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IMPORTANCE: Inconsistent reporting of outcomes in clinical trials of rosacea is impeding and likely preventing accurate data pooling and meta-analyses. There is a need for standardization of outcomes assessed during intervention trials of rosacea. OBJECTIVE: To develop a rosacea core outcome set (COS) based on key domains that are globally relevant and applicable to all demographic groups to be used as a minimum list of outcomes for reporting by rosacea clinical trials, and when appropriate, in clinical practice. EVIDENCE REVIEW: A systematic literature review of rosacea clinical trials was conducted. Discrete outcomes were extracted and augmented through discussions and focus groups with key stakeholders. The initial list of 192 outcomes was refined to identify 50 unique outcomes that were rated through the Delphi process Round 1 by 88 panelists (63 physicians from 17 countries and 25 patients with rosacea in the US) on 9-point Likert scale. Based on feedback, an additional 11 outcomes were added in Round 2. Outcomes deemed to be critical for inclusion (rated 7-9 by ≥70% of both groups) were discussed in consensus meetings. The outcomes deemed to be most important for inclusion by at least 85% of the participants were incorporated into the final core domain set. FINDINGS: The Delphi process and consensus-building meetings identified a final core set of 8 domains for rosacea clinical trials: ocular signs and symptoms; skin signs of disease; skin symptoms; overall severity; patient satisfaction; quality of life; degree of improvement; and presence and severity of treatment-related adverse events. Recommendations were also made for application in the clinical setting. CONCLUSIONS AND RELEVANCE: This core domain set for rosacea research is now available; its adoption by researchers may improve the usefulness of future trials of rosacea therapies by enabling meta-analyses and other comparisons across studies. This core domain set may also be useful in clinical practice.

IMPORTANCE: Extramammary Paget disease (EMPD) is a frequently recurring malignant neoplasm with metastatic potential that presents in older adults on the genital, perianal, and axillary skin. Extramammary Paget disease can precede or occur along with internal malignant neoplasms. OBJECTIVE: To develop recommendations for the care of adults with EMPD. EVIDENCE REVIEW: A systematic review of the literature on EMPD from January 1990 to September 18, 2019, was conducted using MEDLINE, Embase, Web of Science Core Collection, and Cochrane Libraries. Analysis included 483 studies. A multidisciplinary expert panel evaluation of the findings led to the development of clinical care recommendations for EMPD. FINDINGS: The key findings were as follows: (1) Multiple skin biopsies, including those of any nodular areas, are critical for diagnosis. (2) Malignant neoplasm screening appropriate for age and anatomical site should be performed at baseline to distinguish between primary and secondary EMPD. (3) Routine use of sentinel lymph node biopsy or lymph node dissection is not recommended. (4) For intraepidermal EMPD, surgical and nonsurgical treatments may be used depending on patient and tumor characteristics, although cure rates may be superior with surgical approaches. For invasive EMPD, surgical resection with curative intent is preferred. (5) Patients with unresectable intraepidermal EMPD or patients who are medically unable to undergo surgery may receive nonsurgical treatments, including radiotherapy, imiquimod, photodynamic therapy, carbon dioxide laser therapy, or other modalities. (6) Distant metastatic disease may be treated with chemotherapy or individualized targeted approaches. (7) Close follow-up to monitor for recurrence is recommended for at least the first 5 years. CONCLUSIONS AND RELEVANCE: Clinical practice guidelines for EMPD provide guidance regarding recommended diagnostic approaches, differentiation between invasive and noninvasive disease, and use of surgical vs nonsurgical treatments. Prospective registries may further improve our understanding of the natural history of the disease in primary vs secondary EMPD, clarify features of high-risk tumors, and identify superior management approaches.

Paget's disease (PD) is an intraepidermal adenocarcinoma of the skin at the breast (mammary PD) or urogenital locations (extramammary PD [EMPD]). At present, there is lack of clarity on PD’s pathogenesis, the relationship between its subtypes, and its lineage link with the underlying invasive carcinomas. Here we describe that mammary PD and EMPD have similar mutational profiles, with the most frequent recurrent mutations occurring in the chromatin remodeling genes, such as KMT2C (MLL3, 39%) and ARID2 (22%), with additional recurrent somatic mutations detected in genes previously not known to be mutated in cancers, such as CDCC168 (34%), FSIP2 (29%), CASP8AP2 (29%), and BIRC6 (24%). In paired mammary PD and underlying breast carcinoma samples, distinct gene mutations were detected, indicating that they represent independent oncogenic events. Finally, multistage EMPD tissue sequencing revealed KMT2C gene occurring early in EMPD oncogenesis, and that multifocal EMPD samples share the same early gene mutations, suggesting clonal origin of multifocal EMPD. Our results reveal similar genomic landscapes between mammary PD and EMPD, including early aberrations in chromatin remodeling genes. In addition, mammary PD and underlying breast ductal carcinomas represent independent oncogenic events. These findings provide approaches for developing diagnostic tools and therapeutic interventions for PD.

