Search

Funding Information: The authors thank Sabine Fajmann, Petra Kudweis, Philipp Jodl, and Isabella Mayer for excellent experimental support. The authors thank the Biomedical Sequencing Facility at the CeMM Research Center for Molecular Medicine at the Austrian Academy of Sciences and the next-generation sequencing facility at Vienna BioCenter Core Facilities, a member of the Vienna BioCenter, Vienna, Austria. The authors also thank Stephan Hutter and Wecke Walter for bioinformatics support in the analysis of primary patient samples. This work was supported by the Austrian Science Fund (FWF) Special Research Program SFB-F6107 and the European Research Council under the European Union's Horizon 2020 research and innovation program grant agreements (#694354) (V.S.), (#636855) (F.G.), and (#647355) (S.M.). S.M. was supported by the Academy of Finland Heal-Art consortium (314442) and special COVID-19 funding (335527), ERA PerMed (JAKSTAT-TARGET consortium), Sigrid Juselius Foundation, Signe and Ane Gyllenberg Foundation, Helsinki Institute for Life Science, and Cancer Foundation Finland. B.M. was supported by the Fellinger Cancer Research Association. Contribution: V.S. T.B. B.M. and J.S. conceptualized the study; T.B. performed most of the experiments; T.B. and J.S. analyzed all wet laboratory experiments; R.G. T.K. T.E. and J.H. analyzed sequencing data; J.S. E.D. and S.K. performed experiments; G.H. and S.M. provided bioinformatic patient data analysis; J.Z. S.M. G.H. and F.G. were involved in experimental design and scientific discussions; T.B. and V.S. wrote the manuscript; V.S. supervised the study; and all authors revised the manuscript. Gain-of-function mutations in the signal transducer and activator of transcription 3 (STAT3) gene are recurrently identified in patients with large granular lymphocytic leukemia (LGLL) and in some cases of natural killer (NK)/T-cell and adult T-cell leukemia/lymphoma. To understand the consequences and molecular mechanisms contributing to disease development and oncogenic transformation, we developed murine hematopoietic stem and progenitor cell models that express mutated STAT3Y640F. These cells show accelerated proliferation and enhanced self-renewal potential. We integrated gene expression analyses and chromatin occupancy profiling of STAT3Y640F-transformed cells with data from patients with T-LGLL. This approach uncovered a conserved set of direct transcriptional targets of STAT3Y640F. Among these, strawberry notch homolog 2 (SBNO2) represents an essential transcriptional target, which was identified by a comparative genome-wide CRISPR/Cas9-based loss-of-function screen. The STAT3-SBNO2 axis is also present in NK-cell leukemia, T-cell non-Hodgkin lymphoma, and NPM-ALK-rearranged T-cell anaplastic large cell lymphoma (T-ALCL), which are driven by STAT3-hyperactivation/mutation. In patients with NPM-ALK+ T-ALCL, high SBNO2 expression correlates with shorter relapse-free and overall survival. Our findings identify SBNO2 as a potential therapeutic intervention site for STAT3-driven hematopoietic malignancies.

PDF file - 74K, B‐ and C‐Raf are required for PLX‐4032, but not GDC‐0879‐induced ERK phosphorylation in primary wild‐type keratinocytes.

PDF file - 680K, B‐Raf or C‐Raf ablation prevents tumor development in PLX‐4032‐treated KS‐SOS‐F+ mice.

RAF inhibitors achieve unprecedented but mainly transient clinical responses in patients with melanoma whose tumors harbor an activating BRAF mutation. One notable side-effect of RAF inhibitors is the stimulation of cutaneous skin tumors, arising in about 30% of patients receiving these drugs, which are thought to develop as a result of inhibitor-induced activation of wild-type Raf in occult precursor skin lesions. This effect raises the possibility that less manageable tumors might also arise in other epithelial tissues. Here we provide preclinical evidence supporting this disquieting hypothesis by showing that the RAF inhibitors PLX-4032 (vemurafenib) and GDC-0879 precipitate the development of cell-autonomous, Ras-driven tumors in skin and gastric epithelia. The magnitude of the effects correlated with the inhibitors' relative abilities to induce ERK activation. Epidermis-restricted ablation of either B-Raf or C-Raf prevented PLX-4032–induced ERK activation and tumorigenesis. In contrast, GDC-0879 induced ERK activation and tumorigenesis in B-Raf–deficient epidermis, whereas C-Raf ablation blocked GDC-0879–induced tumorigenesis (despite strong ERK activation) by preventing Rokα–mediated keratinocyte dedifferentiation. Thus, inhibitor-induced ERK activation did not require a specific Raf kinase. ERK activation was necessary, but not sufficient for Ras + Raf inhibitor-induced tumorigenesis, whereas C-Raf downregulation of Rokα was essential even in the face of sustained ERK signaling to prevent differentiation and promote tumorigenesis. Taken together, our findings suggest that combination therapies targeting ERK-dependent and -independent functions of Raf may be more efficient but also safer for cancer treatment. Cancer Res; 73(23); 6926–37. ©2013 AACR.

