Armin Zebisch

Raf Family Kinases: Old Dogs Have Learned New Tricks

First identified in the early 1980s as retroviral oncogenes, the Raf proteins have been the objects of intense research. The discoveries 10 years later that the Raf family members (Raf-1, B-Raf, and A-Raf) are bona fide Ras effectors and upstream activators of the ubiquitous ERK pathway increased the interest in these proteins primarily because of the central role that this cascade plays in cancer development. The important role of Raf in cancer was corroborated in 2002 with the discovery of B-Raf genetic mutations in a large number of tumors. This led to intensified drug development efforts to target Raf signaling in cancer. This work yielded not only recent clinical successes but also surprising insights into the regulation of Raf proteins by homodimerization and heterodimerization. Surprising insights also came from the hunt for new Raf targets. Although MEK remains the only widely accepted Raf substrate, new kinase-independent roles for Raf proteins have emerged. These include the regulation of apoptosis by suppressing the activity of the proapoptotic kinases, ASK1 and MST2, and the regulation of cell motility and differentiation by controlling the activity of Rok-α. In this review, we discuss the regulation of Raf proteins and their role in cancer, with special focus on the interacting proteins that modulate Raf signaling. We also describe the new pathways controlled by Raf proteins and summarize the successes and failures in the development of efficient anticancer therapies targeting Raf. Finally, we also argue for the necessity of more systemic approaches to obtain a better understanding of how the Ras-Raf signaling network generates biological specificity.

Functional Roles of Multiple Feedback Loops in Extracellular Signal-Regulated Kinase and Wnt Signaling Pathways That Regulate Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a key event in the generation of invasive tumor cells. A hallmark of EMT is the repression of E-cadherin expression, which is regulated by various signal transduction pathways including extracellular signal-regulated kinase (ERK) and Wnt. These pathways are highly interconnected via multiple coupled feedback loops (CFL). As the function of such coupled feedback regulations is difficult to analyze experimentally, we used a systems biology approach where computational models were designed to predict biological effects that result from the complex interplay of CFLs. Using epidermal growth factor (EGF) and Wnt as input and E-cadherin transcriptional regulation as output, we established an ordinary differential equation model of the ERK and Wnt signaling network containing six feedback links and used extensive computer simulations to analyze the effects of these feedback links in isolation and different combinations. The results show that the feedbacks can generate a rich dynamic behavior leading to various dose-response patterns and have a decisive role in determining network responses to EGF and Wnt. In particular, we made two important findings: first, that coupled positive feedback loops composed of phosphorylation of Raf kinase inhibitor RKIP by ERK and transcriptional repression of RKIP by Snail have an essential role in causing a switch-like behavior of E-cadherin expression; and second, that RKIP expression inhibits EMT progression by preventing E-cadherin suppression. Taken together, our findings provide us with a system-level understanding of how RKIP can regulate EMT progression and may explain why RKIP is downregulated in so many metastatic cancer cells.

Signaling Through RAS-RAF-MEK-ERK: from Basics to Bedside

Aberrant signaling caused by mutations in the RAS-RAF-MEK-ERK pathway and its upstream activators critically contributes to human tumor development. Strategies, which aim at inhibiting hyperactive signaling molecules, appear conceptually straight forward, but their translation into clinical practice has been hampered by many setbacks. Understanding structure, function and regulation of this intracellular pathway as well as its crosstalk with other signaling activities in the cell will be essential to ensure reasonable usage of new therapeutic possibilities. This review provides an understanding of this signaling cascade as revealed by genetic and biochemical approaches and discusses the existing or arising possibilities to interfere with unphysiological activation in cancer. Signaling aberrations and signal transduction therapies will be discussed exemplary for two types of hematological neoplasia, acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). In the future understanding the role of tumor stem cells, both as a source of tumor recurrence and tumor heterogeneity, the signals controlling their fate as well as epigenetic changes in cancer will be the next critical steps to further advance the applicability of these novel therapeutic strategies.

Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia.

Mutations leading to activation of the RAF-mitogen-activated protein kinase/extracellular signal-regulated (ERK) kinase (MEK)-ERK pathway are key events in the pathogenesis of human malignancies. In a screen of 82 acute myeloid leukemia (AML) samples, 45 (55%) showed activated ERK and thus were further analyzed for mutations in B-RAF and C-RAF. Two C-RAF germ-line mutations, S427G and I448V, were identified in patients with therapy-related AML in the absence of alterations in RAS and FLT3. Both exchanges were located within the kinase domain of C-RAF. In vitro and in vivo kinase assays revealed significantly increased activity for (S427G)C-RAF but not for (I448V)C-RAF. The involvement of the S427G C-RAF mutation in constitutive activation of ERK was further confirmed through demonstration of activating phosphorylations on C-RAF, MEK, and ERK in neoplastic cells, but not in nonneoplastic cells. Transformation and survival assays showed oncogenic and antiapoptotic properties for both mutations. Screening healthy individuals revealed a <1/400 frequency of these mutations and, in the case of I448V, inheritance was observed over three generations with another mutation carrier suffering from cancer. Taken together, these data are the first to relate C-RAF mutations to human malignancies. As both mutations are of germ-line origin, they might constitute a novel tumor-predisposing factor.

Therapy-related myeloid neoplasms: pathobiology and clinical characteristics

Therapy‐related myeloid neoplasms (t‐MNs) are serious long‐term consequences of cytotoxic treatments for an antecedent disorder. t‐MNs are observed after ionizing radiation as well as conventional chemotherapy including alkylating agents, topoisomerase‐II‐inhibitors and antimetabolites. In addition, adjuvant use of recombinant human granulocyte‐colony stimulating factor may also increase the risk of t‐MNs. There is clinical and biological overlap between t‐MNs and high‐risk de novo myelodysplastic syndromes and acute myeloid leukaemia suggesting similar mechanisms of leukaemogenesis. Human studies and animal models point to a prominent role of genetic susceptibilty in the pathogenesis of t‐MNs. Common genetic variants have been identified that modulate t‐MN risk, and t‐MNs have been observed in some cancer predisposition syndromes. In either case, establishing a leukaemic phenotype requires acquisition of somatic mutations – most likely induced by the cytotoxic treatment. Knowledge of the specific nature of the initiating exposure has allowed the identification of crucial pathogenetic mechanisms and for these to be modelled in vitro and in vivo. Prognosis of patients with t‐MNs is dismal and at present, the only curative approach for the majority of these individuals is haematopoietic stem cell transplantation, which is characterized by high transplant‐related mortality rates. Novel transplantation strategies using reduced intensity conditioning regimens as well as novel drugs – demethylating agents and targeted therapies – await clinical testing and may improve outcome. Ultimately, individual assessment of genetic risk factors may translate into tailored therapies and establish a strategy for reducing t‐MN incidences without jeopardizing therapeutic success rates for the primary disorders.

Germline mutations in the DNA damage response genes BRCA1, BRCA2, BARD1 and TP53 in patients with therapy related myeloid neoplasms

