Inactivation of CREBBP expands the germinal center B cell compartment, down-regulates MHCII expression and promotes DLBCL growth

The genes encoding the histone acetyl-transferases (HATs) CREB binding protein (CREBBP) and EP300 are recurrently mutated in the activated B cell-like and germinal center (GC) B cell-like subtypes of diffuse large B cell lymphoma (DLBCL). Here, we introduced a patient mutation into a human DLBCL cell line using CRISPR and deleted Crebbp and Ep300 in the GC B cell compartment of mice. CREBBP-mutant DLBCL clones exhibited reduced histone H3 acetylation, expressed significantly less MHCII, and grew faster than wild-type clones in s.c. and orthotopic xenograft models. Mice lacking Crebbp in GC B cells exhibited hyperproliferation of their GC compartment upon immunization, had reduced MHCII surface expression on GC cells, and developed accelerated MYC-driven lymphomas. Ep300 inactivation reproduced some, but not all, consequences of Crebbp inactivation. MHCII deficiency phenocopied the effects of CREBBP loss in spontaneous and serial transplantation models of MYC-driven lymphomagenesis, supporting the idea that the mutational inactivation of CREBBP promotes immune evasion. Indeed, the depletion of CD4(+) T cells greatly facilitated the engraftment of lymphoma cells in serial transplantation models. In summary, we provide evidence that both HATs are bona fide tumor suppressors that control MHCII expression and promote tumor immune control; mutational inactivation of CREBBP, but not of EP300, has additional cell-intrinsic engraftment and growth-promoting effects

Reconstructing promoter activity from Lux bioluminescent reporters

The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a computational approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. This approach is based upon a new mathematical model for Lux activity, that includes the actions of LuxAB, LuxEC and Fre, with improved mechanisms for all reactions, as well as synthesis and turn-over of Lux proteins. The model is calibrated with new experimental data for the LuxAB and Fre reactions from Photorhabdus luminescens --- the source of modern Lux reporters --- while literature data has been used for LuxEC. Importantly, the data show clear evidence for previously unreported product inhibition for the LuxAB reaction. Model simulations show that predicted bioluminescent profiles can be very different from changes in gene expression, with transient peaks of light output, very similar to light output seen in some experimental data sets. By incorporating the calibrated model into a Bayesian inference scheme, we can reverse engineer promoter activity from the bioluminescence. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology

SBML Level 3: an extensible format for the exchange and reuse of biological models

Systems biology has experienced dramatic growth in the number, size, and complexity of computational models. To reproduce simulation results and reuse models, researchers must exchange unambiguous model descriptions. We review the latest edition of the Systems Biology Markup Language (SBML), a format designed for this purpose. A community of modelers and software authors developed SBML Level 3 over the past decade. Its modular form consists of a core suited to representing reaction-based models and packages that extend the core with features suited to other model types including constraint-based models, reaction-diffusion models, logical network models, and rule-based models. The format leverages two decades of SBML and a rich software ecosystem that transformed how systems biologists build and interact with models. More recently, the rise of multiscale models of whole cells and organs, and new data sources such as single-cell measurements and live imaging, has precipitated new ways of integrating data with models. We provide our perspectives on the challenges presented by these developments and how SBML Level 3 provides the foundation needed to support this evolution

The tumor suppressive TGF-β/SMAD1/S1PR2 signaling axis is recurrently inactivated in diffuse large B-cell lymphoma

The sphingosine-1-phosphate receptor S1PR2 and its downstream signaling pathway is commonly silenced in diffuse large B-cell lymphoma (DLBCL), either by mutational inactivation or through negative regulation by the oncogenic transcription factor FOXP1. In this study, we have examined the upstream regulators of S1PR2 expression and have newly identified the TGF-β/TGF-βR2/SMAD1 axis as critically involved in S1PR2 transcriptional activation. Phosphorylated SMAD1 directly binds to regulatory elements in the locus as assessed by chromatin immunoprecipitation, and the CRISPR-mediated genomic editing of , or in DLBCL cell lines renders cells unresponsive to TGF-β-induced apoptosis. DLBCL clones lacking any one of the three factors have a clear growth advantage in vitro, as well as in subcutaneous xenotransplantation models, and in a novel model of orthotopic growth of DLBCL cells in the spleens and bone marrow of MISTRG mice expressing various human cytokines. The loss of induces hyper-proliferation of the germinal center B-cell compartment of immunized mice and accelerates -driven lymphomagenesis in spontaneous and serial transplantation models. The specific loss of in murine GC B-cells phenocopies the effects of loss on GC B-cell hyper-proliferation. Finally, we show that SMAD1 expression is aberrantly downregulated in >85% of analyzed DLBCL patients. The combined results uncover an important novel tumor suppressive function of the TGF-β/TGF-βR2/SMAD1/S1PR2 axis in DLBCL, and show that DLBCL cells have evolved to inactivate the pathway at the level of SMAD1 expression

