Glioblastoma (GBM) is the most common brain tumor in adults and the mesenchymal GBM subtype was reported to be the most malignant, presenting severe hypoxia and necrosis. Here, we investigated the possible role of a hypoxic microenvironment for inducing a mesenchymal and invasive phenotype. The exposure of non-mesenchymal SNB75 and U87 cells to hypoxia induced a strong change in cell morphology that was accompanied by enhanced invasive capacity and the acquisition of mesenchymal marker expression. Further analyses showed the induction of HIF1α and HIF2α by hypoxia and exposure to digoxin, a cardiac glycoside known to inhibit HIF1/2 expression, was able to prevent hypoxia-induced mesenchymal transition. ShRNA-mediated knockdown of HIF1α, and not HIF2α, prevented this transition, as well as the knockdown of the EMT transcription factor ZEB1. We provide further evidence for a hypoxia-induced mesenchymal shift in GBM primary material by showing co-localization of GLUT1, ZEB1 and the mesenchymal marker YKL40 in hypoxic regions of the tumor. Collectively, our results identify a HIF1α-ZEB1 signaling axis that promotes hypoxia induced mesenchymal shift and invasion in GBM in a cell line dependent fashion.

Although NOD-SCID IL2Rγ (NSG) xenograft mice are currently the most frequently used model to study human leukemia in vivo, the absence of a human niche severely hampers faithful recapitulation of the disease. We used NSG mice in which ceramic scaffolds seeded with human mesenchymal stromal cells were implanted to generate a human bone marrow (huBM-sc)-like niche. We observed that, in contrast to the murine bone marrow (mBM) niche, the expression of BCR-ABL or MLL-AF9 was sufficient to induce both primary acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Stemness was preserved within the human niches as demonstrated by serial transplantation assays. Efficient engraftment of AML MLL-AF9 and blast-crisis chronic myeloid leukemia patient cells was also observed, whereby the immature blast-like phenotype was maintained in the huBM-sc niche but to a much lesser extent in mBM niches. We compared transcriptomes of leukemias derived from mBM niches versus leukemias from huBM-like scaffold-based niches, which revealed striking differences in the expression of genes associated with hypoxia, mitochondria and metabolism. Finally, we utilized the huBM-sc MLL-AF9 B-ALL model to evaluate the efficacy of the I-BET151 inhibitor in vivo. In conclusion, we have established human niche models in which the myeloid and lymphoid features of BCR-ABL and MLL-AF9 leukemias can be studied in detail.

The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells.

With the emergence of the concept of the leukemic stem cell (LSC), assays to study them remain pivotal in understanding (leukemic) stem cell biology. Although the in vivo NOD-SCID or NSG xenotransplantation model is currently still the favored assay of choice in most cases, this system has some limitations as well such as its cost-effectiveness, duration, and lack of engraftability of cells from some acute myeloid leukemia (AML) patients. Here, we describe in vitro assays in which long-term expansion and self-renewal of LSCs isolated from AML patients can be evaluated. We have optimized lentiviral transduction procedures in order to stably express genes of interest or stably downmodulate genes using RNAi in primary AML cells, and these approaches are described in detail here. Also, we describe bone marrow stromal coculture systems in which cobblestone area-forming cell activity, self-renewal, long-term expansion, and in vitro myeloid or lymphoid transformation can be evaluated in human CD34(+) cells of fetal or adult origin that are engineered to express oncogenes. Together, these tools should allow a further molecular elucidation of derailed signal transduction in LSCs.

Over the past years, a wide variety of in vivo mouse models have been generated in order to unravel the molecular pathology of Chronic Myeloid Leukemia (CML) and to develop and improve therapeutic approaches. These models range from (conditional) transgenic models, knock-in models, and murine bone marrow retroviral transduction models followed by transplantation. With the advancement of immunodeficient xenograft models, it has become possible to use human stem/progenitor cells for in vivo studies as well as cells directly derived from CML patients. These models not only mimic CML but also have been instrumental in uncovering various fundamental mechanisms of CML disease progression and tyrosine kinase inhibitor (TKI) resistance. With the availability of iPSC technology, it has become feasible to derive, maintain, and expand CML subclones that are at least genetically identical to those in patients. The following review provides an overview of all murine as well as human xenograft models for CML established till date.

