Google Scholar). While these studies suggest that non-canonical signaling components can suppress or accelerate AML, no functional studies have been reported. NIK activates NF-κB non-canonical signaling by directly phosphorylating IKKα, which, in turn, phosphorylates p100 and induces its processing to p52, facilitating the formation and nuclear translocation of RelB/p52 complexes (Ghosh and Hayden, 2008Ghosh S. Hayden M.S. New regulators of NF-kappaB in inflammation.Nat. Rev. Immunol. 2008; 8: 837-848Crossref PubMed Scopus (1042) Google Scholar, Vallabhapurapu and Karin, 2009Vallabhapurapu S. Karin M. Regulation and function of NF-kappaB transcription factors in the immune system.Annu. Rev. Immunol. 2009; 27: 693-733Crossref PubMed Scopus (2043) Google Scholar). In normal, unstimulated cells, NIK protein is continually degraded to prevent unnecessary NF-κB activation (Sun, 2012Sun S.C. The noncanonical NF-κB pathway.Immunol. Rev. 2012; 246: 125-140Crossref PubMed Scopus (504) Google Scholar). NIKΔT3 is a non-degradable NIK mutant lacking the TRAF3-binding domain (Liao et al., 2004Liao G. Zhang M. Harhaj E.W. Sun S.C. Regulation of the NF-kappaB-inducing kinase by tumor necrosis factor receptor-associated factor 3-induced degradation.J. Biol. Chem. 2004; 279: 26243-26250Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). To constitutively or conditionally stabilize NIK in the hematopoietic system, mice carrying a NIKΔT3flSTOP allele were crossed with either Vav-Cre mice (hereinafter termed caNIK) or Rosa-CreERT2 mice (hereinafter termed NIKERT2) (Sasaki et al., 2008Sasaki Y. Calado D.P. Derudder E. Zhang B. Shimizu Y. Mackay F. Nishikawa S. Rajewsky K. Schmidt-Supprian M. NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells.Proc. Natl. Acad. Sci. USA. 2008; 105: 10883-10888Crossref PubMed Scopus (91) Google Scholar, Xiu et al., 2017Xiu Y. Xue W.Y. Lambertz A. Leidinger M. Gibson-Corley K. Zhao C. Constitutive Activation of NIK impairs the self-renewal of hematopoietic stem/progenitor cells and induces bone marrow failure.Stem Cells. 2017; 35: 777-786Crossref PubMed Scopus (11) Google Scholar). We used these mutant mice in combination with the well-characterized MLL-AF9-induced AML mouse model to investigate the role of NIK-induced non-canonical signaling in AML (Krivtsov et al., 2006Krivtsov A.V. Twomey D. Feng Z. Stubbs M.C. Wang Y. Faber J. Levine J.E. Wang J. Hahn W.C. Gilliland D.G. et al.Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.Nature. 2006; 442: 818-822Crossref PubMed Scopus (1163) Google Scholar). Unexpectedly, we found that stabilization of NIK suppressed MLL-AF9-induced AML, which is different from the tumor-promoting role of NIK in B cell neoplasms. We first tested the impact of NIK-induced signaling on the colony-forming ability of MLL-AF9-transduced hematopoietic stem/progenitor cells (HSPCs; Lin−cKit+Sca1+). MLL-AF9-transduced HSPCs from caNIK mice formed fewer colonies in the first plating and exhausted during the second plating (Figure S1A). We then performed a similar assay using HSPCs isolated from NIKERT2 mice to exclude the possibility that the impaired HSPC self-renewal upon NIK stabilization prevents subsequent oncogenic transformation (Xiu et al., 2017Xiu Y. Xue W.Y. Lambertz A. Leidinger M. Gibson-Corley K. Zhao C. Constitutive Activation of NIK impairs the self-renewal of hematopoietic stem/progenitor cells and induces bone marrow failure.Stem Cells. 