TOX is a nuclear factor essential for the development of CD4+ T cells in the thymus. It is normally expressed in low amounts in mature CD4+ T cells of the skin and the peripheral blood. We have recently discovered that the transcript levels of TOX were significantly increased in mycosis fungoides, the most common type of cutaneous T-cell lymphoma (CTCL), as compared to normal skin or benign inflammatory dermatoses. However, its involvement in advanced CTCL and its biological effects on CTCL pathogenesis have not been explored. In this study, we demonstrate that TOX expression is also enhanced significantly in primary CD4+CD7− cells from patients with Sézary syndrome, a leukemic variant of CTCL, and that high TOX transcript levels correlate with increased disease-specific mortality. Stable knockdown of TOX in CTCL cells promoted apoptosis and reduced cell cycle progression, leading to less cell viability and colony-forming ability in vitro and to reduced tumor growth in vivo. Furthermore, TOX knockdown significantly increased 2 cyclin-dependent kinase (CDK) inhibitors, CDKN1B and CDKN1C. Lastly, blocking CDKN1B and CDKN1C reversed growth inhibition of TOX knockdown. Collectively, these findings provide strong evidence that aberrant TOX activation is a critical oncogenic event for CTCL.

TOX is a nuclear factor essential for the development of CD4+T cells in the thymus. It is normally expressed in low amounts in mature CD4+T cells of the skin and the peripheral blood. We have recently discovered that the transcript levels of TOXwere significantly increased in mycosis fungoides, the most common type of cutaneous T-cell lymphoma (CTCL), as compared to normal skin or benign inflammatory dermatoses. However, its involvement in advanced CTCL and its biological effects on CTCL pathogenesis have not been explored. In this study, we demonstrate that TOXexpression is also enhanced significantly in primary CD4+CD7−cells from patients with Sézary syndrome, a leukemic variant of CTCL, and that high TOXtranscript levels correlate with increased disease-specific mortality. Stable knockdown of TOXin CTCL cells promoted apoptosis and reduced cell cycle progression, leading to less cell viability and colony-forming ability in vitro and to reduced tumor growth in vivo. Furthermore, TOXknockdown significantly increased 2 cyclin-dependent kinase (CDK) inhibitors, CDKN1Band CDKN1C. Lastly, blocking CDKN1Band CDKN1Creversed growth inhibition of TOXknockdown. Collectively, these findings provide strong evidence that aberrant TOXactivation is a critical oncogenic event for CTCL.

Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines. In these malignant cell lines we correlate the expression of STAT4 and STAT6 with the T helper (Th) phenotype markers and test the effect of Histone Deacetylase (HDAC) inhibitors and siRNA-mediated knock down of miR-155 on STAT4 expression. Our findings demonstrate that STAT4 expression correlates with Th1 phenotype, while STAT6 is associated with the Th2 phenotype. Our results further document that STAT4 and STAT6 genes are inversely regulated in CTCL. Treatment with HDAC inhibitors upregulates STAT4 expression, while at the same time decreases STAT6 expression in MyLa cells. Also, siRNA-mediated knock down of miR-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA.

Cutaneous CD30+lymphoproliferative disease (CD30+LPD), characterized by the presence of CD30+anaplastic large T cells, comprises the second most common group of cutaneous T-cell lymphoma (CTCL). However, little is known about the pathobiology of the CD30+lymphoma cells, as well as the mechanisms of disease progression. Here we report that Special AT-rich region binding protein 1 (SATB1), a thymocyte specific chromatin organizer, is over-expressed in CD30+lymphoma cells in most CD30+LPDs, and its expression is upregulated during disease progression. Our findings show that SATB1 silencing in CD30+LPD cells leads to G1 cell cycle arrest mediated by p21 activation. Using chromatin immunoprecipitation, luciferase assays, and mutational analysis, we demonstrate that SATB1 directly regulates the transcription of p21 in a p53-independent manner. Moreover, DNA demethylation on a specific CpG-rich region of the SATB1 promoter is associated with the upregulation of SATB1 during disease progression. These experiments define a novel SATB1-p21 pathway in malignant CD30+T lymphocytes, which provides novel molecular insights into the pathogenesis of CD30+LPDs and possibly leads to new therapies.

