IntroductionMultiple myeloma appears to arise from initiating chromosomal translocations and duplications in postgerminal center plasma cells with subsequent secondary mutations contributing to disease progression. 1,2 Six unique myeloma variants can be identified by gene expression profiling with 3 of the 6 groups anchored by an initiating chromosome translocation. It is noteworthy that a unifying event of cyclin D dysregulation is identified in all subgroups. 1,2 One of the initiating chromosomal abnormalities in myeloma involves members of the Maf family of oncogenes. 1 Maf proteins are basic leucine zipper transcription factors in the AP1 family and regulate gene transcription by binding DNA sequences known as MAF responsive elements (MAREs). 3,4 Of the Maf family, c-maf was the first endogenous member identified and is probably the best characterized with respect to function. 5 Genes up-regulated by c-maf include cyclin D2, ␤-integrin 7, ARK 5 and CCR1, all of which are important in the pathogenesis of multiple myeloma. [6][7][8] c-maf is dysregulated in multiple myeloma. For example, approximately 25% of myeloma cell lines have a t(14;16) translocation. 6 In patients with myeloma, approximately 6% have a t(14;16) or t(14;20), translocations that juxtapose IgH with c-maf and its homolog mafB, respectively. 9,10 The frequency of c-maf overexpression in patients who lack the t(14;16) has varied from study to study, depending on the method to assess its overexpression and ranges from 5% to 50%. 6,11,12 In malignant cell lines, including multiple myeloma, overexpression of c-maf augments cell proliferation and increases tumor formation in xenograft models. 6,7 Conversely, inhibition of c-maf with dominant-negative constructs decreases cell proliferation, impairs adhesion to marrow stroma, and delays tumor growth. 6 Overexpression of c-maf is also clinically relevant in that patients with myeloma and the t(14;16) c-maf translocation have a shorter overall survival. 13 Although the functional importance of c-maf has been described, the mechanisms that govern its regulation have not been fully elucidated. Therefore, molecules that decrease c-maf and subsequently its downstream targets, particularly cyclin D2, could further our understanding of the regulation of c-maf. Improved understanding of c-maf will help develop therapies that target this protein.To this end, we developed a high-throughput chemical genomics screen to identify compounds that inhibit c-maf-dependent transactivation of the cyclin D2 promoter. With this assay, we screened libraries of off-patent drugs and chemicals and were surprised to identify glucocorticoids as inhibitors of c-mafdependent cyclin D2 transactivation. Subsequent studies demonstrated that glucocorticoids decrease c-maf protein by promoting its ubiquitination through the up-regulation of ubiquitin C mRNA. Thus, this chemical biology approach has provided insights into a novel mechanism of c-maf regulation. The publication costs of this article were defrayed in part by page charge ...

HOX11 encodes a homeodomain protein that is aberrantly expressed in T-cell acute lymphoblastic leukemia as a consequence of the t(10;14) and t(7;10) chromosomal translocations. We previously reported that HOX11 immortalizes murine hematopoietic progenitors and induces pre-Tcell tumors in mice after long latency. It has been demonstrated in a number of studies that HOX11, similar to other homeodomain proteins, binds DNA and transactivates transcription. These findings suggest that translocation-activated HOX11 functions as an oncogenic transcription factor. Here we report that HOX11 represses transcription through both TATAcontaining and TATA-less promoters. Interestingly, transcriptional repression by HOX11 is independent of its DNA binding capability. Moreover, a systematic mutational analysis indicated that repressor activity was separable from immortalizing function, which requires certain residues within the HOX11 homeodomain that make base-specific or phosphatebackbone contacts with DNA. We further IntroductionThe involvement of homeobox genes in hematologic malignancies is becoming increasingly recognized. 1 HOX11 (TCL-3) is a homeobox gene identified on chromosome 10 at the t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations associated with pediatric T-cell acute lymphoblastic leukemia (T-ALL). [2][3][4][5] As a result of translocation, the T-cell receptor ␦ or ␤ regulatory region is juxtaposed upstream of the HOX11 coding region, resulting in high-level synthesis of a structurally intact HOX11 homeodomain protein. During murine embryogenesis, Hox11 expression has been detected in the bronchial arches, the hindbrain, and the splenic anlage arising from splanchnic mesoderm. [6][7][8] Because HOX11 is not normally expressed in T cells, dysregulated HOX11 expression as a consequence of aberrant recombinational events during T-cell receptor rearrangement is believed to be an early step in the etiology of this subtype of T-ALL. [2][3][4][5] The transforming potential of HOX11 has been confirmed by both in vitro and in vivo studies. We have previously shown that retroviral vector-mediated expression of HOX11 in primary murine bone marrow (BM) cells gives rise to immortalized progenitor lines at high frequency and promotes T-cell tumorigenesis in mice, 9,10 whereas others have reported that targeted expression of HOX11 in thymocytes of transgenic mice resulted in cell-cycle aberration and progression to malignancy. 11 Several possible mechanisms through which inappropriate HOX11 expression might lead to malignant transformation have been proposed. In particular, HOX11 is believed to function as a transcriptional regulator on the basis of its nuclear localization, the DNA binding activity of its homeodomain, and its ability to transactivate transcription of reporter genes. [12][13][14][15] In support of this view, HOX11 induces the up-regulation of an endogenous gene, Aldh-1, in stably transfected NIH3T3 cells; interestingly, an inverse relationship between Hox11 expression and Aldh-1 expression is...

