Pharmacogenetic biomarkers for secukinumab response in psoriasis patients in real-life clinical practice.

BACKGROUND: Prediction of the response to a biological treatment in psoriasis patients would allow efficient treatment allocation. OBJECTIVE: To identify polymorphisms associated with secukinumab response in psoriasis patients in a daily practice setting. METHODS: We studied 180 SNPs in patients with moderate-to-severe plaque psoriasis recruited from 15 Spanish hospitals. Treatment effectiveness was evaluated by absolute PASI ≤3 and ≤1 at 6 and 12 months. Individuals were genotyped using a custom Taqman array. Multiple logistic regression models were generated. Sensitivity, specificity and area under the curve (AUC) were analysed. RESULTS: A total of 173 patients were studied at 6 months, (67% achieved absolute PASI ≤ 3 and 65% PASI ≤ 1) and 162 at 12 months (75% achieved absolute PASI ≤ 3 and 64% PASI ≤ 1). Multivariable analysis showed the association of different sets of SNPs with the response to secukinumab. The model of absolute PASI≤3 at 6 months showed best values of sensitivity and specificity. Four SNPs were associated with the capability of achieving absolute PASI ≤ 3 at 6 months. rs1801274 (FCGR2A), rs2431697 (miR-146a) and rs10484554 (HLCw6) were identified as risk factors for failure to achieve absolute PASI≤3, while rs1051738 (PDE4A) was protective. AUC including these genotypes, weight of patients and history of biological therapy was 0.88 (95% CI 0.83-0.94), with a sensitivity of 48.6% and specificity of 95.7% to discriminate between both phenotypes. CONCLUSION: We have identified a series of polymorphisms associated with the response to secukinumab capable of predicting the potential response/non-response to this drug in patients with plaque psoriasis.

CD133+ cell content correlates with tumour growth in melanomas from skin with chronic sun-induced damage.

BACKGROUND: Melanoma is responsible for almost 80% of the deaths attributed to skin cancer. Stem cells, defined by CD133 expression, have been implicated in melanoma tumour growth, but their specific role is still uncertain. OBJECTIVES: We hypothesized that the phenotypic heterogeneity of human cutaneous melanomas is related to their content of CD133+ cells. METHODS: We compared the percentages of CD133+ cells in 29 tumours from four classic types of melanoma: lentigo maligna melanoma (LMM), superficial spreading melanoma, nodular melanoma and acral lentiginous melanoma (ALM). Also, we compared the percentages of CD133+ cells in melanomas with different degrees of exposure to ultraviolet radiation: 16 melanomas from skin with chronic sun-induced damage and 13 melanomas from skin without such damage. RESULTS: We found a statistically significant increase of CD133+ cells in three different contexts: in melanomas arising on skin with signs of chronic sun-induced damage vs. nonexposed skin, in melanomas in situ vs. invasive melanomas, and in LMM vs. ALM. The proportions of CD133+ cells did not differ among samples of normal skin with different degrees of sun exposure. A distinct subpopulation of CD133+CXCR4+ cancer stem cells (CSCs) was identified and shown to be related to the invasive phenotype of the tumours. CONCLUSIONS: Here, we provide evidence showing, for the first time, that an increase in the CD133+ cell content is associated both with melanomas arising on skin with signs of chronic sun-induced damage and in melanomas in situ with better prognosis. Moreover, our study further confirms the existence of a subpopulation of CD133+CXCR4+ CSCs in cutaneous melanomas with invasive phenotype and poor prognosis.

Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2).

Snai2-deficient cells are radiosensitive to DNA damage. The function of Snai2 in response to DNA damage seems to be critical for its function in normal development and cancer. Here, we applied a functional genomics approach that combined gene-expression profiling and computational molecular network analysis to obtain global dissection of the Snai2-dependent transcriptional response to DNA damage in primary mouse embryonic fibroblasts (MEFs), which undergo p53-dependent growth arrest in response to DNA damage. Although examination of the response showed that overall expression of p53 target gene expression patterns was similarly altered in both control and Snai2-deficient cells, we have identified and validated candidate Snai2 target genes linked to Snai2 gene function in response to DNA damage. This work defines for the first time the effect of Snai2 on p53 target genes in cells undergoing growth arrest, elucidates the Snai2-dependent molecular network induced by DNA damage, points to novel putative Snai2 targets, and suggest a mechanistic model, which has implications for cancer management.

Sustained leukaemic phenotype after inactivation of BCR-ABLp190 in mice.