2017; 35: 777-786Crossref PubMed Scopus (11) Google Scholar). Accordingly, all the following experiments were performed using cells from NIKERT2 mice, unless otherwise specified. After retroviral MLL-AF9 transduction and four rounds of serial plating, MLL-AF9-transformed cells grew in liquid medium supplemented with interleukin (IL)-3 (Figure S1B). Compared to vehicle treatment, stabilization of NIK by 4-hydroxytamoxifen (4-OHT), the active metabolite of tamoxifen, reduced colony and cell numbers in both methylcellulose and liquid cultures and reduced colony size (Figures S1C–S1E). Since NIK promotes cell proliferation in various solid tumors and B cell neoplasms (Cildir et al., 2016Cildir G. Low K.C. Tergaonkar V. Noncanonical NF-κB signaling in health and disease.Trends Mol. Med. 2016; 22: 414-429Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, Gasparini et al., 2014Gasparini C. Celeghini C. Monasta L. Zauli G. NF-kappaB pathways in hematological malignancies.Cell. Mol. Life Sci. 2014; 71: 2083-2102Crossref PubMed Scopus (127) Google Scholar), these unexpected in vitro findings prompted us to test whether NIK suppresses AML in vivo. We transplanted MLL-AF9-transduced Lin−cKit+ bone marrow (BM) cells from NIKERT2 or wild-type (WT) mice into lethally irradiated recipients, along with radioprotective cells, and treated recipients with daily doses of tamoxifen for 4 days, 4–5 weeks post-transplantation (Figure 1A). Mice that received MLL-AF9-transduced WT or NIKERT2 cells developed AML within 3 months. However, the development of AML in recipients of NIKERT2 cells treated with tamoxifen was significantly delayed. In contrast, tamoxifen treatment had no effect on mice transplanted with MLL-AF9-transduced WT cells or HSPCs from CreERT2 (ERT2) mice, indicating that the delayed AML onset is not due to Cre toxicity (Figures 1B, S1F, and S1G). These results suggest that NIK impairs the initiation of MLL-AF9-induced AML, which encouraged us to further investigate whether NIK has a similar effect on fully developed AML. The LSC immunophenotype in this model is well defined as lin−c-kit+Sca1−CD16+CD34+ leukemia cells (Krivtsov et al., 2006Krivtsov A.V. Twomey D. Feng Z. Stubbs M.C. Wang Y. Faber J. Levine J.E. Wang J. Hahn W.C. Gilliland D.G. et al.Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.Nature. 2006; 442: 818-822Crossref PubMed Scopus (1163) Google Scholar). We prospectively isolated LSCs from mice with fully developed AML, transplanted them into sublethally irradiated recipients, and gave tamoxifen 1 week later, daily for 4 days. Strikingly, tamoxifen treatment also significantly delayed full-blown LSC repopulation (Figure 1C). In our system, stabilization of NIK by tamoxifen can be monitored by the expression of GFP. To directly test the effect of NIK on LSC self-renewal, NIK-stabilized LSCs (GFP+lin−c-kit+Sca1−CD16+CD34+) and control LSCs were isolated and re-transplanted into sublethally irradiated recipients. Stabilization of NIK significantly inhibited LSC repopulation capacity and extended the latency of AML development (Figure 1D). No homing defects of NIK-activated LSCs compared with control LSCs were detected (0.099 ± 0.032 versus 0.121 ± 0.037; n = 4, p = 0.403). The aforementioned results differ from those produced from the tumor-promoting NIK in B cell neoplasms, but they are consistent with NIK’s inhibitory role in anaplastic large-cell lymphoma and also are in agreement with the notion that, in certain circumstances, NIK inhibits cell proliferation (Cildir et al., 2016Cildir G. Low K.C. Tergaonkar V. Noncanonical NF-κB signaling in health and disease.Trends Mol. Med. 2016; 22: 414-429Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, Lu et al., 2005Lu L.F. Gondek D.C. Scott Z.A. Noelle R.J. NF kappa B-inducing kinase deficiency results in the development of a subset of regulatory T cells, which shows a hyperproliferative activity upon glucocorticoid-induced TNF receptor family-related gene stimulation.J. Immunol. 2005; 175: 1651-1657Crossref PubMed Scopus (30) Google Scholar, Muro et al., 2014Muro I. Fang G. Gardella K.A. Mahajan I.M. Wright C.W. The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways.Cell Cycle. 