CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide.

There is a strong biological rationale for the augmentation of allogeneic natural killer (NK) cell therapies with a chimeric antigen receptor (CAR) to enhance acute myeloid leukemia (AML) targeting. CD38 is an established immunotherapeutic target in multiple myeloma and under investigation as a target antigen in AML. CD38 expression on NK cells and its further induction during ex vivo NK cell expansion represents a barrier to the development of a CD38 CAR-NK cell therapy. We set out to develop a CD38 CAR-NK cell therapy for AML, first by using an NK cell line which has low baseline CD38 expression and subsequently healthy donor expanded NK cells. To overcome anticipated fratricide due to NK cell CD38 expression when using primary expanded NK cells, we applied CRISPR/Cas9 genome editing to disrupt the CD38 gene during expansion achieving a mean knockdown efficiency of 84%. The resulting CD38 KD expanded NK cells, after expression of an affinity optimized CD38 CAR, showed reduced NK cell fratricide and an enhanced ability to target primary AML blasts. Furthermore, the cytotoxic potential of CD38 CAR-NK cells was augmented by pre-treatment of the AML cells with all-trans retinoic acid which drove enhanced CD38 expression offering a rational combination therapy. These findings support the further investigation of CD38 KD - CD38 CAR-NK cells as a viable immunotherapeutic approach to the treatment of AML.

Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor-natural killer cells.

BACKGROUND: Natural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development. AIMS AND METHODS: We set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein-Barr virus-transformed lymphoblastoid feeder cell-mediated activation and expansion. RESULTS: This approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1-positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype. CONCLUSIONS: This report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.

BCL-2 modulates the unfolded protein response by enhancing splicing of X-box binding protein-1.

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) triggers a highly conserved stress response mechanism termed the unfolded protein response (UPR). The UPR is a complex series of signaling pathways controlled by ER localized transmembrane receptors, PERK, ATF6 and IRE1α. Following activation IRE1α splices XBP-1 mRNA facilitating the formation of a potent transcription factor, spliced XBP-1. The BCL-2 family members, BAX and BAK, in addition to the mitochondrion also localize to the ER and have been demonstrated to directly interact with IRE1α promoting its activity. In this study we show that in addition to BAX and BAK, the anti-apoptotic BCL-2 protein can regulate IRE1α activity. Enhanced splicing of XBP-1 was observed in BCL-2 overexpressing cells implicating BCL-2 in the complex regulation of IRE1α activity.

Co-acting gene networks predict TRAIL responsiveness of tumour cells with high accuracy.

BACKGROUND: Identification of differentially expressed genes from transcriptomic studies is one of the most common mechanisms to identify tumor biomarkers. This approach however is not well suited to identify interaction between genes whose protein products potentially influence each other, which limits its power to identify molecular wiring of tumour cells dictating response to a drug. Due to the fact that signal transduction pathways are not linear and highly interlinked, the biological response they drive may be better described by the relative amount of their components and their functional relationships than by their individual, absolute expression. RESULTS: Gene expression microarray data for 109 tumor cell lines with known sensitivity to the death ligand cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was used to identify genes with potential functional relationships determining responsiveness to TRAIL-induced apoptosis. The machine learning technique Random Forest in the statistical environment "R" with backward elimination was used to identify the key predictors of TRAIL sensitivity and differentially expressed genes were identified using the software GeneSpring. Gene co-regulation and statistical interaction was assessed with q-order partial correlation analysis and non-rejection rate. Biological (functional) interactions amongst the co-acting genes were studied with Ingenuity network analysis. Prediction accuracy was assessed by calculating the area under the receiver operator curve using an independent dataset. We show that the gene panel identified could predict TRAIL-sensitivity with a very high degree of sensitivity and specificity (AUC=0·84). The genes in the panel are co-regulated and at least 40% of them functionally interact in signal transduction pathways that regulate cell death and cell survival, cellular differentiation and morphogenesis. Importantly, only 12% of the TRAIL-predictor genes were differentially expressed highlighting the importance of functional interactions in predicting the biological response. CONCLUSIONS: The advantage of co-acting gene clusters is that this analysis does not depend on differential expression and is able to incorporate direct- and indirect gene interactions as well as tissue- and cell-specific characteristics. This approach (1) identified a descriptor of TRAIL sensitivity which performs significantly better as a predictor of TRAIL sensitivity than any previously reported gene signatures, (2) identified potential novel regulators of TRAIL-responsiveness and (3) provided a systematic view highlighting fundamental differences between the molecular wiring of sensitive and resistant cell types.