PDF file - 185K, Effect of Rok inhibitors on K5‐SOS‐F+; C‐RafKO epithelia.

PDF file - 197K, Effect of GDC‐0879 on wild‐type mice.

PDF file - 213K, C‐Raf, but not B‐Raf ablation, prevents tumor development in GDC‐0879 treated K5‐ SOS‐F+ mice.

PDF file - 1000K, The Mek inhibitor trametinib prevents Ras‐ and Ras+Raf inhibitor‐induced tumorigenesis.

PDF file - 82K, Raf inhibitors activate ERK in human keratinocyte (HaCat) and squamous cell carcinoma (SCC) cell lines.

PDF file - 98K, ERK‐dependent and -independent functions of Raf in Ras + Raf inhibitor‐induced tumorigenesis - a model.

The transcription factors signal transducer and activator of transcription 5A (STAT5A) and STAT5B are critical in hematopoiesis and leukemia. They are widely believed to have redundant functions, but we describe a unique role for STAT5B in driving the self-renewal of hematopoietic and leukemic stem cells (HSCs/LSCs). We find STAT5B to be specifically activated in HSCs and LSCs, where it induces many genes associated with quiescence and self-renewal, including the surface marker CD9. Levels of CD9 represent a prognostic marker for patients with STAT5-driven leukemia, and our findings suggest that anti-CD9 antibodies may be useful in their treatment to target and eliminate LSCs. We show that it is vital to consider STAT5A and STAT5B as distinct entities in normal and malignant hematopoiesis.

The oncogenic protein Bcr-Abl has two major isoforms, p190Bcr-Abl and p210Bcr-Abl. While p210Bcr-Abl is the hallmark of chronic myeloid leukemia (CML), p190Bcr-Abl occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) patients. In CML, p190Bcr-Abl occurs in a minority of patients associating with distinct hematological features and inferior outcomes, yet the pathogenic role of p190Bcr-Abl and potential targeting therapies are largely uncharacterized. We employed next generation sequencing, phospho-proteomic profiling, and drug sensitivity testing to characterize p190Bcr-Abl in CML and hematopoietic progenitor cell line models (Ba/f3 and HPC-LSK). p190Bcr-Abl CML patients demonstrated poor response to imatinib and frequent mutations in epigenetic modifiers genes. In contrast with p210Bcr-Abl, p190Bcr-Abl exhibited specific transcriptional upregulation of interferon, interleukin-1 receptor, and P53 signaling pathways, associated with hyperphosphorylation of relevant signaling molecules including JAK1/STAT1 and PAK1 in addition to Src hyperphosphorylation. Comparable to p190Bcr-Abl CML patients, p190Bcr-Abl cell lines demonstrated similar transcriptional and phospho-signaling signatures. With the drug sensitivity screening we identified targeted drugs with specific activity in p190Bcr-Abl cell lines including IAP-, PAK1-, and Src inhibitors and glucocorticoids. Our results provide novel insights into the mechanisms underlying the distinct features of p190Bcr-Abl CML and promising therapeutic targets for this high-risk patient group.

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situationin vivo. We now report a robust method to generate and maintain LSK (lin-, Sca-1+, c-Kit+) cells which closely resemble MPP1 cells. HPCLSKreconstitute hematopoiesis in lethally irradiated recipient mice over more than eight months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD or MLL-AF9 their leukemic counterparts maintain stem cell propertiesin vitroand recapitulate leukemia formationin vivo. The method to generate HPCLSKcan be applied to transgenic mice and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210transformation,Cdk6-/-HPCLSKsinduce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSKand human BCR/ABL+cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSKsystem represents a unique tool for combinedin vitroandin vivostudies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.Key pointsWe describe the generation of murine cell lines (HPCLSK) which reliably mimic hematopoietic/leukemic progenitor cells.Cdk6-/-BCR/ABLp210HPCLSKsuncover a novel role for CDK6 in homing.

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (Lin−, Sca-1+, c-Kit+) cells, which closely resemble MPP1 cells. HPCLSKs reconstitute hematopoiesis in lethally irradiated recipient mice over >8 months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD, or MLL-AF9, their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSKs can be applied to transgenic mice, and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, HPCLSKsCdk6−/− induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.

Tumor formation is a multi-step process during which cells acquire genetic and epigenetic changes until they reach a fully transformed state. We show that CDK6 contributes to tumor formation by regulating transcriptional responses in a stage-specific manner. In early stages CDK6 kinase induces a complex transcriptional program to block p53 in hematopoietic cells. Cells lacking CDK6 kinase function are required to mutate p53 to achieve a fully transformed immortalized state. CDK6 binds to the promoters of genes including the p53-antagonists PRMT5, PPM1D and MDM4. The findings are relevant to human patients: tumors with low levels of CDK6 have mutations in p53 significantly more often than expected.