Pharmacological inactivation of cancer genes or products is being used as a strategy for therapy in oncology. To investigate the potential role of BCR-ABLp190 cessation in leukaemia development, we generated mice carrying a tetracycline-repressible BCR-ABLp190 transgene. These mice were morphologically normal at birth, and developed leukaemias. Disease was characterized by the presence of B-cell blasts co-expressing myeloid markers, reminiscent of the human counterpart. BCR-ABLp190 activation can initiate leukaemia in both young and adult mice. Transitory expression of BCR-ABLp190 is enough to develop leukaemia. Suppression of the BCR-ABLp190 transgene in leukaemic CombitTA-p190 mice did not rescue the malignant phenotype, indicating that BCR-ABLp190 is not required to maintain the disease in mice. Similar results were obtained by inactivation of BCR-ABLp190 with STI571 (Gleevec; Novartis, East Hanover, NJ, USA) in leukaemic CombitTA-p190 mice. However, gradual suppression of BCR-ABLp190 in leukaemic CombitTA-p190 mice identified a minimum level of BCR-ABLp190 expression necessary to revert the specific block in B-cell differentiation in the leukaemic cells. Overall, the findings indicate that BCR-ABLp190 appears to cause epigenetic and/or genetic changes in tumour-maintaining cells that render them insensitive to BCR-ABLp190 inactivation.

SLUG (SNAI2) overexpression in embryonic development.

The Snail-related zinc-finger transcription factor, SLUG (SNAI2), is critical for the normal development of neural crest-derived cells and loss-of-function SLUG mutations have been proven to cause piebaldism and Waardenburg syndrome type 2 in a dose-dependent fashion. However, little is known about the consequences of SLUG overexpression in embryonic development. We report SLUG duplication in a child with a unique de novo 8q11.2-->q13.3 duplication associated with tetralogy of Fallot, submucous cleft palate, renal anomalies, hypotonia and developmental delay. To investigate the effects of Slug overexpression on development, we analyzed mice carrying a Slug transgene. These mice were morphologically normal at birth, inferring that Slug overexpression is not sufficient to cause overt morphogenetic defects. In the adult mice, there was a 20% incidence of sudden death, cardiomegaly and cardiac failure associated with incipient mesenchymal tumorigenesis. These findings, while not directly implicating Slug in congenital and acquired heart disease, raise the possibility that Slug overexpression may contribute to specific cardiac phenotypes and cancer development.

Understanding mesenchymal cancer: the liposarcoma-associated FUS-DDIT3 fusion gene as a model.

Chromosomal translocations entail the generation of gene fusions in mesenchymal tumors. Despite the successful identification of these specific and consistent genetic events, the nature of the intimate association between the gene fusion and the resulting phenotype still remains to be elucidated. Here these studies are reviewed, using FUS-DDIT3 as a model to illustrate how they have contributed to current understanding in unique and unexpected ways. FUS-DDIT3 is a chimeric oncogene generated by the most common chromosomal translocation t(12;16)(q13;p11) associated with liposarcomas. The application of transgenic methods to the study of this sarcoma-associated FUS-DDIT3 gene fusion has provided insights into their functions in vivo, and suggested mechanisms by which lineage selection may be achieved.

Deletion of the SLUG (SNAI2) gene results in human piebaldism.

Slug is a zinc-finger neural crest transcription factor, encoded by the SLUG gene, which is critical for development of hematopoietic stem cells, germ cells, and melanoblasts in the mouse. In mouse, heterozygous and homozygous slug mutations result in anemia, infertility, white forehead blaze, and depigmentation of the ventral body, tail, and feet. This phenotype is very similar to the heterozygous W (KIT)-mutant mouse phenotype and to human piebaldism, which is characterized by a congenital depigmented patches and poliosis (white forelock). To investigate the possibility that some cases of human piebaldism might result from abnormalities of the human SLUG (SNAI2) gene, we carried out Southern blot analysis of the SLUG gene in 17 unrelated patients with piebaldism, who lack apparent KIT mutations. Three of these patients had evident heterozygous deletions of the SLUG gene encompassing the entire coding region. Real-time PCR confirmed the deletion in all cases. Fluoresence in situ hybridization (FISH) of genomic SLUG probes to metaphase chromosomes independently confirmed the deletion in one of the cases. These findings indicate that some cases of human piebaldism result from mutation of the SLUG gene on chromosome 8, and provide further strong evidence for the role of SLUG in the development of human melanocytes.

Philadelphia-positive B-cell acute lymphoblastic leukemia is initiated in an uncommitted progenitor cell.