2014; 13: 1918-1927Crossref PubMed Scopus (12) Google Scholar). Most importantly, stabilization of NIK has a broad anti-human myeloid leukemia role (Figure S2). To understand the cellular mechanisms underlying the ability for NIK to inhibit AML, we compared the proliferation and apoptosis of NIK-stabilized LSCs to control LSCs. Colony-forming assays showed that NIK-stabilized LSCs formed fewer and smaller colonies and exhausted after the second plating, compared to controls (Figure 2A). The frequency and the proliferation (Ki67+) index of NIK-stabilized LSCs were also significantly decreased compared to those of controls (Figures 2B and 2C). However, annexin V staining showed that apoptosis was only modestly increased in NIK-stabilized leukemic cells isolated from tamoxifen-treated mice with fully developed leukemia (Figure 2D). In addition, stabilization of NIK-associated impaired leukemogenesis was not due to enhanced leukemic cell differentiation (Figure 2E). When cultured in vitro, these cells lost their proliferative capacity and were eventually outcompeted by unrecombinated control leukemic cells (Figure 2F). To elucidate the molecular mechanisms, we performed RNA sequencing (RNA-seq) using sorted LSCs. This analysis demonstrated that stabilization of NIK in LSCs significantly upregulated the expression of NF-κB non-canonical genes, Nfκb2 and Relb, without significantly altering canonical pathway components (Nfkb1, Rela, and Rel), suggesting that NIK mainly activated non-canonical signaling in LSCs (Figure 2G). In addition, RNA-seq results provided additional potential mechanistic insights underlying NIK-associated AML suppression. For example, stabilization of NIK induced upregulation of Gata2, which suppresses MLL-AF9-induced AML (Danis et al., 2015Danis E. Yamauchi T. Echanique K. Haladyna J. Kalkur R. Riedel S. Zhu N. Xie H. Bernt K.M. Orkin S.H. et al.Inactivation of Eed impedes MLL-AF9-mediated leukemogenesis through Cdkn2a-dependent and Cdkn2a-independent mechanisms in a murine model.Exp. Hematol. 2015; 43: 930-935.e6Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Furthermore, downregulation of Bcl2, Flt3, Mef2c, and Cd93 promotes apoptosis, inhibits the proliferation of LSCs, and contributes to AML suppression (Canté-Barrett et al., 2014Canté-Barrett K. Pieters R. Meijerink J.P. Myocyte enhancer factor 2C in hematopoiesis and leukemia.Oncogene. 2014; 33: 403-410Crossref PubMed Scopus (59) Google Scholar, Iwasaki et al., 2015Iwasaki M. Liedtke M. Gentles A.J. Cleary M.L. CD93 marks a non-quiescent human leukemia stem cell population and is required for development of MLL-rearranged acute myeloid leukemia.Cell Stem Cell. 2015; 17: 412-421Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) (Figures 2G and S3A). Consistent with these gene expression data, gene set enrichment analysis (GSEA) further revealed that NIK suppressed the expression of leukemia-maintaining genes controlled by the MLL-AF9 targets, Hoxa9 and Meis1 (Faber et al., 2009Faber J. Krivtsov A.V. Stubbs M.C. Wright R. Davis T.N. van den Heuvel-Eibrink M. Zwaan C.M. Kung A.L. Armstrong S.A. HOXA9 is required for survival in human MLL-rearranged acute leukemias.Blood. 