The endothelium functions as a semipermeable barrier regulating fluid homeostasis, nutrient, and gas supply to the tissue. Endothelial permeability is increased in several pathological conditions including inflammation and tumors; despite its clinical relevance, however, there are no specific therapies preventing vascular leakage. Here, we show that endothelial cell-restricted ablation of BRAF, a kinase frequently activated in cancer, prevents vascular leaking as well metastatic spread. BRAF regulates endothelial permeability by promoting the cytoskeletal rearrangements necessary for the remodeling of VE-Cadherin-containing endothelial cell-cell junctions and the formation of intercellular gaps. BRAF kinase activity and the ability to form complexes with RAS/RAP1 and dimers with its paralog RAF1 are required for proper permeability control, achieved mechanistically by modulating the interaction between RAF1 and the RHO effector ROKα. Thus, RAF dimerization impinges on RHO pathways to regulate cytoskeletal rearrangements, junctional plasticity, and endothelial permeability. The data advocate the development of RAF dimerization inhibitors, which would combine tumor cell autonomous effect with stabilization of the vasculature and antimetastatic spread.

Introduction: The oncoprotein Bcr-Abl has two major isoforms, depending on the breakpoint in BCR gene, p190 and p210. While p210 is the hallmark of chronic myeloid leukemia (CML), p190 occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. p190 occurs as a sole transcript in 1-2% CML patients, associated with distinct features like monocytosis and frequent additional cytogenetic abnormalities (ACA) at diagnosis. It also confers a risk of treatment failure and progression in chronic phase (CP) CML patients. However, the underlying mechanisms are largely unknown. Here we explore the characteristics of p190 and p210 in CML and ALL patients using next generation sequencing, phospho-flowcytometry and high throughput drug testing. Patients and methods: Peripheral blood mononuclear cells (PMNC) were collected at diagnosis from four CP-CML patients harboring p190 isoform from Helsinki University Hospital. Genetic alterations were identified by whole exome sequencing. RNA sequencing was employed to analyze transcriptional profiles of p190 CML (n=3) in contrast to p210 CML patients (n=4). A thorough transcriptional, phosphorylation and drug sensitivity profiling were applied to five p190- and three p210-expressing Ph+ALL patients. Expression alterations were further characterized in two cell line models mimicking BCR-ABL positive leukemia (Ba/F3 and HPCLSK). Phosphorylation profiles were analyzed by flowcytometry and phospho-array (Tyrosine Phosphorylation ProArray, Full Moon Biosystems). For drug sensitivity and resistance testing (DSRT), a custom plate set comprising 75 approved and investigational oncology drugs was used for patient samples and more extensive 528-drugs plates were used for the cell lines. Results: CML patients with p190 had a median age of 72.5 years at the diagnosis (range: 50-80) and all received imatinib as a frontline treatment. Only one patient achieved a fluctuating major molecular response (MMR) by 12 months while the rest of the patients showed primary resistance to treatment and were shifted to a 2nd line TKI, nilotinib (n=2) or proceeded to HSCT (n=1). By exome sequencing we identified 26 variants in p190-CML samples (median per patient=7, range: 2-10), including variants in ASXL1, DNMT3A and KDM4D genes. RNA-sequencing analysis identified 19 and 97 dysregulated genes (Q Conclusions: In CML, p190 isoform of BCR-ABL1 is associated with distinct features and should be considered as a high-risk group. Combining clinical, genomic, phosphorylation and drug sensitivity data, we demonstrated that p190 activates specific cancer pathways, notably Src signaling and interferon pathways. Data also suggests that CML patients with p190 could benefit from broad spectrum TKI with Src inhibiting activity or combination of TKI with MDM2- or IAP-inhibitors. Disclosures Heckman: Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding. Porkka:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Mustjoki:Novartis: Research Funding; Pfizer: Research Funding; BMS: Honoraria, Research Funding.

The mammalian skin is the largest organ of the body and its outermost layer, the epidermis, undergoes dynamic lifetime renewal through the activity of somatic stem cell populations. The EGFR-Ras-Raf pathway has a well-described role in skin development and tumor formation. While research mainly focuses on its role in cutaneous tumor initiation and maintenance, much less is known about Ras signaling in the epidermal stem cells, which are the main targets of skin carcinogenesis. In this review, we briefly discuss the properties of the epidermal stem cells and review the role of EGFR-Ras-Raf signaling in keratinocyte stem cells during homeostatic and pathological conditions.

