Robust temporal map of human in vitro myelopoiesis using single-cell genomics.

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

MicroRNA-155 controls vincristine sensitivity and predicts superior clinical outcome in diffuse large B-cell lymphoma.

A major clinical challenge of diffuse large B-cell lymphoma (DLBCL) is that up to 40% of patients have refractory disease or relapse after initial response to therapy as a result of drug-specific molecular resistance. The purpose of the present study was to investigate microRNA (miRNA) involvement in vincristine resistance in DLBCL, which was pursued by functional in vitro analysis in DLBCL cell lines and by outcome analysis of patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Differential miRNA expression analysis identified miR-155 as highly expressed in vincristine-sensitive DLBCL cell lines compared with resistant ones. Ectopic upregulation of miR-155 sensitized germinal-center B-cell-like (GCB)-DLBCL cell lines to vincristine, and consistently, reduction and knockout of miR-155 induced vincristine resistance, documenting that miR-155 functionally induces vincristine sensitivity. Target gene analysis identified miR-155 as inversely correlated with Wee1, supporting Wee1 as a target of miR-155 in DLBCL. Chemical inhibition of Wee1 sensitized GCB cells to vincristine, suggesting that miR-155 controls vincristine response through Wee1. Outcome analysis in clinical cohorts of DLBCL revealed that high miR-155 expression level was significantly associated with superior survival for R-CHOP-treated patients of the GCB subclass, independent of international prognostic index, challenging the commonly accepted perception of miR-155 as an oncomiR. However, miR-155 did not provide prognostic information when analyzing the entire DLBCL cohort or activated B-cell-like classified patients. In conclusion, we experimentally confirmed a direct link between high miR-155 expression and vincristine sensitivity in DLBCL and documented an improved clinical outcome of GCB-classified patients with high miR-155 expression level.

Anti-Apoptotic Effects of Lentiviral Vector Transduction Promote Increased Rituximab Tolerance in Cancerous B-Cells.

Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness.

High miR-34a expression improves response to doxorubicin in diffuse large B-cell lymphoma.

The standard treatment for patients with diffuse large B-cell lymphoma (DLBCL) is the immunochemotherapy-based R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone). Resistance to treatment, intrinsic or acquired, is observed in approximately 40% of patients with DLBCL, who thus require novel interventions to survive. To identify biomarkers for cytotoxic response assessment, microRNAs (miRNAs) associated with doxorubicin sensitivity were determined by combining global miRNA expression profiling with systematic dose-response screens in 15 human DLBCL cell lines. One candidate, miR-34a, was tested in functional in vitro studies and in vivo in a retrospective clinical cohort. High expression of miR-34a was observed in cell lines sensitive to doxorubicin, and upregulation of miR-34a is documented here to increase doxorubicin sensitivity in in vitro lentiviral transduction assays. High expression of miR-34a had a prognostic impact using overall survival as outcome. With risk stratification of DLBCL samples based on resistance gene signatures (REGS), doxorubicin-responsive samples had statistically significant upregulated miR-34a expression. Classification of the DLBCL samples into subset-specific B cell-associated gene signatures (BAGS) revealed differentiation-specific expression of miR-34a. Our data further support FOXP1 as a target of miR-34a, suggesting that downregulation of FOXP1 may sensitize DLBCL cells to doxorubicin. We conclude that miRNAs, in particular miR-34a, may have clinical utility in DLBCL patients as both predictive and prognostic biomarkers.

Potent microRNA suppression by RNA Pol II-transcribed 'Tough Decoy' inhibitors.

MicroRNAs (miRNAs) are key regulators of gene expression and modulators of diverse biological pathways. Analyses of miRNA function as well as therapeutic managing of miRNAs rely on cellular administration of miRNA inhibitors which may be achieved by the use of viral vehicles. This study explores the miRNA-suppressive capacity of inhibitors expressed intracellularly from lentivirus-derived gene vectors. Superior activity of two decoy-type inhibitors, a "Bulged Sponge" with eight miRNA recognition sites and a hairpin-shaped "Tough Decoy" containing two miRNA recognition sites, is demonstrated in a side-by-side comparison of seven types of miRNA inhibitors transcribed as short RNAs from an RNA Pol III promoter. We find that lentiviral vectors expressing Tough Decoy inhibitors are less vulnerable than Bulged Sponge-encoding vectors to targeting by the cognate miRNA and less prone, therefore, to reductions in transfer efficiency. Importantly, it is demonstrated that Tough Decoy inhibitors retain their miRNA suppression capacity in the context of longer RNA transcripts expressed from an RNA Pol II promoter. Such RNA Pol II-transcribed Tough Decoy inhibitors are new tools in managing of miRNAs and may have potential for temporal and spatial regulation of miRNA activity as well as for therapeutic targeting of miRNAs that are aberrantly expressed in human disease.

Regulation of pro-inflammatory cytokines TNFα and IL24 by microRNA-203 in primary keratinocytes.

Cutaneous homeostasis and innate immunity is procured by a complex circuitry of intercellular cytokine signaling. MicroRNAs are important posttranscriptional regulators of keratinocyte gene expression and assist in modulating the fine balance between cell proliferation and differentiation in skin. A characteristic microRNA profile in inflammatory skin suggests putative functions of microRNAs in perturbed cytokine production and signaling during chronic inflammatory skin conditions such as psoriasis. It remains unclear, however, why certain microRNAs are aberrantly expressed during skin inflammation and if they serve pro- and/or anti-inflammatory functions. In this report, we focus on cytokine regulation by microRNA-203 (miR-203), which is highly abundant in keratinocytes and upregulated in psoriatic lesions. By screening a panel of cytokines that are upregulated in psoriatic skin for regulation by miR-203, we identify the genes encoding the pro-inflammatory cytokines TNFα and IL24 as direct targets of miR-203. Studies of miR-203 overexpression, inhibition, and mutagenesis validate posttranscriptional regulation of TNFα and IL24 by miR-203 in cell lines and primary keratinocytes. Our findings suggest that miR-203 serves to fine-tune cytokine signaling and may dampen skin immune responses by repressing key pro-inflammatory cytokines.