An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance.

Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.

Nucleotide deficiency promotes genomic instability in early stages of cancer development.

Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes significantly decreased the cellular nucleotide levels in the newly transformed cells. Exogenously supplied nucleosides rescued the replication stress and DNA damage and dramatically decreased oncogene-induced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability.

Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6's role in hematopoietic differentiation and leukemia.

BACKGROUND: Long non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources). METHODS: INFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo. RESULTS: The INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo. CONCLUSIONS: Our approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine.

Human Papillomavirus-type distribution in anogenital lesions of prepubertal children.

BACKGROUND: In contrast to adults, only limited data are available on the human papillomavirus (HPV)-type spectrum in anogenital warts (AGW) of children. OBJECTIVE: This study aimed to evaluate the HPV-type spectrum in AGW of prepubertal children. MATERIALS & METHODS: In a retrospective German multicentre study, HPV genotyping was performed in AGW biopsies of 55 1- to 12-year-old children using HPV group-specific PCRs followed by hybridization with type-specific probes or sequence analysis. RESULTS: Human papillomavirus-DNA was found in 53 of the 55 AGW. In 58.5% (31/53) of the HPV-positive AGW, mucosal HPV types were detected. HPV6 (27/53, 50.9%) was the predominant type. 43.4% (23/53) of the lesions were induced by cutaneous HPV types (HPV2, HPV27, HPV57). Mucosal HPV types were significantly more common in children under 5 years of age than in children 5 years of age and older (22/25, 88.0% [95% CI: 70.0-95.8] vs. 9/28, 32.1% [95% CI: 17.9-50.7], P < 0.001). In contrast, cutaneous HPV types were significantly more prevalent in the 5- to 12-year age group (4/25, 16.0% [95% CI 6.4-34.7] vs. 19/28, 67.9% [95% CI 49.3-82.1], P < 0.001). CONCLUSION: Anogenital warts in 5- to 12-year-old children are frequently associated with cutaneous HPV types, possibly due to horizontal transmission. HPV typing, in addition to comprehensive clinical and psychosocial evaluation, can potentially help in the assessment of these cases.

European dermatology forum: Updated guidelines on the use of extracorporeal photopheresis 2020 - Part 2.

BACKGROUND: Following the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma published in 1983, this technology has received continued use and further recognition for additional earlier as well as refractory forms. After the publication of the first guidelines for this technology in the JEADV in 2014, this technology has maintained additional promise in the treatment of other severe and refractory conditions in a multidisciplinary setting. It has confirmed recognition in well-known documented conditions such as graft-vs.-host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection including lung, heart and liver and to a lesser extent inflammatory bowel disease. MATERIALS AND METHODS: In order to further provide recognized expert practical guidelines for the use of this technology for all indications, the European Dermatology Forum (EDF) again proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task. All authors had the opportunity to review each contribution as it was added. RESULTS AND CONCLUSION: These updated 2020 guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion. The guidelines were divided into two parts: PART I covers Cutaneous T-cell lymphoma, chronic graft-vs.-host disease and acute graft-vs.-host disease, while PART II will cover scleroderma, solid organ transplantation, Crohn's disease, use of ECP in paediatric patients, atopic dermatitis, type 1 diabetes, pemphigus, epidermolysis bullosa acquisita and erosive oral lichen planus.

[Anakinra therapy in a patient with multifocal pyoderma gangrenosum and hidradenitis suppurativa]. / Anakinra-Therapie eines multifokalen Pyoderma gangraenosum bei einem Patienten mit Hidradenitis suppurativa.

The case of a patient with pyoderma gangrenosum and exacerbation after an intense massage is reported. After different immunosuppressive therapies and former diagnosis of hidradenitis suppurativa in his medical history the patient underwent therapy with anakinra 100 mg per day and showed good improvement of his ulcers.

European dermatology forum - updated guidelines on the use of extracorporeal photopheresis 2020 - part 1.

BACKGROUND: Following the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma published in 1983, this technology has received continued use and further recognition for additional earlier as well as refractory forms. After the publication of the first guidelines for this technology in the JEADV in 2014, this technology has maintained additional promise in the treatment of other severe and refractory conditions in a multi-disciplinary setting. It has confirmed recognition in well-known documented conditions such as graft-versus-host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection including lung, heart and liver and to a lesser extent inflammatory bowel disease. MATERIALS AND METHODS: In order to further provide recognized expert practical guidelines for the use of this technology for all indications, the European Dermatology Forum (EDF) again proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task. All authors had the opportunity to review each contribution as it was added. RESULTS AND CONCLUSION: These updated 2020 guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion. The guidelines are divided in two parts: PART I covers cutaneous T-cell lymphoma, chronic graft-versus-host disease and acute graft-versus-host disease while PART II will cover scleroderma, solid organ transplantation, Crohn's disease, use of ECP in paediatrics practice, atopic dermatitis, type 1 diabetes, pemphigus, epidermolysis bullosa acquisita and erosive oral lichen planus.

Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development.