Introduction D-type cyclins are critical regulators of the cell cycle that act in a complex with cyclin-dependent kinases (CDKs) to promote the phosphorylation of Rb and initiate cellular transition from G 1 to S phase. 1 Overexpression of D-cyclins occurs in many tumors and leads to increased cell proliferation 2-4 and chemoresistance. 5 In contrast, reducing D-cyclins directly or indirectly can decrease cellular proliferation and induce apoptosis. [6][7][8] Aberrant expression of one or more D-cyclins is virtually universal in multiple myeloma (MM), with 54% of myeloma tumors overexpressing cyclin D1, 48% overexpressing cyclin D2, 3% overexpressing cyclin D3, and 8% overexpressing both cyclin D1 and D2. 9 The overexpression of D-cyclins contributes to the pathogenesis and chemoresistance of this disease. 3,4,9 The t(11;14) involving cyclin D1 is found in 15% to 20% of myeloma patients and is associated with improved survival. 10 However, levels of cyclin D1 protein often do not correlate as well with prognosis. 11 In contrast, patients with myeloma and increased cyclin D2 expression, especially in cooperation with the presence of other oncogenes, such as c-Maf, MafB, and FGFR3/MMSET, have an inferior event-free survival. 12 Likewise, D-cyclins are increased in a subset of patients with acute myeloid leukemia and are associated with poor outcome. 13 To better understand the regulation of D-cyclins and the effects of targeting their expression in myeloma and leukemia cells, we conducted a chemical screen for inhibitors of the human cyclin D2 promoter using NIH3T3 cells engineered to overexpress the cyclin D2 promoter driving firefly luciferase. From this screen, we identified glucocorticoids and demonstrated that these drugs inhibit cyclin D2 transactivation by increasing the proteasomal degradation of c-maf via increased ubiquitin expression. 14 Here we identify that cyproheptadine inhibits the transactivation of cyclin D2 through mechanisms independent of c-maf. Cyproheptadine is a known inhibitor of the H1 histamine and 5-HT serotonin receptors. Previously, cyproheptadine has been evaluated in patients for the treatment of migraines, 15 anorexia, 16 and atopic dermatitis. 17 In clinical trials, the drug was well tolerated without hematologic toxicity. The ability of cyproheptadine to inhibit D-cyclin expression has not been reported previously. In this report, we evaluated the effects of cyproheptadine on myeloma and leukemia cell lines as well as primary patient samples. Cyproheptadine decreased D-cyclin levels and induced cell death in myeloma and leukemia cell lines and patient samples preferentially over normal hematopoietic cells. Finally, cyproheptadine demonstrated activity in mouse models of leukemia and myeloma. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Methods Chemicals and reagentsCyproheptadine hydrochloride, amitriptyline, loratadine, thymidine, histamine, serotonin, and propidium iodine (PI) were purchased from SigmaAldrich (St Louis, M...

Hacs1, a SH3 and SAM domain-containing adaptor protein, is up-regulated by IL-4 in activated B cells and strongly expressed in dendritic cells. To elucidate the function of Hacs1 in immune regulation, we generated Hacs1(-/-) mice by deletion of the SH3 and SAM domains. Hacs1(-/-) mice were viable and fertile and had normal bone marrow B-cell development and normal splenic T- and B-cell populations. However, adult Hacs1(-/-) mice had increased peritoneal B1a cells (IgM(+)CD5(+)). On immunization with T-cell-independent antigen TNP-Ficoll, Hacs1(-/-) mice had increased production of anti-TNP IgM and IgG3. Purified splenic B cells from Hacs1(-/-) mice showed increased cell proliferation on BCR (B-cell receptor) stimulation. We further demonstrate that the Hacs1(-/-) B cells had increased global tyrosine phosphorylation, including tyrosine kinases Lyn and Akt. Both T-helper type 1 (T(h)1) and T-helper type 2 (T(h)2) humoral responses were enhanced in Hacs1(-/-) mice. In vitro bone marrow-derived Hacs1(-/-) dendritic cells showed increased IL-12 production on stimulation with ovalbumin (OVA). This study suggests that Hacs1 is an immunoinhibitory adaptor that might be a useful target for immune suppression therapy.-Wang, D., Stewart, A. K., Zhuang, L., Zhu, Y., Wang, Y., Shi, C., Keating, A., Slutsky, A., Zhang, H., Wen, X.-Y. Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1.

Although immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, are widely used in the treatment of multiple myeloma (MM), the molecular mechanism of IMiDs' action is largely unknown. In this review, we will summarize recent advances in the application of IMiDs in MM cancer treatment as well as their effects on immunomodulatory activities, anti-angiogenic activities, intervention of cell surface adhesion molecules between myeloma cells and bone marrow stromal cells, anti-inflammatory activities, anti-proliferation, pro-apoptotic effects, cell cycle arrest, and inhibition of cell migration and metastasis. In addition, the potential IMiDs' target protein, IMiDs' target protein's functional role, and the potential molecular mechanisms of IMiDs resistance will be discussed. We wish, by presentation of our naive discussion, that this review article will facilitate further investigation in these fields.