Kinetics in signal transduction pathways involving promiscuous oligomerizing receptors can be determined by receptor specificity: apoptosis induction by TRAIL.

Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows down the formation of active receptor complexes and thus can block signal transduction. Our model predicts that increasing the receptor specificity of the ligand without changing its binding parameters should result in faster receptor activation and enhanced signaling. We experimentally validated this hypothesis using the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its four membrane-bound receptors as an example. Bypassing ligand-induced receptor hetero-oligomerization by receptor-selective TRAIL variants enhanced the kinetics of receptor activation and augmented apoptosis. Our results suggest that control of signaling pathways by promiscuous ligands could result in apparent slow biological kinetics and blocking signal transmission. By modulating the relative amount of the different receptors for the ligand, signaling processes like apoptosis can be accelerated or decelerated and even inhibited. It also implies that more effective treatments using protein therapeutics could be achieved simply by altering specificity.

CCPlotR: an R package for the visualization of cell-cell interactions.

Summary: We present CCPlotR-an R package that generates visualizations of cell-cell interactions. CCPlotR is designed to work with the output of tools that predict cell-cell interactions from single-cell gene expression data and requires only a table of predicted interactions as input. The package can generate a comprehensive set of publication-ready figures such as heatmaps, dotplots, circos plots and network diagrams, providing a useful resource for researchers working on cell-cell interactions. Availability and implementation: CCPlotR is available to download and install from GitHub (https://github.com/Sarah145/CCPlotR) and comes with a toy dataset to demonstrate the different functions. Support for users will be provided via the GitHub issues tracker (https://github.com/Sarah145/CCPlotR/issues).

Cell-cell interactome of the hematopoietic niche and its changes in acute myeloid leukemia.

The bone marrow (BM) is a complex microenvironment, coordinating the production of billions of blood cells every day. Despite its essential role and its relevance to hematopoietic diseases, this environment remains poorly characterized. Here we present a high-resolution characterization of the niche in health and acute myeloid leukemia (AML) by establishing a single-cell gene expression database of 339,381 BM cells. We found significant changes in cell type proportions and gene expression in AML, indicating that the entire niche is disrupted. We then predicted interactions between hematopoietic stem and progenitor cells (HSPCs) and other BM cell types, revealing a remarkable expansion of predicted interactions in AML that promote HSPC-cell adhesion, immunosuppression, and cytokine signaling. In particular, predicted interactions involving transforming growth factor ß1 (TGFB1) become widespread, and we show that this can drive AML cell quiescence in vitro. Our results highlight potential mechanisms of enhanced AML-HSPC competitiveness and a skewed microenvironment, fostering AML growth.

Natural killer cell-mimic nanoparticles can actively target and kill acute myeloid leukemia cells.