Met and its ligand HGF are associated with clinical outcome in breast cancer

Few biomarkers exist to predict radiotherapy response in breast cancer. In vitro studies suggest a role for Met and its ligand HGF. To study this suggested role, MET and HGF gene copy numbers were determined by droplet digital PCR in tumours from 205 pre-menopausal and 184 post-menopausal patients, both cohorts randomised to receive either chemo-or radiotherapy. MET amplification was found in 8% of the patients in both cohorts and HGF amplification in 7% and 6% of the patients in the pre-and post-menopausal cohort, respectively. Met, phosphorylated Met (pMet), and HGF protein expression was determined by immunohistochemistry in the premenopausal cohort. Met, pMet, and HGF was expressed in 33%, 53%, and 49% of the tumours, respectively. MET amplification was associated with increased risk of distant recurrence for patients receiving chemotherapy. For the pre-menopausal patients, expression of cytoplasmic pMet and HGF significantly predicted benefit from radiotherapy in terms of loco-regional recurrence. Similar trends were seen for MET and HGF copy gain. In the post-menopausal cohort, no significant association of benefit from radiotherapy with neither genes nor proteins was found. The present results do not support that inhibition of Met prior to radiotherapy would be favourable for pre-menopausal breast cancer, as previously suggested.Funding Agencies|Swedish Cancer Society; Swedish Research Council; LiU Cancer Foundation</p

Pharmacological DNA demethylation restores SMAD1 expression and tumor suppressive signaling in diffuse large B-cell lymphoma

The sphingosine-1-phosphate (S1P) receptor S1PR2 and its downstream adaptor Gα13 are recurrently mutationally inactivated in the germinal center B-cell subtype of diffuse large B-cell lymphoma (DLBCL) and are silenced by the S1PR2 repressor FOXP1 in the activated B-cell like subtype of the disease. Loss of S1PR2 signaling relieves the germinal center confinement that is maintained by an S1P gradient and allows cells to resist S1P-induced apoptosis. We have shown previously that S1PR2 expression is induced in normal B cells through a newly described transforming growth factor-β (TGF-β)/TGF-βRII/SMAD1 signaling axis that is inactivated in >85% of DLBCL patients. DLBCL cell lines lacking S1PR2, TGFBRII, or SMAD1 as the result of genomic editing all have a strong growth advantage in vitro, as well as in subcutaneous and orthotopic xenotransplantation models. Here, we show that the TGF-β signaling pathway in DLBCL is blocked at the level of SMAD1 in DLBCL cell lines and patient samples by hypermethylation of CpG-rich regions surrounding the SMAD1 transcription start site. The pharmacologic restoration of SMAD1 expression by the demethylating agent decitabine (DAC) sensitizes cells to TGF-β-induced apoptosis and reverses the growth of initially SMAD1- cell lines in ectopic and orthotopic models. This effect of DAC is reduced in a SMAD1-knockout cell line. We further show that DAC restores SMAD1 expression and reduces the tumor burden in a novel patient-derived orthotopic xenograft model. The combined data lend further support to the concept of an altered epigenome as a major driver of DLBCL pathogenesis

Inactivation of CREBBP expands the germinal center B cell compartment, down-regulates MHCII expression and promotes DLBCL growth

The genes encoding the histone acetyl-transferases (HATs) CREB binding protein (CREBBP) and EP300 are recurrently mutated in the activated B cell-like and germinal center (GC) B cell-like subtypes of diffuse large B cell lymphoma (DLBCL). Here, we introduced a patient mutation into a human DLBCL cell line using CRISPR and deleted Crebbp and Ep300 in the GC B cell compartment of mice. CREBBP-mutant DLBCL clones exhibited reduced histone H3 acetylation, expressed significantly less MHCII, and grew faster than wild-type clones in s.c. and orthotopic xenograft models. Mice lacking Crebbp in GC B cells exhibited hyperproliferation of their GC compartment upon immunization, had reduced MHCII surface expression on GC cells, and developed accelerated MYC-driven lymphomas. Ep300 inactivation reproduced some, but not all, consequences of Crebbp inactivation. MHCII deficiency phenocopied the effects of CREBBP loss in spontaneous and serial transplantation models of MYC-driven lymphomagenesis, supporting the idea that the mutational inactivation of CREBBP promotes immune evasion. Indeed, the depletion of CD4(+) T cells greatly facilitated the engraftment of lymphoma cells in serial transplantation models. In summary, we provide evidence that both HATs are bona fide tumor suppressors that control MHCII expression and promote tumor immune control; mutational inactivation of CREBBP, but not of EP300, has additional cell-intrinsic engraftment and growth-promoting effects

The IL-6 signaling complex is a critical driver, negative prognostic factor, and therapeutic target in diffuse large B-cell lymphoma

Interleukin-6 (IL-6) is a growth factor for normal B cells and plasma cell-derived malignancies. Here, we show that the IL-6 signaling pathway is also active in a subset of diffuse large B-cell lymphoma (DLBCL) patients with particularly poor prognosis. Primary DLBCL cells and DLBCL cell lines expressing IL-6R engraft and form orthotopic lymphomas in humanized mice that ectopically produce human IL-6, and in mice reconstituted with a human immune system. We show that a subset of DLBCL cases have evolved mechanisms that ensure constitutive activation of the IL-6 signaling pathway, i.e., the expression of both chains of the IL-6R, the expression of the cytokine itself, and the mutational inactivation of a negative regulator of IL-6 signaling, SOCS1. IL-6 signaling promotes MYC-driven lymphomagenesis in a genetically engineered model, and treatment with the IL-6R-specific antibody tocilizumab reduces growth of primary DLBCL cells and of DLBCL cell lines in various therapeutic settings. The combined results uncover the IL-6 signaling pathway as a driver and negative prognosticator in aggressive DLBCL that can be targeted with a safe and well-tolerated biologic