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive “Fas apoptosis” to “Fas multisignals” paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases., Signalling by the tumor necrosis factor receptor (TNFR) superfamily member Fas can promote either survival or death of a cell, but the mechanism underlying this choice is unclear. This study reveals that the outcome of Fas signalling (death versus survival) is determined by the tyrosine phosphorylation status of its death domain., Author Summary The versatility of the tumor necrosis factor receptor superfamily members in cell fate regulation is well illustrated by the dual signaling generated by one of the most extensively studied members of the family, Fas (CD95/TNFSFR6). Upon binding its ligand, Fas is able to elicit both pro-death and pro-survival signals. Until now, we have lacked mechanistic knowledge about when and how one signaling output of Fas is favored over the other. We demonstrate here that the outcome of Fas signaling is determined by the phosphorylation status of two tyrosine residues (Y232 and Y291) within the death domain. Dephosphorylation of Fas tyrosines by SHP-1 tyrosine phosphatase turns on the pro-apoptotic signal whereas the tyrosine phosphorylation by Src family kinases (SFKs) turns off the pro-apoptotic signal and turns on the pro-survival signal. Furthermore, we provide evidence that Fas tyrosine phosphorylation status may vary among different cancer types and influence the response to anti-cancer treatments. This information reveals an opportunity to use the screening of Fas tyrosine phosphorylation, a newly discovered direct molecular indicator of Fas functional output, to aid the design of Fas-related cancer therapies.

RAF inhibitors achieve unprecedented but mainly transient clinical responses in patients with melanoma whose tumors harbor an activating BRAF mutation. One notable side-effect of RAF inhibitors is the stimulation of cutaneous skin tumors, arising in about 30% of patients receiving these drugs, which are thought to develop as a result of inhibitor-induced activation of wild-type Raf in occult precursor skin lesions. This effect raises the possibility that less manageable tumors might also arise in other epithelial tissues. Here we provide preclinical evidence supporting this disquieting hypothesis by showing that the RAF inhibitors PLX-4032 (vemurafenib) and GDC-0879 precipitate the development of cell-autonomous, Ras-driven tumors in skin and gastric epithelia. The magnitude of the effects correlated with the inhibitors' relative abilities to induce ERK activation. Epidermis-restricted ablation of either B-Raf or C-Raf prevented PLX-4032–induced ERK activation and tumorigenesis. In contrast, GDC-0879 induced ERK activation and tumorigenesis in B-Raf–deficient epidermis, whereas C-Raf ablation blocked GDC-0879–induced tumorigenesis (despite strong ERK activation) by preventing Rokα–mediated keratinocyte dedifferentiation. Thus, inhibitor-induced ERK activation did not require a specific Raf kinase. ERK activation was necessary, but not sufficient for Ras + Raf inhibitor-induced tumorigenesis, whereas C-Raf downregulation of Rokα was essential even in the face of sustained ERK signaling to prevent differentiation and promote tumorigenesis. Taken together, our findings suggest that combination therapies targeting ERK-dependent and -independent functions of Raf may be more efficient but also safer for cancer treatment. Cancer Res; 73(23); 6926–37. ©2013 AACR.

International audience; Cellular fibronectin (cFN) variants harboring extra FN type 3 repeats, namely extra domains B and A, are major constituents of the extracellular matrix around newly forming blood vessels during development and angiogenesis. Their expression is induced by angiogenic stimuli and their assembly into fibrillar arrays is driven by cell-generated tension at α5β1 integrin-based adhesions. Here, we examined the role and functional redundancy of cFN variants in cultured endothelial cells by isoform-selective RNA interference. We show that FN fibrillogenesis is a cell-autonomous process whereby basally directed secretion and assembly of cellular FN are tightly coupled events that play an important role not only in signaling at cell-matrix adhesions but also at cell-cell contacts. Silencing of cFN variants differentially affects integrin usage, cell spreading, motility and capillary morphogenesis in vitro. cFN-deficient cells undergo a switch from α5β1- to αvβ3-based adhesion, accompanied by a Src-regulated disruption of adherens junctions. These studies identify a crucial role for autocrine FN in subendothelial matrix assembly and junctional integrity that provides spatially and temporally restricted control of endothelial plasticity during angiogenic blood vessel remodeling.

International audience; The activation of cysteine-aspartic proteases or caspases and the dynamic arrangement of cytoskeletal components are crucial during apoptosis. Here we describe the fate of Fas downstream of the FasL-induced internalization step, including formation of caspase-dependent SDS-stable Fas complexes, which is mediated by cytoskeleton integrity. We show, in particular, that following FasL treatment, the Fas lower aggregate complex can be co-immunoprecipitated with tubulin and an active form of caspase-8 and that this interaction contributes to the propagation of FasL-induced cell death. The importance of cytoskeletal components during FasL-induced apoptosis is highlighted by our detection of a pool of microtubule-associated Fas complexes.