Qingyun Wu

RNA interference-mediated silencing of NANOG leads to reduced proliferation and self-renewal, cell cycle arrest and apoptosis in T-cell acute lymphoblastic leukemia cells via the p53 signaling pathway.

NANOG is critical for maintaining the self-renewal and proliferative properties of embryonic stem cells. Here we found that cultured T-cell acute lymphoblastic leukemia (T-ALL) cells, as well as human primary T-ALL cells, express a functional variant of NANOG. NANOG mRNA is derived predominantly from a retrogene locus termed NANOGP8. Furthermore, we showed that RNA interference-mediated NANOG knockdown inhibited cell proliferation, reduced self-renewal, promoted apoptosis and arrested the cell cycle through a p53-mediated pathway in leukemic cells. These findings demonstrate the oncogenic potential of this pluripotent gene in human T-ALL cells.

Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-κB p65 subunit nuclear import

Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys(38) to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has been reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumines mechanism of action and novel approach to ABC-DLBCL target therapy.

Alantolactone induces G1 phase arrest and apoptosis of multiple myeloma cells and overcomes bortezomib resistance

Abstract Several sesquiterpene lactones have been extracted and demonstrated to exert various pharmacological functions in a variety of cancers. Here, we investigated anti-tumor effect of alantolactone, an allergenic sesquiterpene lactone, on human multiple myeloma (MM) and showed alantolactone inhibited growth of MM cells, both in the presence or absence of bone marrow (BM)-derived stromal cells (HS-5), and subsequent G1 phase arrest, and apoptosis as demonstrated by increased Annexin-V/7-AAD binding, caspase-3 or caspase-9 activation and down-modulation of activation of extracellular signal-regulated kinases 1/2. In addition, alantolactone reduced the secretion of MM survival and growth-related cytokines, vascular endothelial growth factor, from MM cells or HS-5 cells, and inhibited cytokine-induced osteoclastogenesis. Notably, alantolactone also inhibited cell proliferation in bortezomib-resistant MM cells. Taken together, alantolactone exerted anti-tumor effect on MM by suppressing cell proliferation, triggering apoptosis, partly damaging the BM microenvironment and overcoming proteasome inhibitor resistance, suggesting alantolactone may be a novel therapeutic approach for the treatment of human MM.

MicroRNA‐181a, a potential diagnosis marker, alleviates acute graft versus host disease by regulating IFN‐γ production

Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is a valuable therapeutic strategy for a wide variety of diseases. Acute graft‐versus‐host disease (aGVHD) is a major complication in up to 75% of allo‐HSCT. The absence of a reliable predicative marker for aGVHD onset prevents preemptive treatment and impedes widespread and successful application of this therapy. In this study we found that after allo‐HSCT, the levels of miR‐181a were reduced significantly prior to the onset of aGVHD. More importantly, the degree of its reduction correlated with the severity of aGVHD. Mechanistically, miR‐181a affects the function of T lymphocytes by down‐regulating IFN‐γ in a dose‐dependent manner. Meanwhile, we confirmed that miR‐181a can effectively preserve the anti‐leukemic effect in vitro. Using a murine allo‐HSCT model, we demonstrated that murine miR‐181b, the human miR‐181a homolog, served as an effective predictor of aGVHD. Moreover, expression of this microRNA ameliorated the severity of aGVHD. Collectively, these results show that the level of miR‐181a may serve as a reliable marker for the diagnosis and prognosis the onset of aGVHD. Am. J. Hematol. 90:998–1007, 2015.

The identification and characteristics of IL-22-producing T cells in acute graft-versus-host disease following allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) remains the major obstacle for allogeneic bone marrow transplantation, in which many proinflammatory cytokines secreted by alloreactive donor T cells are involved. Role of IL-22 as a member of IL-10 family in GVHD is still disputed and the properties of IL-22-producing cells are unclear. We demonstrated here that CD4⁺ T cells but not CD8⁺ T cells involved in GVHD were the main cellular source of donor-derived IL-22. Th1 and Th17 cells were detected not only express classical cytokine IFN-γ or IL-17, but also contributed to IL-22 secretion in GVHD. Th22 cells characterized by the independent secretion of IL-22 were identified and occupied almost half percentage of IL-22-producing CD4⁺ T cells. The frequency of IL-22-producing CD4⁺ T cells showed dynamic changes with the development of GVHD. Finally, we observed that IL-22-producing CD4⁺ T cells in GVHD mouse carried CD62L⁻CD44(high/low) surface markers. In conclusion, we illuminate the characteristics of donor-derived IL-22-producing CD4⁺ T cells, which may have potent implication for further study of pathogenesis of GVHD.