[Treatment of mycosis fungoides and Sézary syndrome]. / Therapie der Mycosis fungoides und des Sézary-Syndroms.

Adequate therapeutic management of cutaneous T-cell lymphoma (CTCL) requires the identification of the exact CTCL stage and entity within the current WHO classification. There is no curative therapy for CTCL yet, so that treatment currently aims at improving symptoms and quality of life as well as reducing relapse rates. The treatment has to be stage-adapted. Therapeutic options comprise skin-directed as well as systemic treatment. In early stages, phototherapy and local steroids are the first-line therapeutic options. For the therapy of higher stages, interferon alpha and the RXR-specific retinoid bexarotene are used as first-line medications. Second-line treatment comprises monochemotherapy with agents like gemcitabine or liposomal doxorubicine. Nevertheless, the high relapse rates in higher stages make novel alternative treatment options necessary. As future therapy, especially the fusion protein brentuximab-vedotin directed against CD30 shows promising potential in clinical studies.

The complex basis underlying common fragile site instability in cancer.

Common fragile sites (CFSs) were characterized almost 30 years ago as sites undergoing genomic instability in cancer. Recently, in vitro studies have found that oncogene-induced replication stress leads to CFS instability. In vivo, CFSs were found to be preferentially unstable during early stages of cancer development and to leave a unique signature of instability. It is now increasingly clear that, along the spectrum of replication features characterizing CFSs, failure of origin activation is a common feature. This and other features of CFSs, together with the replication stress characterizing early stages of cancer development, lead to incomplete replication that results in genomic instability preferentially at CFSs. Here, we review the shared and unique characteristics of CFSs, their underlying causes and their implications, particularly with respect to the development of cancer.

Histopathological and immunophenotypical criteria for the diagnosis of Sézary syndrome in differentiation from other erythrodermic skin diseases: a European Organisation for Research and Treatment of Cancer (EORTC) Cutaneous Lymphoma Task Force Study of 97 cases.

BACKGROUND: Patients with erythrodermic disease are a diagnostic challenge regarding the clinical and histological differential diagnosis. OBJECTIVES: To evaluate histopathological and immunohistochemical diagnostic markers for Sézary syndrome. METHODS: Ninety-seven erythrodermic cases [Sézary syndrome (SS), n = 57; erythrodermic inflammatory dermatoses (EIDs), n = 40] were collected by the EORTC Cutaneous Lymphoma Task Force histopathology group. Evaluation criteria were (i) epidermal and dermal changes; (ii) morphology of the infiltrate; (iii) immunohistochemical analysis of marker loss (CD2, CD3, CD4, CD5 and CD7); (iv) bystander infiltrate by staining for CD8, FOXP3 and CD25; and (v) expression of Ki-67, CD30, PD-1 and MUM-1. RESULTS: The workshop panel made a correct diagnosis of SS in 51% of cases (cutaneous T-cell lymphoma 81%) and of EID in 80% without clinical or laboratory data. Histology revealed a significantly increased degree of epidermotropism (P < 0.001) and more intraepidermal atypical lymphocytes (P = 0.0014) in SS biopsies compared with EID. Pautrier microabscesses were seen only in SS (23%) and not in EID (P = 0.0012). SS showed significantly more dermal cerebriform and blastic lymphocytes than EID. Immunohistochemistry revealed a significant loss of CD7 expression (< 50%) in 33 of 51 (65%) cases of SS compared with two of 35 (6%) EID (P < 0.001). The lymphocytic infiltrate in SS skin samples was found significantly to express PD-1 (P = 0.0053), MUM-1 (P = 0.0017) and Ki-67 (P < 0.001), and showed less infiltration of CD8(+) lymphocytes (P < 0.001). A multivariate analysis identified CD7 loss, increased numbers of small cerebriform lymphocytes, low numbers of CD8(+) lymphocytes and increased proliferation (Ki-67(+) lymphocytes) as the strongest indicators for the diagnosis of SS. CONCLUSIONS: A number of different histological and immunophenotypical criteria are required to differentiate between SS and EIDs.

Interplay between genetic and epigenetic factors governs common fragile site instability in cancer.

Common fragile sites (CFSs) are regions within the normal chromosomal structure that were characterized as hotspots for genomic instability in cancer almost 30 years ago. In recent years, many efforts have been made to understand the basis of CFS fragility and their involvement in the genomic signature of instability found in malignant tumors. CFSs are among the first regions to undergo genomic instability during cancer development because of their intrinsic sensitivity to replication stress conditions, which result from oncogene expression. The preferred sensitivity of CFSs to replication stress stems from various mechanisms including: replication fork arrest at AT-rich repeats, origin paucity along large genomic regions, failure in activation of dormant origins, late replication timing, collision between replication and transcription along large genes, all leading to incomplete replication of the CFS region and resulting in chromosomal instability. Here we review shared and unique characteristics of CFSs, their underlying causes and implications, particularly for the development of cancer.

Guidelines on the use of extracorporeal photopheresis.