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the c-ABLgene on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, BCR-ABLp190 and BCR-ABLp210, are produced that are characteristic of chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph1-ALL) respectively. In CML, it is evident that the transformation occurs at the level of pluripotent stem cells. However, Ph1-ALL has been thought to affect progenitor cells with lymphoid differentiation. Recently, it has been demonstrated that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation in Ph1-ALL. In this review, we discuss what is known about the relationship between the specific BCR-ABLp190 oncogene, the target cell and the characteristics of the subsequent disease process it causes. We also discuss how this information may be applied to the establishment of new directions in therapy.

RNase P: from biological function to biotechnological applications.

The M1 RNA subunit of Escherichia coli RNase P is a ribozyme responsible for the catalytic activity of the complex. It removes the 5' leader sequence from tRNA precursors to form mature tRNAs. M1 recognizes its target mainly on the basis of its structure and this allows the design of modified ribozymes engineered to destroy other molecules without the need for special sequences in the targeted mRNAs. M1 is thus an ideal tool to eliminate the tumourigenic chimeric messengers created after chromosomal translocations. These results have direct implications for cancer therapeutics and molecular biology in general.

Selective destruction of tumor cells through specific inhibition of products resulting from chromosomal translocations.

A key problem in the effective treatment of patients with cancer (both leukemia and solid tumors) is to distinguish between tumor and normal cells. This problem is the main reason why current treatments for cancer are often ineffective. There have been remarkable advances in our understanding of the molecular biology of cancer that provides new selective tumor destruction mechanisms. The molecular characterization of the tumor-specific chromosomal abnormalities has revealed that fusion proteins are the consequence in the majority of cancers. These fusion proteins result from chimeric genes created by the translocations, which form chimeric mRNA species that contain exons from the genes involved in the translocation. Obviously, these chimeric molecules are attractive therapeutic targets since they are unique to the disease (they only exist in the tumor cells but not in the normal cells of the patient), allowing the design of specific anti-tumor drugs. Inhibition of chimeric gene expression by anti-tumor agents specifically kills leukemic cells without affecting normal cells. As therapeutic agents targeting chimeric genes, zinc-finger proteins, antisense RNAs or hammerhead-based ribozymes have been used. All of these agents have some limitations, indicating that new therapeutic tools are required as gene inactivating agents that should be able to inhibit any chimeric fusion gene product. Recently, we have used the catalytic RNA subunit of RNase P from Escherichia coli, which can be specifically directed to cut any mRNA sequence, to specifically destroy tumor-specific fusion genes created as a result of chromosomal translocations. In this chapter, we will review the advances made to selectively destroy tumor cells through specific inhibition of products resulting from chromosomal translocations.

The chimeric FUS/TLS-CHOP fusion protein specifically induces liposarcomas in transgenic mice.

The characteristic t(12;16)(q13;p11) chromosomal translocation, which leads to gene fusion that encodes the FUS-CHOP chimeric protein, is associated with human liposarcomas. The altered expression of FUS-CHOP has been implicated in a characteristic subgroup of human liposarcomas. We have introduced the FUS-CHOP transgene into the mouse genome in which the expression of the transgene is successfully driven by the elongation factor 1alpha (EF1alpha) promoter to all tissues. The consequent overexpression of FUS-CHOP results in most of the symptoms of human liposarcomas, including the presence of lipoblasts with round nuclei, accumulation of intracellular lipid, induction of adipocyte-specific genes and a concordant block in the differentiation program. We have demonstrated that liposarcomas in the FUS-CHOP transgenic mice express high levels of the adipocyte regulatory protein PPARgamma, whereas it is not expressed in embryonic fibroblasts from these animals following induction to differentiation toward the adipocyte lineage, indicating that the in vitro system does not really reflect the in vivo situation and the developmental defect is downstream of PPARgamma expression. No tumors of other tissues were found in these transgenic mice despite widespread activity of the EF1alpha promoter. This establishes FUS-CHOP overexpression as a key determinant of human liposarcomas and provide the first in vivo evidence for a link between a fusion gene created by a chromosomal translocation and a solid tumor.

In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment.

One major obstacle to the effective treatment of cancer is to distinguish between tumor cells and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities are ideal therapeutic targets because they are unique to the disease. We describe the use of a novel approach based on the catalytic RNA subunit of RNase P to destroy specifically the tumor-specific fusion genes created as a result of chromosome abnormalities. Using as a target model the abnormal BCR-ABL p190 and p210 products, we constructed M1-RNA with guide sequences that recognized the oncogenic messengers at the fusion point (M1-p190-GS and M1-p210-GS). To test the effectiveness and the specificity of M1-p190-GS and M1-p210-GS, we studied in vitro and in vivo effects of these RNA enzymes against BCR-ABL(p190) and BCR-ABL(p210), bearing in mind that both fusion genes share the ABL sequence but differ in the sequence coming from the BCR gene. We showed that M1-p190-GS and M1-p210-GS can act as sequence-specific endonucleases and can exclusively cleave target RNA that forms a base pair with the guide sequence (GS). We also demonstrated that when M1-p190-GS and M1-p210-GS were expressed in proper mammalian cell models, they abolished the effect of BCR-ABL by specifically decreasing the amount of the target BCR-ABL mRNA and preventing the function of the BCR-ABL oncogenes. These data clearly demonstrate the usefulness of the catalytic activity of M1-GS RNA to cleave specifically the chimeric molecules created by chromosomal abnormalities in human cancer and to represent a novel approach to cancer treatment.