2009; 113: 2375-2385Crossref PubMed Scopus (258) Google Scholar) (Figure 2H). This is likely due to non-canonical signaling directly regulating the expression of Hoxa9/Meis1 target genes in NIK-stabilized LSCs, given that the expression of Hoxa9 and Meis1 themselves was not significantly altered. In addition, stabilization of NIK upregulated Notch signaling, which has been show to inhibit MLL-AF9-induced AML (Lobry et al., 2013Lobry C. Ntziachristos P. Ndiaye-Lobry D. Oh P. Cimmino L. Zhu N. Araldi E. Hu W. Freund J. Abdel-Wahab O. et al.Notch pathway activation targets AML-initiating cell homeostasis and differentiation.J. Exp. Med. 2013; 210: 301-319Crossref PubMed Scopus (123) Google Scholar) (Figures S3B and S3C). To further understand the underlying molecular mechanisms, we assessed the expression of NF-κB proteins. NIK protein was ∼2-fold increased in NIK-stabilized AML cells and was associated with increased IKKα phosphorylation, upregulation of RelB, and activation of p100 processing to p52 (Figure 3A). Consistent with activation of non-canonical signaling, nuclear localization of RelB and p52 was increased (Figure 3B). Interestingly, the expression and nuclear localization of RelA protein were markedly reduced (Figures 3A and 3B). The decrease in nuclear RelA was at least partially due to NIK-induced upregulation of the canonical pathway inhibitors Nfkbia/IκBα and Tnfaip3/A20, which form a negative-feedback control of canonical NF-κB signaling (Figures 2G and 3A) (O’Dea and Hoffmann, 2010O’Dea E. Hoffmann A. The regulatory logic of the NF-kappaB signaling system.Cold Spring Harb. Perspect. Biol. 2010; 2: a000216Crossref PubMed Scopus (85) Google Scholar). These results are consistent with a previous report that NIK can antagonize canonical signaling in a context-dependent manner (Mao et al., 2016Mao X. Phanavanh B. Hamdan H. Moerman-Herzog A.M. Barger S.W. NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.J. Neurochem. 2016; 137: 154-163Crossref PubMed Scopus (9) Google Scholar). It has been suggested that inhibition of canonical NF-κB signaling leads to robust anti-leukemia effects, phenocopying loss of functional MLL oncoprotein (Kuo et al., 2013Kuo H.P. Wang Z. Lee D.F. Iwasaki M. Duque-Afonso J. Wong S.H. Lin C.H. Figueroa M.E. Su J. Lemischka I.R. Cleary M.L. Epigenetic roles of MLL oncoproteins are dependent on NF-κB.Cancer Cell. 2013; 24: 423-437Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). However, overexpression of RelA, confirmed by real-time PCR and western blot, failed to restore the reduced colony formation and the delay in leukemogenesis caused by NIK stabilization (Figures S4A–S4D). To test whether activation of non-canonical signaling suppresses AML directly, we overexpressed RelB or RelA in MLL-AF9 leukemic cells. Strikingly, overexpression of RelB significantly repressed—while overexpression of RelA significantly accelerated—AML (Figure 3C). The latter is consistent with RelA overexpression, which accelerates AML with MLL mutations (Kuo et al., 2013Kuo H.P. Wang Z. Lee D.F. Iwasaki M. Duque-Afonso J. Wong S.H. Lin C.H. Figueroa M.E. Su J. Lemischka I.R. Cleary M.L. Epigenetic roles of MLL oncoproteins are dependent on NF-κB.Cancer Cell. 2013; 24: 423-437Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Consistent with activation of NIK-reduced RelA protein, overexpression of RelB also repressed RelA protein expression (Figure S4A). We further investigated the expression of NIK, RelA, RelB, and their target, cyclin D1, in control and RelA-overexpressing leukemia cells in the presence or absence of 4-OHT. The expression of cytoplasmic and nuclear RelA was increased in RelA-overexpressing cells, correlated with the expression of its target cyclin D1. However, when NIK was stabilized by 4-OHT, the nuclear translocation of RelA was blocked with only slightly decreased expression of cyclin D1 (Figure 3D), possibly due to increased expression of RelB (Demicco et al., 2005Demicco E.G. Kavanagh K.T. Romieu-Mourez R. Wang X. Shin S.R. Landesman-Bollag E. Seldin D.C. Sonenshein G.E. RelB/p52 NF-kappaB complexes rescue an early delay in mammary gland development in transgenic mice with targeted superrepressor IkappaB-alpha expression and promote carcinogenesis of the mammary gland.Mol. Cell. Biol. 2005; 25: 10136-10147Crossref PubMed Scopus (74) Google Scholar). Collectively, these data indicate that non-canonical and canonical signaling have opposite roles in AML. The inactivating DNMT3A mutation enhances HSPC self-renewal and is a driver mutation in about 30% of AML (Yang et al., 2015Yang L. Rau R. Goodell M.A. DNMT3A in haematological malignancies.Nat. Rev. Cancer. 