TNFα secreted by glioma associated macrophages promotes endothelial activation and resistance against anti-angiogenic therapy.

One of the most prominent features of glioblastoma (GBM) is hyper-vascularization. Bone marrow-derived macrophages are actively recruited to the tumor and referred to as glioma-associated macrophages (GAMs) which are thought to provide a critical role in tumor neo-vascularization. However, the mechanisms by which GAMs regulate endothelial cells (ECs) in the process of tumor vascularization and response to anti-angiogenic therapy (AATx) is not well-understood. Here we show that GBM cells secrete IL-8 and CCL2 which stimulate GAMs to produce TNFα. Subsequently, TNFα induces a distinct gene expression signature of activated ECs including VCAM-1, ICAM-1, CXCL5, and CXCL10. Inhibition of TNFα blocks GAM-induced EC activation both in vitro and in vivo and improve survival in mouse glioma models. Importantly we show that high TNFα expression predicts worse response to Bevacizumab in GBM patients. We further demonstrated in mouse model that treatment with B20.4.1.1, the mouse analog of Bevacizumab, increased macrophage recruitment to the tumor area and correlated with upregulated TNFα expression in GAMs and increased EC activation, which may be responsible for the failure of AATx in GBMs. These results suggest TNFα is a novel therapeutic that may reverse resistance to AATx. Future clinical studies should be aimed at inhibiting TNFα as a concurrent therapy in GBMs.

CIC protein instability contributes to tumorigenesis in glioblastoma.

Capicua (CIC) is a transcriptional repressor that counteracts activation of genes downstream of receptor tyrosine kinase (RTK)/Ras/ERK signaling. It is well-established that tumorigenesis, especially in glioblastoma (GBM), is attributed to hyperactive RTK/Ras/ERK signaling. While CIC is mutated in other tumors, here we show that CIC has a tumor suppressive function in GBM through an alternative mechanism. We find that CIC protein levels are negligible in GBM due to continuous proteasome-mediated degradation, which is mediated by the E3 ligase PJA1 and show that this occurs through binding of CIC to its DNA target and phosphorylation on residue S173. PJA1 knockdown increased CIC stability and extended survival using in-vivo models of GBM. Deletion of the ERK binding site resulted in stabilization of CIC and increased therapeutic efficacy of ERK inhibition in GBM models. Our results provide a rationale to target CIC degradation in Ras/ERK-driven tumors, including GBM, to increase efficacy of ERK inhibitors.

Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells.

PURPOSE: Hexokinase II (HK2) protein expression is elevated in glioblastoma (GBM), and we have shown that HK2 could serve as an effective therapeutic target for GBM. Here, we interrogated compounds that target HK2 effectively and restrict tumor growth in cell lines, patient-derived glioma stem cells (GSCs), and mouse models of GBM.Experimental Design: We performed a screen using a set of 15 drugs that were predicted to inhibit the HK2-associated gene signature. We next determined the EC50 of the compounds by treating glioma cell lines and GSCs. Selected compounds showing significant impact in vitro were used to treat mice and examine their effect on survival and tumor characteristics. The effect of compounds on the metabolic activity in glioma cells was also assessed in vitro. RESULTS: This screen identified the azole class of antifungals as inhibitors of tumor metabolism. Among the compounds tested, ketoconazole and posaconazole displayed the greatest inhibitory effect on GBM both in vitro and in vivo. Treatment of mice bearing GBM with ketoconazole and posaconazole increased their survival, reduced tumor cell proliferation, and decreased tumor metabolism. In addition, treatment with azoles resulted in increased proportion of apoptotic cells. CONCLUSIONS: Overall, we provide evidence that azoles exert their effect by targeting genes and pathways regulated by HK2. These findings shed light on the action of azoles in GBM. Combined with existing literature and preclinical results, these data support the value of repurposing azoles in GBM clinical trials.

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

The genomic landscape of schwannoma.

Schwannomas are common peripheral nerve sheath tumors that can cause debilitating morbidities. We performed an integrative analysis to determine genomic aberrations common to sporadic schwannomas. Exome sequence analysis with validation by targeted DNA sequencing of 125 samples uncovered, in addition to expected NF2 disruption, recurrent mutations in ARID1A, ARID1B and DDR1. RNA sequencing identified a recurrent in-frame SH3PXD2A-HTRA1 fusion in 12/125 (10%) cases, and genomic analysis demonstrated the mechanism as resulting from a balanced 19-Mb chromosomal inversion on chromosome 10q. The fusion was associated with male gender predominance, occurring in one out of every six men with schwannoma. Methylation profiling identified distinct molecular subgroups of schwannomas that were associated with anatomical location. Expression of the SH3PXD2A-HTRA1 fusion resulted in elevated phosphorylated ERK, increased proliferation, increased invasion and in vivo tumorigenesis. Targeting of the MEK-ERK pathway was effective in fusion-positive Schwann cells, suggesting a possible therapeutic approach for this subset of tumors.

DICER governs characteristics of glioma stem cells and the resulting tumors in xenograft mouse models of glioblastoma.