Cutaneous CD30+ lymphoproliferative disease (CD30+LPD), characterized by the presence of CD30+ anaplastic large T cells, comprises the second most common group of cutaneous T-cell lymphoma (CTCL). However, little is known about the pathobiology of the CD30+ lymphoma cells, as well as the mechanisms of disease progression. Here we report that Special AT-rich region binding protein 1 (SATB1), a thymocyte specific chromatin organizer, is over-expressed in CD30+ lymphoma cells in most CD30+LPDs, and its expression is upregulated during disease progression. Our findings show that SATB1 silencing in CD30+LPD cells leads to G1 cell cycle arrest mediated by p21 activation. Using chromatin immunoprecipitation, luciferase assays, and mutational analysis, we demonstrate that SATB1 directly regulates the transcription of p21 in a p53-independent manner. Moreover, DNA demethylation on a specific CpG-rich region of the SATB1 promoter is associated with the upregulation of SATB1 during disease progression. These experiments define a novel SATB1-p21 pathway in malignant CD30+ T lymphocytes, which provides novel molecular insights into the pathogenesis of CD30+LPDs and possibly leads to new therapies.

Skin lesions from mycosis fungoides (MF) patients display an increased expression of interleukin-15 (IL-15), IL-17F, and other cytokines implicated in inflammation and malignant cell proliferation in cutaneous T-cell lymphoma (CTCL). In the leukemic variant of CTCL, Sézary syndrome (SS), IL-2 and IL-15 trigger activation of the Jak-3/STAT3 pathway and transcription of IL17A gene, whereas it is unknown what causes IL-15 expression, Jak3/STAT3 activation, and production of IL-17F in MF. Here, we studied the expression and regulation of IL-15 and its relation to IL-17F in MF cell lines and skin lesions from 60 MF patients. We show that: (1) the spontaneous IL-15 mRNA expression is resistant to Jak3 and STAT3 inhibitors at concentrations that profoundly inhibit STAT3 activation and IL-17F mRNA expression; (2) anti-IL-15 antibody blocks STAT3 activation induced by exogenous IL-15 in non-malignant MF T cells, whereas the spontaneous STAT3 activation and IL-17F expression in malignant T cells is not inhibited; (3) patients display heterogeneous IL-15/IL-17F mRNA expression patterns in skin lesions; and (4) IL-15 expression (in contrast to IL-17F) is not associated with progressive disease. Taken together, these findings indicate that IL-15 and IL-17F are differentially regulated and expressed in MF. We propose that IL-15 and IL-17F are markers for different inflammatory environments and play distinct roles in the development and progression of MF.

Inappropriately regulated expression of interleukin (IL)-17A is associated with the development of inflammatory diseases and cancer. However, little is known about the role of other IL-17 family members in carcinogenesis. Here, we show that a set of malignant T-cell lines established from patients with cutaneous T-cell lymphoma (CTCL) spontaneously secrete IL-17F and that inhibitors of Janus kinases and Signal transducer and activator of transcription 3 are able to block that secretion. Other malignant T-cell lines produce IL-17A but not IL-17F. Upon activation, however, some of the malignant T-cell lines are able to coexpress IL-17A and IL-17F, leading to formation of IL-17A/F heterodimers. Clinically, we demonstrate that IL-17F messenger RNA expression is significantly increased in CTCL skin lesions compared with healthy donors and patients with chronic dermatitis. IL-17A expression is also increased and a significant number of patients express high levels of both IL-17A and IL-17F. Concomitantly, we observed that the expression of the IL-17 receptor is significantly increased in CTCL skin lesions compared with control subjects. Importantly, analysis of a historic cohort of 60 CTCL patients indicates that IL-17F expression is associated with progressive disease. These findings implicate IL-17F in the pathogenesis of CTCL and suggest that IL-17 cytokines and their receptors may serve as therapeutic targets.

Inappropriately regulated expression of interleukin (IL)-17A is associated with the development of inflammatory diseases and cancer. However, little is known about the role of other IL-17 family members in carcinogenesis. Here, we show that a set of malignant T-cell lines established from patients with cutaneous T-cell lymphoma (CTCL) spontaneously secrete IL-17F and that inhibitors of Janus kinases and Signal transducer and activator of transcription 3 are able to block that secretion. Other malignant T-cell lines produce IL-17A but not IL-17F. Upon activation, however, some of the malignant T-cell lines are able to coexpress IL-17A and IL-17F, leading to formation of IL-17A/F heterodimers. Clinically, we demonstrate that IL-17F messenger RNA expression is significantly increased in CTCL skin lesions compared with healthy donors and patients with chronic dermatitis. IL-17A expression is also increased and a significant number of patients express high levels of both IL-17A and IL-17F. Concomitantly, we observed that the expression of the IL-17 receptor is significantly increased in CTCL skin lesions compared with control subjects. Importantly, analysis of a historic cohort of 60 CTCL patients indicates that IL-17F expression is associated with progressive disease. These findings implicate IL-17F in the pathogenesis of CTCL and suggest that IL-17 cytokines and their receptors may serve as therapeutic targets.

Sézary syndrome (SS) is an aggressive subtype of cutaneous T-cell lymphoma that is characterized by circulating leukemic Sézary cells. The accumulation of these malignant cells has been shown to be the result of the resistance to apoptosis, in particular, activation-induced cell death. However, the mechanism of apoptosis resistance remains unknown. By characterizing the gene transcription profiles of purified CD4+CD7−Sézary cells from patients with SS and cultured Sézary cells, it was found that Sézary cells are deficient in the expression of special AT-rich region binding protein 1 (SATB1), a key regulator of T-cell development and maturation. Retrovirus-mediated gene transduction revealed that SATB1restoration in cultured Sézary cells (Hut78) triggered spontaneous cell death and sensitized Hut78 cells to activation-induced cell death, with associated activation of caspase 8 and caspase 3. Furthermore, endogenous expression of FasL in Sézary cells was increased in transcriptional and translational levels on restoration of SATB1expression in cultured Sézary cells. These results suggest that deficiency in SATB1expression in Sézary cells plays an important role in SS pathogenesis by causing apoptosis resistance. Thus, restoration of SATB1expression may represent a potential molecular targeted therapy for SS, which does not have a cure at present.

Sézary syndrome (SS) is an aggressive subtype of cutaneous T-cell lymphoma that is characterized by circulating leukemic Sézary cells. The accumulation of these malignant cells has been shown to be the result of the resistance to apoptosis, in particular, activation-induced cell death. However, the mechanism of apoptosis resistance remains unknown. By characterizing the gene transcription profiles of purified CD4+CD7− Sézary cells from patients with SS and cultured Sézary cells, it was found that Sézary cells are deficient in the expression of special AT-rich region binding protein 1 (SATB1), a key regulator of T-cell development and maturation. Retrovirus-mediated gene transduction revealed that SATB1 restoration in cultured Sézary cells (Hut78) triggered spontaneous cell death and sensitized Hut78 cells to activation-induced cell death, with associated activation of caspase 8 and caspase 3. Furthermore, endogenous expression of FasL in Sézary cells was increased in transcriptional and translational levels on restoration of SATB1 expression in cultured Sézary cells. These results suggest that deficiency in SATB1 expression in Sézary cells plays an important role in SS pathogenesis by causing apoptosis resistance. Thus, restoration of SATB1 expression may represent a potential molecular targeted therapy for SS, which does not have a cure at present.

AHI-1is an oncogene often targeted by provirus insertional mutagenesis in murine leukemias and lymphomas. Aberrant expression of human AHI-1occurs in cutaneous T-cell lymphoma (CTCL) cells and in CD4+CD7−Sezary cells from patients with Sezary syndrome. Stable knockdown of AHI-1using retroviral-mediated RNA interference in CTCL cells inhibits their transforming activity in vitro and in vivo. To identify genes involved in AHI-1–mediated transformation, microarray analysis was performed to identify differentially expressed genes in AHI-1–suppressed CTCL cells. Fifteen up-regulated and 6 down-regulated genes were identified and confirmed by quantitative reverse transcription-polymerase chain reaction. Seven were further confirmed in a microarray analysis of CD4+CD7−Sezary cells from Sezary syndrome patients. HCKand BIN1emerged as new candidate cooperative genes, with differential protein expression, which correlates with observed transcript changes. Interestingly, changes in HCK phosphorylation and biologic response to its inhibitor, dasatinib, were observed in AHI-1–suppressed or –overexpressed cells. The tumor suppressor BIN1 physically interacts with MYC in CTCL cells, which also exhibit differential MYC protein expression. In addition, aberrant expression of alternative splicing forms of BIN1was observed in primary and transformed CTCL cells. These findings indicate that HCK and BIN1 may play critical roles in AHI-1–mediated leukemic transformation of human CTCL cells.