Background Therapy related myeloid neoplasms (t-MNs) are complex diseases originating from an interplay between exogenous toxicities and a susceptible organism. It has been hypothesised that in a subset of cases t-MNs develop in the context of hereditary cancer predisposition syndromes. Methods The study systematically evaluated pedigrees of patients with t-MNs for cancer incidences and the possibility of a hereditary cancer predisposition syndrome. In addition, mutational analyses were performed using constitutional DNA from index patients, and deleterious heterozygous germline mutations were assessed for loss of heterozygosity in sorted leukaemic cells by single nucleotide polymorphism array. Results A nuclear pedigree was obtained in 51/53 patients with t-MNs resulting in a total of 828 individuals analysed. With a standardised incidence ratio of 1.03 (95% CI 0.74 to 1.39), the tumour incidence of first- degree relatives was not increased. However, six pedigrees were suggestive for a hereditary breast and ovarian cancer syndrome, three of a Li-Fraumeni like syndrome, and three index patients showed multiple primary neoplasms. Mutational analysis revealed two BRCA1 (c.3112G→T, c.5251C→T), one BRCA2 (c.4027A→G), two BARD1 (C557S) and four TP53 germline mutations (g.18508_18761delinsGCC, c.847C→T, c.845_848dupGGCG, c.1146delA) in nine of 53 (17%) index patients with t-MNs. Loss of heterozygosity in leukaemic cells was demonstrated for the BRCA1c.3112G→T and TP53c.845_848dupGGCG mutations, respectively. Conclusion It is concluded that a proportion of patients with t-MNs carry cancer susceptibility mutations which are likely to contribute to therapy related leukaemogenesis.

Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X

Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4AV78M demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

Rituximab plus subcutaneous cladribine in patients with extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue: a phase II study by the Arbeitsgemeinschaft Medikamentöse Tumortherapie

Currently, there is no standard systemic treatment for extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue. Both rituximab and cladribine have shown some activity in this disease, but the combination has not been tested so far. In view of this, we initiated a phase II study to assess the activity and safety of rituximab and cladribine in patients with histologically verified mucosa-associated lymphoid tissue lymphoma. Treatment consisted of rituximab 375 mg/m2 i.v. day 1 and cladribine 0.1 mg/kg s.c. days 1 – 4 every 21 days. In case of complete remission after two courses, another two cycles of therapy were administered, while patients with a partial response or stable disease were scheduled to receive six cycles of treatment. Out of 40 evaluable patients (14 female, 26 male), 39 received treatment as scheduled while one patient died before initiation of therapy and was rated as having progressive disease in the intent-to-treat analysis. Twenty-one patients had gastric lymphoma, while 19 suffered from extragastric mucosa-associated lymphoid tissue lymphoma. Side effects consisted mainly of hematologic toxicity including leukopenia, lymphopenia, anemia and thrombocytopenia. Twenty-three patients had a complete remission (58%) and nine had a partial remission (23%) for an overall response rate of 81%, while five had stable disease (13%) and two progressed during therapy. After a median follow-up of 16.7 months (interquartile range: 15.9 – 18.7 months), 35 patients are alive (88%) while four patients have died and one patient withdrew consent and did not allow further follow up. Our data demonstrate that rituximab plus cladribine is active and safe in patients with mucosa-associated lymphoid tissue lymphoma.

Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.

Evaluation of potential risk factors for early infectious complications after autologous peripheral blood stem cell transplantation in patients with lymphoproliferative diseases.

A number of risk factors for the occurrence of neutropaenic fever after haematopoietic stem cell transplantation (HSCT) have been proposed. We were interested in whether these factors remain valid for several early infection-related outcomes when applied to a homogeneous group of patients in uni- and multivariate analyses. Therefore, we analysed 144 consecutive patients with lymphoproliferative disorders receiving autologous peripheral blood HSCT. Variables tested as potential risk factors for the occurrence of fever, documented infection (DI), microbiologically documented infection (MDI) or failure of first-line antimicrobial therapy were sex, conditioning regimen, prolonged neutropaenia, low number of CD34+ cells transplanted, purging, lack of selective gut decontamination, higher age and increased body mass index. In uni- and multivariate analyses, conditioning including total body irradiation was the only risk factor for the occurrence of fever, and neutropaenia ≥10 days was the only factor associated with failure of first-line antimicrobial therapy. None of the variables tested was associated with an increased risk for DI or MDI. This analysis suggests that a number of previously proposed risk factors actually are of minor clinical relevance for early infections in the majority of patients receiving autologous HSCT.