Evidence that the amino acid residue Ile121 is involved in arginine kinase activity and structural stability

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Domain-domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of amino acid residues located in the linker between the N- and C-terminal domains in the structural stability and functions of multidomain proteins. In this research, A series mutation of conserved residue Ile121 located in the linker were mutated to explore its roles in the activity and structural stability of AK. The mutations I121D and I121K led to pronounced loss of activity and structural stability. Furthermore, these mutations also led to serious aggregation during heat-and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, all the mutantions except I121L could not successfully recover their activities by dilution-initiated refolding, and showed significant decreased rate constant during AK refolding. While the mutation I121L almost had no effect on AK activity and structural stability. These results suggested that mutations of the residue I121 in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the N- and C-terminal domains.

Acute ablation of DP thymocytes induces up-regulation of IL-22 and Foxn1 in TECs.

Thymic epithelial cells (TECs) provide the basic architecture for the development of thymocytes. TEC is regenerative after impairment in thymus of young mice. However, how this regeneration program is governed remains unclear. Transcription factor Foxn1 is a central mediator of the differentiation and function of TEC. We examined the relation between thymic injury and Foxn1 in TEC. Total body irradiation (TBI) treatments induced up-regulation of Foxn1 in TEC, which was abolished when thymic function recovered. Specific depletion of double positive (DP) thymocytes triggered the up-regulation of Foxn1. On the other hand, extracellular IL-22 is a potential regulator of homeostasis of TEC. We demonstrated that TBI treatments also induced the up-regulation of intrathymic IL-22. Expression pattern of Foxn1 shares similar characteristics with IL-22. Furthermore, Foxn1 related genes that regulate the function of TEC were also up-regulated. Thus, our data reveal that TBI treatment triggers regeneration program of TEC.

The effects of R683S (G) genetic mutations on the JAK2 activity, structure and stability

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in the hematopoietic and immune response. The acquired JAK2 R683S (G) mutations are presumed to be a biomarker for B-cell acute lymphoblastic leukemia (B-ALL). However, how these mutations leading to the B-ALL is still unclear. The crystal structure of JAK2 JH2 domain suggests that the residue R683 locating in the linker between the N and C lobes of JH2 domain is important for keeping the compact structure, activity and structural stability of this domain. Mutations R683S, R683G and R683E significantly increase JAK2 activity and decrease its structural stability. While the R683K and R683H mutations almost have no effects on the JAK2 activity and structural stability. Furthermore, the spectroscopic experiments imply that mutations R683S, R683G and R683E impair the structure of JAK2 JH2 domain, and lead JAK2 to partially unfolded state. It may be this partially unfolded state that caused JAK2 R683S (G) constitutive activation. This study provides clues in understanding the mechanism of JAK2 R683S (G) mutations caused B-ALL.

Amino acid residue E543 in JAK2 C618R is a potential therapeutic target for myeloproliferative disorders caused by JAK2 C618R mutation

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays a key role in the hematopoietic and immune responses. The acquired JAK2 C618R somatic mutation is detected in a subset of myeloproliferative disorders (MPDs) patients and presumed to be a biomarker for MPDs. However, how JAK2 C618R mutation causes MPDs is still unclear. Our results indicate that the amino acid residue E543 in JAK2 C618R is indispensable for its constitutive activation. When the glutamic acid at this position was mutated to alanine (E543A) in the JAK2 C618R, its activity significantly decreased. However when the glutamic acid was mutated to the acidic amino acid, aspartic acid, JAK2 C618R activity changed little. These results suggest that there is an interaction between the amino acid residue R618 and E543, and that this interaction is crucial to sustain the constitutive activation of JAK2 C618R. More importantly, the E543 single mutation had no effects on the function of wild type JAK2 (WT JAK2). This study suggests that the amino acid residue E543 might be a potential target for specific inhibitors to treat MPDs caused by the JAK2 C618R mutation.

Scriptaid inhibits cell survival, cell cycle, and promotes apoptosis in multiple myeloma via epigenetic regulation of p21

Multiple myeloma (MM) is an extremely serious plasma cell malignancy. Despite the recent introduction of chemotherapies such as bortezomib and lenalidomide, it remains an incurable disease due to the high rate of relapse and the development of drug resistance. Epigenetic regulation is closely related to MM progression, but the epigenetic modification mechanism of MM cell apoptosis has remained unclear. As a novel histone deacetylase inhibitor (HDACi), Scriptaids possible roles in MM progression have not been explored. Herein, we found that Scriptaid decreased several human MM cell viabilities in a dose-dependent manner. Scriptaid was also able to dose dependently and significantly induce MM cell cycle arrest at the G2/M phase. Moreover, Scriptaid facilitates p21 transcriptional activities by mediating H3Ac gene-activated modification, eventually leading to MM cell apoptosis. Overall, our results show that Scriptaid is an inducer of MM cell death, suggesting the possibility for Scriptaid-mediated therapeutics to cure refractory/relapsed MM.