AHI-1 is an oncogene often targeted by provirus insertional mutagenesis in murine leukemias and lymphomas. Aberrant expression of human AHI-1 occurs in cutaneous T-cell lymphoma (CTCL) cells and in CD4+CD7− Sezary cells from patients with Sezary syndrome. Stable knockdown of AHI-1 using retroviral-mediated RNA interference in CTCL cells inhibits their transforming activity in vitro and in vivo. To identify genes involved in AHI-1–mediated transformation, microarray analysis was performed to identify differentially expressed genes in AHI-1–suppressed CTCL cells. Fifteen up-regulated and 6 down-regulated genes were identified and confirmed by quantitative reverse transcription-polymerase chain reaction. Seven were further confirmed in a microarray analysis of CD4+CD7− Sezary cells from Sezary syndrome patients. HCK and BIN1 emerged as new candidate cooperative genes, with differential protein expression, which correlates with observed transcript changes. Interestingly, changes in HCK phosphorylation and biologic response to its inhibitor, dasatinib, were observed in AHI-1–suppressed or –overexpressed cells. The tumor suppressor BIN1 physically interacts with MYC in CTCL cells, which also exhibit differential MYC protein expression. In addition, aberrant expression of alternative splicing forms of BIN1 was observed in primary and transformed CTCL cells. These findings indicate that HCK and BIN1 may play critical roles in AHI-1–mediated leukemic transformation of human CTCL cells.

Cell migrations play a critical role in animal development and organogenesis. Here, we describe a mechanism by which the migration behaviour of a particular cell type is regulated temporally and coordinated with over-all development of the organism. The hermaphrodite distal tip cells (DTCs) of Caenorhabditis elegans migrate along the body wall in three sequential phases distinguished by the orientation of their movements, which alternate between the anteroposterior and dorsoventral axes. The ventral-to-dorsal second migration phase requires the UNC-6 netrin guidance cue and its receptors UNC-5 and UNC-40, as well as additional, UNC-6-independent guidance systems. We provide evidence that the transcriptional upregulation of unc-5 in the DTCs is coincident with the initiation of the second migration phase, and that premature UNC-5 expression in these cells induces precocious turning in an UNC-6-dependent manner. The DAF-12 steroid hormone receptor, which regulates developmental stage transitions in C. elegans, is required for initiating the first DTC turn and for coincident unc-5 upregulation. We also present evidence for the existence of a mechanism that opposes or inhibits UNC-5 function during the longitudinal first migration phase and for a mechanism that facilitates UNC-5 function during turning. The facilitating mechanism presumably does not involve transcriptional regulation of unc-5 but may represent an inhibition of the phase 1 mechanism that opposes or inhibits UNC-5. These results, therefore, reveal the existence of two mechanisms that regulate the UNC-5 receptor that are critical for responsiveness to the UNC-6 netrin guidance cue and for linking the directional guidance of migrating distal tip cells to developmental stage advancements.

We have examined the termination efficiency of T7 and T3 RNA polymerases (RNAPs) at a variety of termination signals. In agreement with previous investigators we find that termination occurs after the synthesis of an RNA product with a stable secondary structure followed by a run of U residues. Stem-loop structures that lack a 3' U-tract fail to terminate the phage enzyme. The distance (or the sequence) between the start site for transcription and the termination signal may also be important, as placing the terminator at different locations downstream from the promoter, or changing the promoter sequence, results in alterations in termination efficiency. We have explored termination at extended runs of homopolymers in the absence of an apparent stem-loop structure, and have observed that the enzyme terminates (inefficiently) when synthesizing U-rich transcripts, but not A- or C-rich transcripts. This is especially true at low concentrations of UTP. Strikingly, when an elongation complex (EC) encounters a dA-tract in the template strand it is able to slide on the template, resulting in the synthesis of products that have more or fewer U residues than predicted by the sequence of the DNA. This observation suggests that the formation of an RNA : DNA hybrid may be important to the lateral stability of the EC (its ability to maintain proper register with the DNA template). We have also explored the termination properties of a proteolytically nicked form of T7 RNAP. The nicked enzyme forms a less stable EC than the intact RNAP and dissociates more readily from the template in regions that encode inherently destabilizing RNAs (e.g. stem-loop structures, poly(U)-tracts). However, the nicked enzyme terminates less efficiently at the late T7 terminator (T7-TΦ) or at a termination signal in the human preproparathyroid hormone gene. These results suggest that termination is a highly specific event, and not merely a consequence of decreased stability of the EC. Our observations are not consistent with previous models of termination by the phage RNAP and indicate that revisions to these models may be required. Copyright 1993, 1999 Academic Press

No relevant conflicts of interest to declare.