Intrinsic and extrinsic signals together contribute to determine self renewal, quiescence or the specific metabolic status of leukemic stem cells (LSC) in BCR-ABL mediated chronic myeloid leukemia (CML). Our previous studies have shown that expression of BCR-ABL together with the polycomb repression complex 1 member BMI1 in human CD34+ cells is sufficient to induce a serially transplantable lymphoid leukemia in vivo while a myeloid phenotype was never observed. Yet in vitro, both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in CML patients. Since NSG models are typically lymphoid biased due to the absence of species-specific myeloid growth factors, we hypothesized that extrinsic factors might dictate lineage fate. Using a “humanized” NSG mouse model in which scaffolds seeded with human mesenchymal stromal cells were implanted we observed that, in contrast to the murine niche, BCR-ABL overexpression alone was sufficient to induce a serially transplantable leukemia of both the lymphoid and myeloid lineage. Using myeloid blast-crisis CML patient cells, engraftment was also observed whereby the immature blast-like phenotype was predominantly maintained in the humanized scaffold niche, and to a much lesser extent in the murine niche. This distinction could also be demonstrated functionally by using in vitro long-term self-renewing cultures. Blast cells retrieved from the human scaffold niche could readily be established while no long-term cultures could be initiated from cells retrieved from the murine bone marrow niche. Genome-wide transcriptome analyses of leukemic cells retrieved from the mouse BM niche and from the human scaffold niche revealed striking differences in gene expression imposed on BCR-ABL+ cells by these different environments. For example, endogenous BMI1 levels were significantly higher in BCR-ABL cells retrieved from human scaffold niche as compared to murine BM harvested cells suggesting that BMI1 might still be required as additional factor to prevent oncogene-induced senescence. Apart from epigenetic modifiers, we hypothesized that the hypoxic microenvironment might play an important role in maintaining CML LSCs and studied that in detail. Hypoxia inducible factor 1α (HIF1) and HIF2 act as transcription factors that are stabilized under hypoxic conditions. HIF1 has been characterized as an important factor that controls cellular metabolism while the role of HIF2 is still less clear. Earlier we identified HIF2 as downstream target of STAT5 and observed elevated glucose uptake in STAT5 activated HSCs. Several genes associated with glucose metabolism were upregulated by STAT5 in an HIF2 dependent manner, including SLC2A1 and GYS2. Here, we investigated metabolic changes in BCR-ABL expressing human stem/progenitor cells and focused on the role on HIF1 and HIF2. Genome-wide transcriptome analyses were performed on human CB CD34+ cells transduced with BCR-ABL as well as on BCR-ABL-positive CML and B-ALL patient samples. GSEA analyses indicated that these transcriptome changes were strongly enriched for STAT5 and MYC signatures as well as for hypoxia, embryonic stem cell and glucose metabolism gene signatures which included upregulation of e.g. SLC2A3, SLC2A1 and HIF1 and HIF2. These data suggest that BCR-ABL imposes hypoxic signaling under normoxic conditions. Moreover, downregulation of HIF1 and HIF2 using a shRNA approach impaired proliferation and reduced progenitor frequencies of BCR-ABL+ cells. Next we studied metabolic changes in BCR-ABL+ cells using NMR spectroscopy. We observed striking differences in uptake and secretion of metabolites when BCR-ABL CB CD34+ cells were compared to normal CB CD34+ cells under normoxia and hypoxia. As expected, BCR-ABL cells exhibited enhanced glycolysis as determined by an increased production and secretion of lactate under both normoxic and hypoxic conditions. Interestingly, glutamine levels were strongly enhanced in BCR-ABL+ cells, in a HIF1/2-dependent manner, possibly via enhanced glutamine import or glutamine production via upregulation/activation of Glutamine Synthase. Our current hypothesis is that BCR-ABL+ cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells, and studies focus on whether targeting this pathway might provide alternative means to eradicate LSCs. Disclosures No relevant conflicts of interest to declare.

No abstract

Background Immune checkpoint inhibitors (ICIs) targeting PD-1 or its ligand PD-L1 have shown clinical activity in patients with metastatic non-small cell lung cancer (mNSCLC). However, only subgroups of mNSCLC patients respond to ICI, while their robust and accurate identification using PD-L1 as a biomarker remains challenging. Typically, PD-L1 expression is assessed by pathologist scoring of immunohistochemically (IHC) stained tissue, e.g. using the tumor proportion score (TPS). However, this manual process is subjective and semiquantitative. To this end, we aim to develop robust quantitative continuous scoring of PD-L1 expression via IHC (PD-L1 QCS), relying on digitized image analysis, with the aim of improving robustness of patient selection. Methods QCS of PD-L1 (Ventana SP263) on digitized whole slide images (WSI) is approached by segmenting the tumor epithelium within a given region of interest. Here, a deep learning (DL) region segmentation model is applied which was enriched with additional training data; expanding previous work. 1,2 A second DL model segments individual tumor cells and their membranes. By applying color deconvolution, the resulting Optical Density (OD) provides a continuous measurement of PD-L1 intensity on each cell membrane. The percentage of positive cells is derived by thresholding the OD, whereas the specific cut-point for stratification was obtained by optimizing on an exploratory cohort (samples from 163 mNSCLC patients treated with anti-PD-L1; NCT01693562) and validated for its robustness using an independent cohort (samples from 252 patients treated with anti-PD-L1; NCT02453282), for which IHC staining and WSI scanning were completed at a contract research organization (CRO). 3,4 Results On the exploratory cohort, pathologist TPS correlated favorably against PD-L1 QCS (Spearman R=0.86), confirming the validity of image analysis. PD-L1 QCS yielded a group of responders to anti-PD-L1 treatment with a significantly increased median overall survival (mOS) by 9.2 months (logrank p=0.0017, HR=0.54, prevalence=46%). On the independent validation cohort, this finding was confirmed with an mOS increase of 9.9 months (log-rank p=0.0001, HR=0.55, prevalence=40%), although IHC for the second cohort was completed in a different laboratory and slides digitized with a different scanner. Conclusions We describe a computational pathology approach for precise quantification of PD-L1 expression and selection of mNSCLC patients for anti-PD-L1 treatment using the Ventana SP263 assay. Importantly, we successfully validated the performance of our PD-L1 QCS solution in two independent clinical trial datasets, which were processed by different CROs using different scanners revealing broad applicability and thereby underscoring the potential of PD-L1 QCS to transform pathology. Trial Registration NCT01693562, NCT02453282Ethics Approval Clinical studies NCT01693562 and NCT02453282, from which data in this report were obtained, were carried out in accordance with the Declaration of Helsinki and GoodClinic...