A primitive hematopoietic cell is the target for the leukemic transformation in human philadelphia-positive acute lymphoblastic leukemia.

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the chromosomal counterpart (c-ABL gene) on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, BCR-ABL(p190) and BCR-ABL(p210), are produced that are characteristic of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(1)-ALL). In CML, the transformation occurs at the level of pluripotent stem cells. However, Ph(1)-ALL is thought to affect progenitor cells with lymphoid differentiation. Here we demonstrate that the cell capable of initiating human Ph(1)-ALL in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID), termed SCID leukemia-initiating cell (SL-IC), possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all Ph(1)-ALL analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34(+ )CD38(-), which is similar to the cell-surface phenotype of normal SCID-repopulating cells. This indicates that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation in Ph(1)-ALL.

Liposarcoma initiated by FUS/TLS-CHOP: the FUS/TLS domain plays a critical role in the pathogenesis of liposarcoma.

The most common chromosomal translocation in liposarcomas, t(12;16)(q13;p11), creates the FUS/TLS-CHOP fusion gene. We previously developed a mouse model of liposarcoma by expressing FUS-CHOP in murine mesenchymal stem cells. In order to understand how FUS-CHOP can initiate liposarcoma, we have now generated transgenic mice expressing altered forms of the FUS-CHOP protein. Transgenic mice expressing high levels of CHOP, which lacks the FUS domain, do not develop any tumor despite its tumorigenicity in vitro and widespread activity of the EF1alpha promoter. These animals consistently show the accumulation of a glycoprotein material within the terminally differentiated adipocytes, a characteristic figure of liposarcomas associated with FUS-CHOP. On the contrary, transgenic mice expressing the altered form of FUS-CHOP created by the in frame fusion of the FUS domain to the carboxy end of CHOP (CHOP-FUS) developed liposarcomas. No tumors of other tissues were found in these transgenic mice despite widespread activity of the EF1alpha promoter. The characteristics of the liposarcomas arising in the CHOP-FUS mice were very similar to those previously observed in our FUS-CHOP transgenic mice indicating that the FUS domain is required not only for transformation but also influences the phenotype of the tumor cells. These results provide evidence that the FUS domain of FUS-CHOP plays a specific and critical role in the pathogenesis of liposarcoma.

Chromosomal abnormalities and tumor development: from genes to therapeutic mechanisms.

This article highlights the recent advances in our understanding of the molecular structure and function of proteins that are activated or created by chromosomal abnormalities and discusses their possible role in tumor development. The molecular characterization of these proteins has revealed that tumor-specific fusion proteins are the consequence of the majority of chromosomal translocations associated with leukemias and solid tumors. A common theme that emerges is that creation of these proteins disrupts the normal development of tumor-specific target cells by blocking apoptosis. These insights identify these chromosomal translocation-associated genes as potential targets for improved cancer therapies.

Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells.

A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouracil-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis.

Promoter-specific activation of gene expression directed by bacteriophage-selected zinc fingers.

It has been shown that sequence-specific DNA-binding domains containing zinc fingers can be selected from libraries displayed on filamentous bacteriophage. The affinity and specificity of these peptides are well characterised in vitro, but few data are available to demonstrate specific DNA binding and discrimination between closely related DNA sequences in vivo. Transient transactivation assays were performed in mammalian cells, using expression plasmids which produce different amounts of a model transcription factor containing a phage-selected zinc finger DNA-binding domain, and reporter plasmids which carry systematic variations of the promoter sequence. When the intracellular concentration of the transcription factor was appropriate, activation of gene expression was absolutely dependent on a promoter having the same DNA sequence as that originally used to select the zinc finger domain by phage display. However, excessive intracellular concentrations of the transcription factor resulted in some less-specific DNA binding, leading to gene activation from similar promoters containing a maximum of two base changes. Thus, provided delivery is carefully controlled, highly specific control of gene expression in vivo can be achieved using artificial transcription factors containing phage-selected zinc finger DNA-binding domains.