Cutaneous T cell lymphoma (CTCL) consists of two main subtypes: mycosis fungoides (MF), which primarily affects the skin; and Sézary syndrome (SS), which is characterized by the presence of circulating malignant Sézary cells. Although defective apoptosis and cell cycle progression have been implicated in CTCL disease development, the precise molecular mechanism underlying these abnormalities is only partially understood. Recently our group showed that TOX, a transcription factor transiently expressed during thymocyte development, was ectopically expressed in the malignant CD4+T cells in MF skin lesions. Normally in mature CD4+T cells, TOX is expressed at very low levels. Not only is TOX highly expressed in the infiltrating CD4+T cells in MF skin lesions, but its expression levels also inversely correlate with the clinical outcome of CTCL patients. However, its involvement in other types of CTCL remains unknown and its biological effects on CTCL pathogenesis have not been explored. In this study, we sought to examine if aberrant TOX expression occurs in SS (a leukemic variant of CTCL), and whether it contributes to CTCL pathogenesis. We found that TOX expression is also enhanced significantly in primary CD4+CD7-cells from SS patients (n=12), and its enhanced transcript levels correlate with higher disease-specific mortality (P=0.039). To investigate the role of TOX in CTCL, we corrected the ectopic TOX expression by lentiviral-shRNA-mediated TOX gene knock-down in CTCL cell lines (Hut78, HH, and SZ4). In all three cell lines, this resulted in markedly increased apoptosis (measured using Annexin V apoptosis assay, 2-3 fold, P<0.05) and reduced cellular proliferation (assessed by BrdU incorporation assay and colony formation in 3D cultures, 1.5-12 fold, P<0.05). In line with these phenotypes, TOX knock-down led to an increase in cleaved caspases (3, 9) and enhanced expression of multiple cell cycle inhibitors (CDKN1B, CDKN1C). Moreover, to test the effect of TOX suppression on the tumor-inducing ability of CTCL cells in vivo, we injected TOX-suppressed cells or control CTCL cells (1x106per injection site) subcutaneously into NOD-SCID IL2rγ null (NSG) mice and monitored local tumor formation. Control CTCL cells (Hut78, HH) induced local tumor growth in six out of six mice within 2 weeks of injection, with enlargement progressively to 1.5cm in diameter within 4 weeks of injection. In contrast, TOX silencing effectively reduced the tumor volume induced by HH cells (n=6) and abolished the tumor-forming ability of Hut78 cells (n=6). Since TOX is a DNA-binding nuclear protein, we next sought to explore the potential downstream molecular partners of TOX by performing comparative transcriptome analysis between TOX-suppressed cells and control CTCL cells (Hut78, HH). The expression of several tumor suppressor genes, including SMAD3 and FOXO3, was increased upon TOX knock-down in both Hut78 and HH cell lines. Pathway analyses of the differentially-expressed genes uncovered activation of apoptotic pathways and cell cycle arrest processes upon TOX suppression. These findings were validated using quantitative RT-PCR. In conclusion, our results indicate that ectopic expression of TOX in CTCL cells causes the proliferation/apoptosis balance to shift toward proliferation through suppression of the transcription of multiple tumor suppressors. Therefore abolishing TOX activity may be a promising treatment strategy for CTCL.

Cutaneous T-cell lymphoma (CTCL) is a potentially devastating malignancy. The pathogenesis of this cancer remains poorly elucidated. Previous studies focused on analysis of expression and function of known oncogenes and tumor suppressor genes. However, emerging reports highlight that it is also important to analyze the expression of genes that are ectopically expressed in CTCL (e.g., embryonic stem cell genes (ESC), cancer testis (CT) genes, etc.). Currently, it is not known whether ESC genes are expressed in CTCL. In the current work, we analyze by RT-PCR the expression of 26 ESC genes, many of which are known to regulate pluripotency and promote cancer stem cell-like phenotype, in a historic cohort of 60 patients from Boston and in a panel of 11 patient-derived CTCL cell lines and compare such expression to benign inflammatory dermatoses that often clinically mimic CTCL. Our findings document that many critical ESC genes including NANOG, SOX2, OCT4(POU5F1) and their upstream and downstream signaling members are expressed in CTCL. Similarly, polycomb repressive complex 2 (PRC2) genes (i.e., EZH2, EED, and SUZ12) are also expressed in CTCL lesional skin. Furthermore, select ESC genes (OCT4, EED, TCF3, THAP11, CHD7, TIP60, TRIM28) are preferentially expressed in CTCL samples when compared to benign skin biopsies. Our work suggests that ESC genes are ectopically expressed together with CT genes, thymocyte development genes and B cell-specific genes and may be working in concert to promote tumorigenesis. Specifically, while ESC genes may be promoting cancer stem cell-like phenotype, CT genes may be contributing to aneuploidy and genomic instability by producing aberrant chromosomal translocations. Further analysis of ESC expression and function in this cancer will greatly enhance our fundamental understanding of CTCL and will help us identify novel therapeutic targets.

Neutralizing CXCL10 reverses established vitiligo.

No relevant conflicts of interest to declare.