2015; 15: 152-165Crossref PubMed Scopus (314) Google Scholar). It has recently been shown that overexpression of DNMT3A inhibits AML (Lu et al., 2016Lu R. Wang P. Parton T. Zhou Y. Chrysovergis K. Rockowitz S. Chen W.Y. Abdel-Wahab O. Wade P.A. Zheng D. Wang G.G. Epigenetic perturbations by Arg882-mutated DNMT3A potentiate aberrant stem cell gene-expression program and acute leukemia development.Cancer Cell. 2016; 30: 92-107Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In addition, Mef2c is a known MLL-AF9 downstream target, and deletion of Mef2c impairs MLL-AF9-induced leukemogenesis (Canté-Barrett et al., 2014Canté-Barrett K. Pieters R. Meijerink J.P. Myocyte enhancer factor 2C in hematopoiesis and leukemia.Oncogene. 2014; 33: 403-410Crossref PubMed Scopus (59) Google Scholar). To determine the direct downstream targets of non-canonical signaling in NIK-stabilized leukemic cells, we focused and tested Dnmt3a, Mef2c, and Tifab using chromatin immunoprecipitation. We found that RelB directly binds to Mef2c and one of the Dnmt3a promoters in front of exon 7, which functionally regulates the expression of an active DNMT3a variant (Chen et al., 2002Chen T. Ueda Y. Xie S. Li E. A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation.J. Biol. Chem. 2002; 277: 38746-38754Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), but not Tifab promoter (Figure 3E). Consistent with previous reports (Canté-Barrett et al., 2014Canté-Barrett K. Pieters R. Meijerink J.P. Myocyte enhancer factor 2C in hematopoiesis and leukemia.Oncogene. 2014; 33: 403-410Crossref PubMed Scopus (59) Google Scholar, Lu et al., 2016Lu R. Wang P. Parton T. Zhou Y. Chrysovergis K. Rockowitz S. Chen W.Y. Abdel-Wahab O. Wade P.A. Zheng D. Wang G.G. Epigenetic perturbations by Arg882-mutated DNMT3A potentiate aberrant stem cell gene-expression program and acute leukemia development.Cancer Cell. 2016; 30: 92-107Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), overexpression of MEF2C and DNMT3A significantly enhanced and suppressed AML development, respectively (Figures 3F and 3G). However, neither knockdown of Dnmt3a (Figure S4E) or overexpression of MEF2C (data not shown) in AML cells restored NIK-induced delayed leukemogenesis. In contrast, knockdown of Relb partially restored NIK’s anti-leukemic effect, suggesting that upregulation of DNMT3a and downregulation of MEF2C, along with other RelB-regulated molecules, contribute to AML suppression (Figure 3H). As NIK also impairs the self-renewal of normal HSPCs, a key challenge is the need to stabilize NIK, specifically in leukemia cells (Xiu et al., 2017Xiu Y. Xue W.Y. Lambertz A. Leidinger M. Gibson-Corley K. Zhao C. Constitutive Activation of NIK impairs the self-renewal of hematopoietic stem/progenitor cells and induces bone marrow failure.Stem Cells. 2017; 35: 777-786Crossref PubMed Scopus (11) Google Scholar). One potential, but currently unavailable, method would be to develop specific NIK activators/stabilizers and deliver them via LSC-specific surface receptors (e.g., Tim3 and CD93) (Iwasaki et al., 2015Iwasaki M. Liedtke M. Gentles A.J. Cleary M.L. CD93 marks a non-quiescent human leukemia stem cell population and is required for development of MLL-rearranged acute myeloid leukemia.Cell Stem Cell. 2015; 17: 412-421Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, Jan et al., 2011Jan M. Chao M.P. Cha A.C. Alizadeh A.A. Gentles A.J. Weissman I.L. Majeti R. Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker.Proc. Natl. Acad. Sci. USA. 2011; 108: 5009-5014Crossref PubMed Scopus (201) Google Scholar). We noted that NIK impaired the function of HSPCs and LSCs through different sets of genes. One prevailing concept for identifying therapeutic targets for the treatment of cancer is that a transcriptional program can be used to identify druggable targets (Ashton et al., 2012Ashton J.M. Balys M. Neering S.J. Hassane D.C. Cowley G. Root D.E. Miller P.G. Ebert B.L. McMurray H.R. Land H. Jordan C.T. Gene sets identified with oncogene cooperativity analysis regulate in vivo growth and survival of leukemia stem cells.Cell Stem Cell. 2012; 11: 359-372Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, Lamb et al., 2006Lamb J. Crawford E.D. Peck D. Modell J.W. Blat I.C. Wrobel M.J. Lerner J. Brunet J.P. Subramanian A. Ross K.N. et al.The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease.Science. 2006; 313: 1929-1935Crossref PubMed Scopus (3509) Google Scholar, Li et al., 2017Li F. He B. Ma X. Yu S. Bhave R.R. Lentz S.R. Tan K. Guzman M.L. Zhao C. Xue H.H. Prostaglandin E1 and its analog misoprostol inhibit human CML stem cell self-renewal via EP4 receptor activation and repression of AP-1.Cell Stem Cell. 2017; 21: 359-373.e5Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The Connectivity Map (CMAP) database comprises a large reference catalog of gene expression profiles from cultured human cells stimulated with various chemicals. The database can be queried with a gene signature of interest to identify those compounds that induce desired gene expression changes. We queried CMAP using genes that are exclusively and significantly up- and downregulated in LSCs and identified verteporfin as a candidate for the treatment of AML (Figures 4A and 4B ). Verteporfin is a Food and Drug Administration (FDA)-approved photosensitizer for eliminating abnormal blood vessels in macular degeneration by increasing cellular reactive oxygen species levels. It inhibits tumor growth in various models, including acute lymphoblastic leukemia, with minimal effects on normal hematopoiesis (Gibault et al., 2016Gibault F. Corvaisier M. Bailly F. Huet G. Melnyk P. Cotelle P. Non-photoinduced biological properties of verteporfin.Curr. Med. Chem. 2016; 23: 1171-1184Crossref PubMed Scopus (68) Google Scholar, Morishita et al., 2016Morishita T. Hayakawa F. Sugimoto K. Iwase M. Yamamoto H. Hirano D. Kojima Y. Imoto N. Naoe T. Kiyoi H. The photosensitizer verteporfin has light-independent anti-leukemic activity for Ph-positive acute lymphoblastic leukemia and synergistically works with dasatinib.Oncotarget. 2016; 7: 56241-56252Crossref PubMed Scopus (18) Google Scholar). We found that verteporfin dose-dependently suppressed leukemic cell growth in liquid culture and colony formation and delayed AML development in vivo (Figures 4B–4F). Verteporfin treatment transiently upregulated the expression of non-canonical NF-κB signaling components at the mRNA and protein levels (Figures 4G and 4H). Interestingly, verteporfin downregulated RelA protein in primary MLL-AF9 leukemia cells, mimicking the effects of NIK stabilization (Figures 3A and 4H). Importantly, the inhibitory effect of verteporfin was largely attenuated by knockdown of RelB and has no additive effects on NIK-stabilized AML cells (Figure 4I). Similarly, verteporfin had a moderate additive inhibitory effect on RelB-overexpressing cells only at a higher (0.5-μM) dose, not at a lower (0.2-μM) dose, and overexpression of RelA minimally antagonized low-dose verteporfin treatm" @default.
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• W2783452613 title "Stabilization of NF-κB-Inducing Kinase Suppresses MLL-AF9-Induced Acute Myeloid Leukemia" @default.
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