Natural killer (NK) cells play a crucial role in recognizing and killing emerging tumor cells. However, tumor cells develop mechanisms to inactivate NK cells or hide from them. Here, we engineered a modular nanoplatform that acts as NK cells (NK cell-mimics), carrying the tumor-recognition and death ligand-mediated tumor-killing properties of an NK cell, yet without being subject to tumor-mediated inactivation. NK cell mimic nanoparticles (NK.NPs) incorporate two key features of activated NK cells: cytotoxic activity via the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and an adjustable tumor cell recognition feature based on functionalization with the NK cell Fc-binding receptor (CD16, FCGR3A) peptide, enabling the NK.NPs to bind antibodies targeting tumor antigens. NK.NPs showed potent in vitro cytotoxicity against a broad panel of cancer cell lines. Upon functionalizing the NK.NPs with an anti-CD38 antibody (Daratumumab), NK.NPs effectively targeted and eliminated CD38-positive patient-derived acute myeloid leukemia (AML) blasts ex vivo and were able to target and kill CD38-positive AML cells in vivo, in a disseminated AML xenograft system and reduced AML burden in the bone marrow compared to non-targeted, TRAIL-functionalized liposomes. Taken together, NK.NPs are able to mimicking key antitumorigenic functions of NK cells and warrant their development into nano-immunotherapeutic tools.

TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype and is associated with increased survival in cancer patients with high tumor macrophage content.

Background: TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an imbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known. Methods: Primary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes in vitro. The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients. Results: TRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells in vitro. Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content. Conclusions: TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization.

TRAIL in the Treatment of Cancer: From Soluble Cytokine to Nanosystems.

The death ligand tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine superfamily, has long been recognized for its potential as a cancer therapeutic due to its low toxicity against normal cells. However, its translation into a therapeutic molecule has not been successful to date, due to its short in vivo half-life associated with insufficient tumor accumulation and resistance of tumor cells to TRAIL-induced killing. Nanotechnology has the capacity to offer solutions to these limitations. This review provides a perspective and a critical assessment of the most promising approaches to realize TRAIL's potential as an anticancer therapeutic, including the development of fusion constructs, encapsulation, nanoparticle functionalization and tumor-targeting, and discusses the current challenges and future perspectives.

Macromolecular crowding in the development of a three-dimensional organotypic human breast cancer model.

Although cell-derived matrices are at the forefront of scientific research and technological innovation for the development of in vitro tumour models, their two-dimensional structure and low extracellular matrix composition restrict their capacity to accurately predict toxicity of candidate molecules. Herein, we assessed the potential of macromolecular crowding (a biophysical phenomenon that significantly enhances and accelerates extracellular matrix deposition, resulting in three-dimensional tissue surrogates) in improving cell-derived matrices in vitro tumour models. Among the various decellularisation protocols assessed (NH4OH, DOC, SDS/EDTA, NP40), the NP40 appeared to be the most effective in removing cellular matter and the least destructive to the deposited matrix. Among the various cell types (mammary, skin, lung fibroblasts) used to produce the cell-derived matrices, the mammary fibroblast derived matrices produced under macromolecular crowding conditions and decellularised with NP40 resulted in significant increase in focal adhesion molecules, matrix metalloproteinases and proinflammatory cytokines, when seeded with MDA-MB-231 cells. Further, macromolecular crowding derived matrices significantly increased doxorubicin resistance and reduced the impact of intracellular reactive oxygen species mediated cell death. Collectively our data clearly illustrate the potential of macromolecular crowding in the development of cell-derived matrices-based in vitro tumour models that more accurately resemble the tumour microenvironment.

Targeting AML through DR4 with a novel variant of rhTRAIL.

Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL-induced apoptosis. Here we show that TRAIL-induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL-C3) that is a strong inducer of DR4-mediated apoptosis. TRAIL-C3 demonstrated much stronger pro-apoptotic activity than wild-type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro-apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS-345541, a selective inhibitor of inhibitor-κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL-C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death-inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.

An unfractionated fucoidan from Ascophyllum nodosum: extraction, characterization, and apoptotic effects in vitro.