Cutaneous T-cell lymphomas (CTCLs) represent a group of lymphoproliferative disorders that are characterized by homing of malignant T-cells to the surface of skin. There are two main types of CTCL: Mycosis Fungoides (MF) and its leukemic variant Sezary Syndrome (SS), which together represent about 65-70% of all CTCL cases. The precise genetic pathogenesis of these diseases remains largely undetermined. Recently, our research group has demonstrated that AHI-1(Abelson Helper Integration site-1) oncogene is involved in CTCL. Expression of AHI-1is increased in human leukemia cell lines, with marked upregulation (up to 40 fold) in CTCL lines (Hut78 and Hut102). Moreover, in FACS-purified CD4+CD7-Sezary cells from patients with Sezary Syndrome, AHI-1 expression is higher at both the RNA and protein levels compared to normal CD4+cells. Furthermore, stable suppression of endogenous AHI-1in Hut78 cells using small interfering RNA, normalizes their transforming activity both in vitroand in vivo. Thus, lymphomagenic activity of Hut78 cells is partially dependent on the expression of AHI-1. Interestingly, BIN1(Bridging integrator 1) was identified through microarray analysis as one of the genes that may be involved in AHI-1-mediated leukemic transformation in CTCL cells. BIN1 is a nucleocytosolic adaptor protein with more than ten isoforms; some isoforms, including the BIN1isoform (+10, +13), act as tumor suppressors, whereas the BIN1(+12A) behaves as a cancer-related isoform in solid tumor models. However, the role of BIN1in regulation of normal hematopoiesis and lymphomagenesis remains unknown. We have recently demonstrated that transcript levels of BIN1isoforms are significantly lower in patients with MF or SS compared to controls. Four isoforms of BIN1have been identified in Hut78 and primary CD4+CD7-Sezary cells. To investigate the role of BIN1in CTCL, the BIN1isoforms (+10, +13) and BIN1(+12A) lentiviral constructs were transduced into two CTCL cell lines, Hut78 and HH cells. Overexpression of BIN1isoforms led to a significant reduction in cell proliferation, as assessed by colony forming cell assays and 3H-Thymidine uptake assays (2-3 fold, p<0.05). Furthermore, a significant increase in spontaneous and specific apoptosis was observed in BIN1-transduced cells, with and without exogenous FAS-ligand (2-3 fold, p<0.05). Interestingly, a significant reduction in protein expression of c-FLIP (inhibitor of the FAS-mediated apoptosis pathway) and upregulation of downstream cleaved caspase-8 and caspase-3 was demonstrated in BIN1-transduced cells, suggesting that BIN1isoforms induce apoptosis by downregulating the expression of c-FLIP, which leads to activation of the FAS-mediated apoptosis pathway. These findings show anti-proliferative and pro-apoptotic roles for BIN1 isoforms in human CTCL cells. In addition, subcellular fractionation and confocal microscopy further indicated that both the BIN1 (+10, +13) and BIN1 (+12A) isoforms are mainly located in the nucleus in Hut78 and HH cells. Furthermore, to investigate the effects of overexpression of BIN1isoforms on the ability to induce tumors in vivo, we tested their leukemogenic potential by injecting transduced HH cells into non-obese diabetic/severe-combined immunodeficiency (NOD/SCID) mice. Mice injected subcutaneously with either parental HH or control empty vector cells (2 x 107/per mouse), showed local tumor formation in 6 of 6 mice within 4 days post-injection. The local tumors enlarged progressively and were often 1.5–2 cm in diameter by 3 weeks after injection. In contrast, no local tumors formed in mice given injections of equal numbers of BIN1-transduced HH cells after 14 days, in 12 out of 12 mice. Tumor formation was only observed in BIN1-transduced HH cells after 3 weeks post-injection. However, the local tumors were significantly smaller in BIN1-transduced HH cells compared to controls (∼4-fold). These findings further indicate that the two BIN1isoforms have tumor suppressor activities in NOD/SCID mice and can significantly delay tumor formation and reduce tumor size in vivo.

