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.

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 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.

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.

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.

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.

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.

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.

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.

Involvement of planned cell death of necroptosis in cancer treatment by nanomaterials: Recent advances and future perspectives.

With the development of the field of nanomedicine, the application of nanomaterials/NPs in cancer treatment has raised questions about their potential effects as well as thier unpredictable adverse effects. To date, the cytotoxic effects of nanomaterials have been investigated based on cell survival and cellular functionality, such as membrane integrity, mitochondrial activity and cell morphology. It is increasingly noted that more detailed analysis of RCD triggered by nanomaterials is essential to understand their full mechanism of action. One the one hand, this knowledge helps us to design safe therapeutics and also increases the therapeutic potential of NP-based anti-cancer drugs. The most common pathways of RCD in cancer cells include apoptosis, necrosis, necroptosis and autophagy with the latter two often act as secondary death pathways in cancer cells when the apoptotic and necrotic pathways are non-functional. This article reviews the recent developments and future perspectives in the ability of nanomaterials/NPs to induce the above forms of RCD especially necroptosis.

Repression of Mcl-1 expression by the CDC7/CDK9 inhibitor PHA-767491 overcomes bone marrow stroma-mediated drug resistance in AML.

Acute myeloid leukaemia (AML) is an aggressive cancer with 50-75% of patients relapsing even after successful chemotherapy. The role of the bone marrow microenvironment (BMM) in protecting AML cells from chemotherapeutics and causing consequent relapse is increasingly recognised. However the role that the anti-apoptotic Bcl-2 proteins play as effectors of BMM-mediated drug resistance are less understood. Here we show that bone marrow mesenchymal stromal cells (BMSC) provide resistance to AML cells against BH3-mimetics, cytarabine and daunorubicin, but this is not mediated by Bcl-2 and/or Bcl-XL as previously thought. Instead, BMSCs induced Mcl-1 expression over Bcl-2 and/or Bcl-XL in AML cells and inhibition of Mcl-1 with a small-molecule inhibitor, A1210477, or repressing its expression with the CDC7/CDK9 dual-inhibitor, PHA-767491 restored sensitivity to BH3-mimetics. Furthermore, combined inhibition of Bcl-2/Bcl-XL and Mcl-1 could revert BMSC-mediated resistance against cytarabine + daunorubicin. Importantly, the CD34+/CD38- leukemic stem cell-encompassing population was equally sensitive to the combination of PHA-767491 and ABT-737. These results indicate that Bcl-2/Bcl-XL and Mcl-1 act in a redundant fashion as effectors of BMM-mediated AML drug resistance and highlight the potential of Mcl-1-repression to revert BMM-mediated drug resistance in the leukemic stem cell population, thus, prevent disease relapse and ultimately improve patient survival.

N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

TRAIL receptor gene editing unveils TRAIL-R1 as a master player of apoptosis induced by TRAIL and ER stress.

TRAIL induces selective tumor cell death through TRAIL-R1 and TRAIL-R2. Despite the fact that these receptors share high structural homologies, induction of apoptosis upon ER stress, cell autonomous motility and invasion have solely been described to occur through TRAIL-R2. Using the TALEN gene-editing approach, we show that TRAIL-R1 can also induce apoptosis during unresolved unfolded protein response (UPR). Likewise, TRAIL-R1 was found to co-immunoprecipitate with FADD and caspase-8 during ER stress. Its deficiency conferred resistance to apoptosis induced by thaspigargin, tunicamycin or brefeldin A. Our data also demonstrate that tumor cell motility and invasion-induced by TRAIL-R2 is not cell autonomous but induced in a TRAIL-dependant manner. TRAIL-R1, on the other hand, is unable to trigger cell migration owing to its inability to induce an increase in calcium flux. Importantly, all the isogenic cell lines generated in this study revealed that apoptosis induced TRAIL is preferentially induced by TRAIL-R1. Taken together, our results provide novel insights into the physiological functions of TRAIL-R1 and TRAIL-R2 and suggest that targeting TRAIL-R1 for anticancer therapy is likely to be more appropriate owing to its lack of pro-motile signaling capability.

Generation of Tumour-stroma Minispheroids for Drug Efficacy Testing.

The three-dimensional organisation of cells in a tissue and their interaction with adjacent cells and extracellular matrix is a key determinant of cellular responses, including how tumour cells respond to stress conditions or therapeutic drugs (Elliott and Yuan, 2011). In vivo, tumour cells are embedded in a stroma formed primarily by fibroblasts that produce an extracellular matrix and enwoven with blood vessels. The 3D mixed cell type spheroid model described here incorporates these key features of the tissue microenvironment that in vivo tumours exist in; namely the three-dimensional organisation, the most abundant stromal cell types (fibroblasts and endothelial cells), and extracellular matrix. This method combined with confocal microscopy can be a powerful tool to carry out drug sensitivity, angiogenesis and cell migration/invasion assays of different tumour types.

The Janus Face of Death Receptor Signaling during Tumor Immunoediting.

Cancer immune surveillance is essential for the inhibition of carcinogenesis. Malignantly transformed cells can be recognized by both the innate and adaptive immune systems through different mechanisms. Immune effector cells induce extrinsic cell death in the identified tumor cells by expressing death ligand cytokines of the tumor necrosis factor ligand family. However, some tumor cells can escape immune elimination and progress. Acquisition of resistance to the death ligand-induced apoptotic pathway can be obtained through cleavage of effector cell expressed death ligands into a poorly active form, mutations or silencing of the death receptors, or overexpression of decoy receptors and pro-survival proteins. Although the immune system is highly effective in the elimination of malignantly transformed cells, abnormal/dysfunctional death ligand signaling curbs its cytotoxicity. Moreover, DRs can also transmit pro-survival and pro-migratory signals. Consequently, dysfunctional death receptor-mediated apoptosis/necroptosis signaling does not only give a passive resistance against cell death but actively drives tumor cell motility, invasion, and contributes to consequent metastasis. This dual contribution of the death receptor signaling in both the early, elimination phase, and then in the late, escape phase of the tumor immunoediting process is discussed in this review. Death receptor agonists still hold potential for cancer therapy since they can execute the tumor-eliminating immune effector function even in the absence of activation of the immune system against the tumor. The opportunities and challenges of developing death receptor agonists into effective cancer therapeutics are also discussed.