BACKGROUND: After the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma was published in 1983 with its subsequent recognition by the FDA for its refractory forms, the technology has shown significant promise in the treatment of other severe and refractory conditions in a multi-disciplinary setting. Among the major studied conditions are graft versus host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection and inflammatory bowel disease. MATERIALS AND METHODS: In order to provide recognized expert practical guidelines for the use of this technology for all indications the European Dermatology Forum (EDF) proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task. RESULTS AND CONCLUSION: These guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion.

The tumour suppressor p53 is frequently nonfunctional in Sézary syndrome.

BACKGROUND: Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group with Sézary syndrome (SS) as one of the most aggressive variants. Recently, we identified a loss of E2A as a recurrent event in SS, which enhanced proliferation via upregulation of the proto-oncogene MYC. MYC-induced transformation usually requires deleterious alterations of key apoptotic genes including p53; however, p53 functionality and mutation status in SS are unclear. OBJECTIVES: We investigated functionality of p53 signalling by pharmacological treatment with the MDM2 antagonist nutlin-3, which might result in p53 activation. Furthermore, we analysed the TP53 mutation status in CTCL cell lines and highly purified tumour cells from patients with SS by mRNA and DNA sequencing. METHODS: We analysed the apoptosis induction due to nutlin-3 treatment in various SS cell lines and primary patient samples by annexin V/propidium iodide staining. Induction of p53 target genes was analysed by immunoblotting, and TP53 was sequenced at the mRNA and DNA level. RESULTS: We identified various TP53 mutations and an impaired p53 signalling in the vast majority of the investigated cell lines and primary SS cells. CONCLUSIONS: In accordance with the importance of MYC deregulation in SS, p53 signalling is frequently nonfunctional in SS. However, although most likely ineffective as exclusive treatment in SS, it remains possible that pharmacological p53 activation could be beneficial in combination with other approaches including classical chemotherapeutics.

[Cutaneous malignant lymphomas. Update on diagnosis and therapy of cutaneous T-cell lymphomas]. / Maligne Lymphome der Haut. Update zu Diagnostik und Therapie kutaner T-Zell-Lymphome.

Cutaneous T-cell lymphomas represent extranodal non-Hodgkin lymphomas of mature T-cells, which accumulate in the skin. They have been recognized as a heterogeneous group with distinct variability in clinical presentation and histopathology, with divergent biological behaviour and prognosis. Therefore the exact diagnosis is an important prerequisite for an adequate and stage-adapted treatment.

[cFLIP diminishes CD95-induced apoptosis of CD30-stimulated cutaneous anaplastic large T cell lymphoma (cALCL) cells]. / Kutanes anaplastisches großzelliges Lymphom. Apoptoseresistenz durch CD30-induzierte cFLIP-Expression.

Stimulation of the TNF receptors CD30 and CD95 exerts opposite effects. Crosstalk of both receptors is unknown. We aimed to reveal regulatory mechanisms of CD30-induced effects on CD95 signaling of cALCL cell lines. "CD30/CD95 crosstalk analysis" was performed in cALCL cell lines by comparison of CD30 or CD95 stimulation and CD30/CD95 costimulation. Receptor expression and induction of apoptosis was investigated by flow cytometry. mRNA expression of CD30-inducible genes (cFLIP, TRAF1, cIAP2, and A20) was compared by semiquantitative reverse transcription (RT-RQ-) PCR in stimulated and unstimulated cells. Protein expression of IκBα, p100/p52, caspase-8, caspase-3, and cFLIP was analyzed by immunoblotting. A lentiviral-based shRNA-mediated approach was used to inhibit cFLIP expression. CD30/CD95 crosstalk experiments revealed that CD30 ligation leads to NFκB-mediated cFLIP upregulation in cALCL cells, which in turn enhanced resistance to CD95-mediated apoptosis. This effect is based on the CD30-induced upregulation of cFLIP. Knockdown of cFLIP restores sensitivity to CD95-mediated apoptosis. We conclude that the anti-apoptotic function of CD30 antibodies should be kept in mind if CD30 antibody-based therapeutic concepts for ALCL lymphoma are considered.

Diagnostic tools in Sezary syndrome.

Primary cutaneous T-cell lymphomas (CTCL) mycosis fungoides (Mf) and Sézary syndrome (SS) belong to the group of non-Hodgkin lymphomas which are characterized by clonally proliferating CD4+ cells localized in the skin. SS is a leukemic variant of CTCL and is characterized by erythroderma, generalized lymphadenopathy, and circulating atypical T-cells with cerebriform nuclei, so-called Sézary cells. Palmoplantar hyperkeratosis, generalized alopecia, and severe pruritus are additional symptoms that are associated with SS. Patients have a poor prognosis with an estimated five year survival of 12.5 to 27 percent and estimated median survival of 14.5 to 18 months. The incidence of MF and also SS has increased with time and may be in part due to improved clinical awareness and especially advances in diagnostic testing.

Germline NPM1 mutations lead to altered rRNA 2'-O-methylation and cause dyskeratosis congenita.

RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.