An unfractionated fucoidan was extracted from the brown alga Ascophyllum nodosum. Extraction of fucoidan from seaweed was carried out using an innovative low-chemical process. A combinational approach involving compositional analysis, HPAEC, IR analysis, GPC, and NMR was employed to elucidate the composition and structure of an unfractionated fucoidan from A. nodosum. This fucoidan is composed mainly of fucose (52.1%), and also galactose (6.1%), glucose (21.3%), and xylose (16.5%). Sulfate content was determined to be 19%. GPC data indicated a polydisperse fucoidan containing two main size fractions (47 and 420 kDa). NMR analyses revealed a fucoidan displaying broad, complex signals as expected for such a high molecular weight and heterogeneous polymer with resonances consistent with a fucoidan isolated previously from A. nodosum. The effects of fucoidan on the apoptosis of human colon carcinoma cells and fucoidan-mediated signaling pathways were also investigated. Fucoidan decreased cell viability and induced apoptosis of HCT116 colon carcinoma cells. Fucoidan treatment of HCT116 cells induced activation of caspases-9 and -3 and the cleavage of PARP, led to apoptotic morphological changes, and altered mitochondrial membrane permeability. These results detail the structure and biological activity of an unfractionated fucoidan from A. nodosum.

Hematopoietic versus leukemic stem cell quiescence: Challenges and therapeutic opportunities.

Hematopoietic stem cells (HSC) are responsible for the production of mature blood cells. To ensure that the HSC pool does not get exhausted over the lifetime of an individual, most HSCs are in a state of quiescence with only a small proportion of HSCs dividing at any one time. HSC quiescence is carefully controlled by both intrinsic and extrinsic, niche-driven mechanisms. In acute myeloid leukemia (AML), the leukemic cells overtake the hematopoietic bone marrow niche where they acquire a quiescent state. These dormant AML cells are resistant to chemotherapeutics. Because they can re-establish the disease after therapy, they are often termed as quiescent leukemic stem cells (LSC) or leukemia-initiating cells. While advancements are being made to target particular driver mutations in AML, there is less focus on how to tackle the drug resistance of quiescent LSCs. This review summarises the current knowledge on the biochemical characteristics of quiescent HSCs and LSCs, the intracellular signaling pathways and the niche-driven mechanisms that control quiescence and the key differences between HSC- and LSC-quiescence that may be exploited for therapy.

Recreating the Bone Marrow Microenvironment to Model Leukemic Stem Cell Quiescence.

The main challenge in the treatment of acute myeloid leukemia (AML) is relapse, as it has no good treatment options and 90% of relapsed patients die as a result. It is now well accepted that relapse is due to a persisting subset of AML cells known as leukemia-initiating cells or leukemic stem cells (LSCs). Hematopoietic stem cells (HSCs) reside in the bone marrow microenvironment (BMM), a specialized niche that coordinates HSC self-renewal, proliferation, and differentiation. HSCs are divided into two types: long-term HSCs (LT-HSCs) and short-term HSCs, where LT-HSCs are typically quiescent and act as a reserve of HSCs. Like LT-HSCs, a quiescent population of LSCs also exist. Like LT-HSCs, quiescent LSCs have low metabolic activity and receive pro-survival signals from the BMM, making them resistant to drugs, and upon discontinuation of therapy, they can become activated and re-establish the disease. Several studies have shown that the activation of quiescent LSCs may sensitize them to cytotoxic drugs. However, it is very difficult to experimentally model the quiescence-inducing BMM. Here we report that culturing AML cells with bone marrow stromal cells, transforming growth factor beta-1 and hypoxia in a three-dimensional system can replicate the quiescence-driving BMM. A quiescent-like state of the AML cells was confirmed by reduced cell proliferation, increased percentage of cells in the G0 cell cycle phase and a decrease in absolute cell numbers, expression of markers of quiescence, and reduced metabolic activity. Furthermore, the culture could be established as co-axial microbeads, enabling high-throughput screening, which has been used to identify combination drug treatments that could break BMM-mediated LSC quiescence, enabling the eradication of quiescent LSCs.

Aspartic Aminopeptidase Is a Novel Biomarker of Aggressive Chronic Lymphocytic Leukemia.