The RNAse III endonuclease DICER is a key regulator of microRNA (miRNA) biogenesis and is frequently decreased in a variety of malignancies. We characterized the role of DICER in glioblastoma (GB), specifically demonstrating its effects on the ability of glioma stem-like cells (GSCs) to form tumors in a mouse model of GB. DICER silencing in GSCs reduced their stem cell characteristics, while tumors arising from these cells were more aggressive, larger in volume, and displayed a higher proliferation index and lineage differentiation. The resulting tumors, however, were more sensitive to radiation treatment. Our results demonstrate that DICER silencing enhances the tumorigenic potential of GSCs, providing a platform for analysis of specific relevant miRNAs and development of potentially novel therapies against GB.

CD24 tracks divergent pluripotent states in mouse and human cells.

Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture.

Divergent reprogramming routes lead to alternative stem-cell states.

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Genome-wide characterization of the routes to pluripotency.

Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

An epigenomic roadmap to induced pluripotency reveals DNA methylation as a reprogramming modulator.

Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement of the epigenetic landscape. To characterize this epigenomic roadmap, we have performed MethylC-seq, ChIP-seq (H3K4/K27/K36me3) and RNA-Seq on samples taken at several time points during murine secondary reprogramming as part of Project Grandiose. We find that DNA methylation gain during reprogramming occurs gradually, while loss is achieved only at the ESC-like state. Binding sites of activated factors exhibit focal demethylation during reprogramming, while ESC-like pluripotent cells are distinguished by extension of demethylation to the wider neighbourhood. We observed that genes with CpG-rich promoters demonstrate stable low methylation and strong engagement of histone marks, whereas genes with CpG-poor promoters are safeguarded by methylation. Such DNA methylation-driven control is the key to the regulation of ESC-pluripotency genes, including Dppa4, Dppa5a and Esrrb. These results reveal the crucial role that DNA methylation plays as an epigenetic switch driving somatic cells to pluripotency.

Small RNA changes en route to distinct cellular states of induced pluripotency.

MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent 'F-class' state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs.

An expanded population of CD34+ cells from frozen banked umbilical cord blood demonstrate tissue repair mechanisms of mesenchymal stromal cells and circulating angiogenic cells in an ischemic hind limb model.

Peripheral vascular disease affects ~20 % of the population over 50 years of age and is a complication of type 2 diabetes. Cell therapy studies revealed that cells from older or diabetic donors have a reduced capacity to induce tissue repair compared to healthy and younger cells. This fact greatly impedes the use of autologous cells for treatment. Umbilical cord blood CD34+ cells are a source of angiogenic cells but unlike bone marrow CD34+ angiogenic cells, achieving clinically significant cell numbers has been difficult without in vitro expansion. We report here that culturing CD34+/CD45+ blood cells from frozen umbilical cord blood units in a medium supplemented with FGF4, SCF and FLT3-ligand produced a population of cells that remain CD34+/CD45+ but have an increased capacity for tissue healing. The cultured CD34+ cells were compared directly to non-cultured CD34+ cells in a mouse model of ischemia. Cultured CD34+ cells demonstrated strong paracrine signaling as well as the capacity to differentiate into endothelial cells, smooth muscle and striated muscle. We observed an improvement in blood flow and a significant reduction in foot necrosis. A second study was completed to assess the safety of the cells. No adverse effects were associated with the injection of the cultured cells. Our method described here for culturing umbilical cord blood cells resulted in cells with a strong paracrine effect that induces substantial tissue repair in a murine model of hind limb ischemia and evidence of engraftment and differentiation of the cultured cells into new vasculature and muscle.

The transfer of host MHC class I protein protects donor cells from NK cell and macrophage-mediated rejection during hematopoietic stem cell transplantation and engraftment in mice.

Human hematopoietic stem cell engraftment has been studied extensively using xenograft transplant models with immunocompromised mice. It is standard practice to incorporate mouse models, such as the limiting dilution assay, to accurately assess the number of repopulating stem cells in bone marrow or umbilical cord blood collections or to confirm the long-term repopulating ability of cultured hematopoietic stem cells. In a previous study using a standard NOD/SCID mouse model to assess human hematopoietic stem cell engraftment we observed that all human cells had mouse MHC class I protein on their surface, suggesting that this is a mechanism adopted by the cells to evade host immune surveillance. To determine whether this was a xenograft phenomenon we studied host MHC transfer in an intraspecies mouse model and observed similar results. The transfer of MHC class I proteins has implications for antigen presentation and immune modulation. In this report, we used a standard mouse model of bone marrow transplantation to demonstrate that surface protein transfer between cells plays an important role in protecting donor hematopoietic cells from NK cell and macrophage-mediated rejection. The transfer of intact MHC class I antigens from host cells to transplanted donor cells confers a self identity on these otherwise foreign cells. This gives them the ability to evade detection by the host NK cells and macrophages. Once full donor chimerism is established, transplanted cells no longer require host MHC class I protein transfer to survive.