Ahi-1 (Abelson helper integration site 1) is a novel gene that is commonly activated by provirus insertional mutagenesis in v-abl and myc-induced murine leukemias and lymphomas. It encodes a unique protein with SH3 and WD40-repeat domains suggesting novel signaling activities. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and lymphomas and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemia cell lines, particularly in the cutaneous T-cell leukemia cell lines, Hut 78 and Hut 102, where increases in AHI-1 transcripts of 40-fold are seen. To test directly whether the deregulated expression of AHI-1 in leukemic cells contributes to their transformed properties, knockdown of AHI-1 expression in Hut 78 cells, a cell line derived from peripheral blood of a patient with Sezary syndrome, was performed using retroviral-mediated RNA interference (RNAi). In a screen of 9 constructs that produce specific short hairpin AHI-1 transcripts, one was found to specifically inhibit AHI-1 expression in transduced Hut 78 cells by 80%, as evaluated by quantitative real-time RT-PCR, Northern and Western blot analyses. Retroviral-mediated suppression of AHI-1 also reduced the autocrine production of IL-2, IL-4 and TNFalpha in Hut 78 cells by up to 85% and caused a significant reduction in their growth factor independence in semi-solid cultures (up to 10-fold) and in single cell cultures (4-fold) by comparison to cells transduced with a control vector. Interestingly, although addition of IL-4, TNFalpha or a combination of 3 growth factors restored colony formation from the shRNA-transduced Hut 78 cells in semi-solid cultures, this was not achieved if only IL-2 was added, even though AHI-1 expression was inhibited. The ability of Hut 78 cells to produce tumors in NOD/SCID-β2microglobulin−/− mice within 3 weeks was also lost when AHI-1 expression was suppressed. Microarray analysis on RNA from Hut 78 cells with the suppression of AHI-1, using the Affymetrix Human Genome U133 plus 2.0 Arrays, identified differentially expressed molecules critical in T-cell activation, signal transduction, as well as cell proliferation and differentiation. Q-RT-PCR analysis revealed that the transcript levels of AHI-1 and its isoforms were significantly increased in CD4+CD7− Sezary cells, in which more than 85% of these cells are leukemic cells, in 5 of 6 blood samples obtained from patients with Sezary syndrome as compared to T cells similarly isolated from 8 healthy individuals. Elevated AHI-1 transcript levels were not found in 3 patient samples containing less than 35% leukemic Sezary cells. Taken together, these findings provide strong evidence of the oncogenic activity of AHI-1 in human T-cell leukemic cells and its deregulation can contribute to the development of human cutaneous T-cell lymphomas, including Sezary syndrome.

Ahi-1 (Abelson helper integration site 1) is a novel gene that is commonly activated by provirus insertional mutagenesis in v-abl and myc-induced murine leukemias and lymphomas. It encodes a unique protein with SH3 and WD40-repeat domains suggesting novel signaling activities. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and lymphomas and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemia cell lines, particularly in the cutaneous T-cell leukemia cell lines, Hut 78 and Hut 102, where increases in AHI-1 transcripts of 40-fold are seen. To test directly whether the deregulated expression of AHI-1 in leukemic cells contributes to their transformed properties, knockdown of AHI-1 expression in Hut 78 cells, a cell line derived from peripheral blood of a patient with Sezary syndrome, was performed using retroviral-mediated RNA interference (RNAi). In a screen of 9 constructs that produce specific short hairpin AHI-1 transcripts, one was found to specifically inhibit AHI-1 expression in transduced Hut 78 cells by 80%, as evaluated by quantitative real-time RT-PCR, Northern and Western blot analyses. Retroviral-mediated suppression of AHI-1 also reduced the autocrine production of IL-2, IL-4 and TNFalpha in Hut 78 cells by up to 85% and caused a significant reduction in their growth factor independence in semi-solid cultures (up to 10-fold) and in single cell cultures (4-fold) by comparison to cells transduced with a control vector. Interestingly, although addition of IL-4, TNFalpha or a combination of 3 growth factors restored colony formation from the shRNA-transduced Hut 78 cells in semi-solid cultures, this was not achieved if only IL-2 was added, even though AHI-1 expression was inhibited. The ability of Hut 78 cells to produce tumors in NOD/SCID-β2microglobulin−/−mice within 3 weeks was also lost when AHI-1 expression was suppressed. Microarray analysis on RNA from Hut 78 cells with the suppression of AHI-1, using the Affymetrix Human Genome U133 plus 2.0 Arrays, identified differentially expressed molecules critical in T-cell activation, signal transduction, as well as cell proliferation and differentiation. Q-RT-PCR analysis revealed that the transcript levels of AHI-1 and its isoforms were significantly increased in CD4+CD7−Sezary cells, in which more than 85% of these cells are leukemic cells, in 5 of 6 blood samples obtained from patients with Sezary syndrome as compared to T cells similarly isolated from 8 healthy individuals. Elevated AHI-1 transcript levels were not found in 3 patient samples containing less than 35% leukemic Sezary cells. Taken together, these findings provide strong evidence of the oncogenic activity of AHI-1 in human T-cell leukemic cells and its deregulation can contribute to the development of human cutaneous T-cell lymphomas, including Sezary syndrome.

Methylated DNA-binding protein (MDBP) from human placement recognizes specific DNA sequences containing 5-methylcytosine (m5c) residues. Comparisous of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains ∼/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferense. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an ∼20-bp long region may be recognized by MDBP.