Treatment of chronic lymphocytic leukemia has advanced substantially as our understanding of the kinase signal transduction pathways driven by the B cell receptor (BcR) has developed. Particularly, understanding the role of Bruton tyrosine kinase and phosphatidyl inositol 3 kinase delta in driving prosurvival signal transduction in chronic lymphocytic leukemia (CLL) cells and their targeting with pharmacological inhibitors (ibrutinib and idelalisib, respectively) has improved patient outcomes significantly. The kinase signaling pathway induced by the BcR is highly complex and has multiple interconnecting branches mediated by tyrosine and serine/threonine kinases activated downstream of the BcR. There is a high level of redundancy in the biological responses, with several BcR-signaling kinases driving nuclear factor kappa B activation or inducing antiapoptotic Bcl-2 genes. Accordingly, common gene targets of BcR-signaling kinases may serve as biomarkers indicating enhanced BCR-signaling and aggressive disease progression. This study used a gene expression correlation analysis of malignant B cell lines and primary CLL cells to identify genes whose expression correlated with BCR-signaling kinases overexpressed and/or overactivated in CLL, namely: AKT1, AKT2, BTK, MAPK1, MAPK3, PI3KCD and ZAP70. The analysis identified a 32-gene signature with a strong prognostic potential and DNPEP, the gene coding for aspartic aminopeptidase, as a predictor of aggressive CLL. DNPEP gene expression correlated with MAPK3, PI3KCD, and ZAP70 expression and, in the primary CLL test dataset, showed a strong prognostic potential. The inhibition of DNPEP with a pharmacological inhibitor enhanced the cytotoxic potential of idelalisib and ibrutinib, indicating a biological functionality of DNPEP in CLL. DNPEP, as an aminopeptidase, contributes to the maintenance of the free amino acid pool in CLL cells found to be an essential process for the survival of many cancer cell types, and thus, these results warrant further research into the exploitation of aminopeptidase inhibitors in the treatment of drug-resistant CLL.

Enhancement of antitumor properties of rhTRAIL by affinity increase toward its death receptors.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent and selective inducer of apoptosis in various tumor types, raising enthusiasm for TRAIL as a potential anticancer agent. TRAIL-induced apoptosis is mediated by death receptors 4 (DR4) and DR5. The design of rhTRAIL variants either with improved affinity or selectivity toward one or both death-inducing receptors is thought to enhance the therapeutical potential of TRAIL. Here we demonstrate that a single amino acid mutation at the position of glycine 131 to lysine or arginine in wild-type rhTRAIL significantly improved the affinity of rhTRAIL toward its death receptors, with the highest affinity increase observed for the DR4 receptor. These variants were able to induce higher in vitro levels of apoptosis in cancer cells responsive to only DR4 or to both death receptors and could therefore increase the potential use of rhTRAIL as an anticancer therapeutic agent.

The mitochondrial death pathway: a promising therapeutic target in diseases.

The mitochondrial pathway to apoptosis is a major pathway of physiological cell death in vertebrates. The mitochondrial cell death pathway commences when apoptogenic molecules present between the outer and inner mitochondrial membranes are released into the cytosol by mitochondrial outer membrane permeabilization (MOMP). BCL-2 family members are the sentinels of MOMP in the mitochondrial apoptotic pathway; the pro-apoptotic B cell lymphoma (BCL)-2 proteins, BCL-2 associated x protein and BCL-2 antagonist killer 1 induce MOMP whereas the anti-apoptotic BCL-2 proteins, BCL-2, BCL-x(l) and myeloid cell leukaemia 1 prevent MOMP from occurring. The release of pro-apoptotic factors such as cytochrome c from mitochondria leads to formation of a multimeric complex known as the apoptosome and initiates caspase activation cascades. These pathways are important for normal cellular homeostasis and play key roles in the pathogenesis of many diseases. In this review, we will provide a brief overview of the mitochondrial death pathway and focus on a selection of diseases whose pathogenesis involves the mitochondrial death pathway and we will examine the various pharmacological approaches that target this pathway.