SUMMARYEarly T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients.

The cytokine tumor necrosis factor (TNF) has pleiotropic functions both in normal physiology and disease. TNF signals by the virtue of two cell surface receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Exogenous TNF promotes experimental metastasis in some models, yet the underlying mechanisms are poorly understood. To study the contribution of host TNFR1 and TNFR2 on tumor cell progression and metastasis, we employed a syngeneic B16F10 melanoma mouse model of lung metastasis combined with in vivo bioluminescence imaging. Treatment of tumor-bearing mice with recombinant human TNF resulted in a significant increase in tumor burden and metastatic foci. This correlated with an increase in pulmonary regulatory CD4(+)/Foxp3(+) T cells. TNF caused an expansion of regulatory T (Treg) cells in vitro in a TNFR2-dependent manner. To assess the contribution of immune cell expression of endogenous TNF and its two receptors on B16F10 metastasis, we generated bone marrow chimeras by reconstituting wild-type mice with bone marrow from different knockout mice. Loss of either TNF or TNFR2 on immune cells resulted in decreased B16F10 metastasis and lower numbers of Treg cells within the lungs of these animals. Selective depletion of Treg cells attenuated metastasis even in conjunction with TNF treatment. We propose a novel mechanism in which TNF activates TNFR2 on Treg cells and thereby expands this immunosuppressive immune cell population. Loss of either TNF or TNFR2 prevents the accumulation of Treg cells and results in a less tolerogenic environment, enabling the immune system to control B16F10 tumor metastasis and growth.

SummaryThe heat shock transcription factor 1 (HSF1) has recently been reported to promote malignant transformation and growth. Here we provide experimental evidence for a role of HSF1 in the pathogenesis of multiple myeloma (MM). Immunohistochemical analyses revealed that HSF1 was overexpressed in half of the investigated MM samples, including virtually all cases with extramedullary manifestations or anaplastic morphology. HSF1 function was inhibited either by siRNA-mediated knockdown or pharmacologically through treatment with triptolide. Both approaches caused depletion of HSF1, lowered the constitutively high expression of a multitude of protective HSPs (such as HSP90, HSP70, HSP40 and HSP27), induced apoptosis in human MM cells in vitro, and strongly reduced MM tumour growth in vivo. Furthermore, we observed that treatment-induced upregulation of HSPs after proteasome or HSP90 inhibition was critically dependent on HSF1. Importantly, the apoptotic effects of the HSP90 inhibitor NVP-AUY922 or the proteasome inhibitor bortezomib were strongly enhanced in combination with triptolide, suggesting a salvage role of HSF1-dependent HSP induction in response to drug treatment. Collectively, our data indicate that inhibition of HSF1 affects multiple protective HSPs and might therefore represent a therapeutic strategy -in particular in combination with proteasome or HSP90 inhibitors.

(2014) A novel llama antibody targeting Fn14 exhibits anti-metastatic activity in vivo, mAbs, 6:1, 297-308,

Understanding the spatiotemporal changes of cellular and molecular events within an organism is crucial to elucidate the complex immune processes involved in infections, autoimmune disorders, transplantation, and neoplastic transformation and metastasis. Here we introduce a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and T cell responses in Peyer's patches following stimulation of the immune system. In addition, we employed LSFM to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. Thus, we present a versatile imaging technology that should be highly beneficial in biomedical research.

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BackgroundMultiple myeloma (MM) is a B-cell malignancy, where malignant plasma cells clonally expand in the bone marrow of older people, causing significant morbidity and mortality. Typical clinical symptoms include increased serum calcium levels, renal insufficiency, anemia, and bone lesions. With standard therapies, MM remains incurable; therefore, the development of new drugs or immune cell-based therapies is desirable. To advance the goal of finding a more effective treatment for MM, we aimed to develop a reliable preclinical MM mouse model applying sensitive and reproducible methods for monitoring of tumor growth and metastasis in response to therapy.Material and MethodsA mouse model was created by intravenously injecting bone marrow-homing mouse myeloma cells (MOPC-315.BM) that expressed luciferase into BALB/c wild type mice. The luciferase in the myeloma cells allowed in vivo tracking before and after melphalan treatment with bioluminescence imaging (BLI). Homing of MOPC-315.BM luciferase+ myeloma cells to specific tissues was examined by flow cytometry. Idiotype-specific myeloma protein serum levels were measured by ELISA. In vivo measurements were validated with histopathology.ResultsStrong bone marrow tropism and subsequent dissemination of MOPC-315.BM luciferase+ cells in vivo closely mimicked the human disease. In vivo BLI and later histopathological analysis revealed that 12 days of melphalan treatment slowed tumor progression and reduced MM dissemination compared to untreated controls. MOPC-315.BM luciferase+ cells expressed CXCR4 and high levels of CD44 and α4β1 in vitro which could explain the strong bone marrow tropism. The results showed that MOPC-315.BM cells dynamically regulated homing receptor expression and depended on interactions with surrounding cells.ConclusionsThis study described a novel MM mouse model that facilitated convenient, reliable, and sensitive tracking of myeloma cells with whole body BLI in living animals. This model is highly suitable for monitoring the effects of different treatment regimens.

BackgroundAcute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention.MethodsMurine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles.ResultsWe identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4β7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD.ConclusionsBased on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention.