Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL.

Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

Interleukin-27 potentiates CD8+ T-cell-mediated antitumor immunity in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells are highly dependent on interactions with the immunosuppressive tumor microenvironment (TME) for survival and proliferation. In the search for novel treatments, pro-inflammatory cytokines have emerged as candidates to reactivate the immune system. Among those, interleukin 27 (IL-27) has recently gained attention, but its effects differ among malignancies. Here, we utilized the Eµ-TCL1 and EBI3 knock-out mouse models as well as clinical samples from patients to investigate the role of IL-27 in CLL. Characterization of murine leukemic spleens revealed that the absence of IL-27 leads to enhanced CLL development and a more immunosuppressive TME in transgenic mice. Gene-profiling of T-cell subsets from EBI3 knock-out highlighted transcriptional changes in the CD8+ T-cell population associated with T-cell activation, proliferation, and cytotoxicity. We also observed an increased anti-tumor activity of CD8+ T cells in the presence of IL-27 ex vivo with murine and clinical samples. Notably, IL-27 treatment led to the reactivation of autologous T cells from CLL patients. Finally, we detected a decrease in IL-27 serum levels during CLL development in both pre-clinical and patient samples. Altogether, we demonstrated that IL-27 has a strong anti-tumorigenic role in CLL and postulate this cytokine as a promising treatment or adjuvant for this malignancy.

Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) including Variant Analysis by Mass Spectrometry in Placental Tissue.

Among neonates, tested positive for SARS-CoV-2, the majority of infections occur through postpartum transmission. Only few reports describe intrauterine or intrapartum SARS-CoV-2 infections in newborns. To understand the route of transmission, detection of the virus or virus nucleic acid in the placenta and amniotic tissue are of special interest. Current methods to detect SARS-CoV-2 in placental tissue are immunohistochemistry, electron microscopy, in-situ hybridization, polymerase chain reaction (PCR) and next-generation sequencing. Recently, we described an alternative method for the detection of viral ribonucleic acid (RNA), by combination of reverse transcriptase-PCR and mass spectrometry (MS) in oropharyngeal and oral swabs. In this report, we could detect SARS-CoV-2 in formal-fixed and paraffin-embedded (FFPE) placental and amniotic tissue by multiplex RT-PCR MS. Additionally, we could identify the British variant (B.1.1.7) of the virus in this tissue by the same methodology. Combination of RT-PCR with MS is a fast and easy method to detect SARS-CoV-2 viral RNA, including specific variants in FFPE tissue.

Method for the Analysis of the Tumor Microenvironment by Mass Cytometry: Application to Chronic Lymphocytic Leukemia.

In the past 20 years, the interest for the tumor microenvironment (TME) has exponentially increased. Indeed, it is now commonly admitted that the TME plays a crucial role in cancer development, maintenance, immune escape and resistance to therapy. This stands true for hematological malignancies as well. A considerable amount of newly developed therapies are directed against the cancer-supporting TME instead of targeting tumor cells themselves. However, the TME is often not clearly defined. In addition, the unique phenotype of each tumor and the variability among patients limit the success of such therapies. Recently, our group took advantage of the mass cytometry technology to unveil the specific TME in the context of chronic lymphocytic leukemia (CLL) in mice. We found the enrichment of LAG3 and PD1, two immune checkpoints. We tested an antibody-based immunotherapy, targeting these two molecules. This combination of antibodies was successful in the treatment of murine CLL. In this methods article, we provide a detailed protocol for the staining of CLL TME cells aiming at their characterization using mass cytometry. We include panel design and validation, sample preparation and acquisition, machine set-up, quality control, and analysis. Additionally, we discuss different advantages and pitfalls of this technique.

Purification of Leukemia-Derived Exosomes to Study Microenvironment Modulation.

Exosomes are membrane-enclosed vesicles released by different cell types into the extracellular space. As mediators of intercellular communication, they are involved in multiple physiological processes, but they are also associated with the pathogenesis of human malignancies including leukemia. Isolation of exosomes enables the characterization of their role in microenvironment modulation as well as their participation in disease pathology. A variety of strategies and techniques exists to purify exosomes from many biological fluids (e.g., blood, urine, and saliva). Here, we describe the efficient production of large quantities of exosomes from leukemic cell lines by using CELLine bioreactors based on two-compartment technology, as well as their isolation and purification by combining differential centrifugation and ultracentrifugation through a density gradient (17% OptiPrep™ cushion). Thus, exosomes are appropriately prepared for characterization by western blotting to detect exosome markers or imaging flow cytometry (ImageStream), and for downstream analyses such as the internalization in microenvironmental cells by confocal imaging or flow cytometry, methods which are also described in this chapter.

High-dimensional mass cytometry analysis revealed microenvironment complexity in chronic lymphocytic leukemia.

In this study, we extensively dissected the phenotypic complexity of the splenic tumor microenvironment (TME) in chronic lymphocytic leukemia (CLL) by high-dimensional mass cytometry (CyTOF). As a result, we identified potential new targets and tested a dual immune checkpoint blockade targeting the TME in pre-clinical mouse models of CLL.

Transgenic expression of the RNA binding protein IMP2 stabilizes miRNA targets in murine microsteatosis.

Adult expression of IMP2 is often associated with several types of disease and cancer. The RNA binding protein IMP2 binds and stabilizes the IGF2 mRNA as well as hundreds of other transcripts during development. To gain insight into the molecular action of IMP2 and its contribution to disease in context of adult cellular metabolism, we analyze transgenic overexpression of IMP2 in mouse livers, which has been shown to induce a steatosis-like phenotype and enhanced risk to develop hepatocellular carcinoma (HCC). Our data show up-regulation of several HCC marker genes and miRNAs (miR438-3p and miR151-5p). To characterize the impact of miRNAs to their targets, integrative analysis of transcriptome-and miRNAome-dynamics in combination with IMP2 target prediction was carried out. Our analyses show that targets of expressed miRNAs become accumulated in the case that these transcripts have positive IMP2 binding prediction. Therefore, our data indicates that overexpression of IMP2 alters the regulatory capacity of many miRNAs and we conclude that IMP2 competes with miRNAs for binding sites on thousands of transcripts. As a result, our data implicates that overexpression of IMP2 has distinct effects to the regulatory capacity of miRNAs with yet unknown consequences for translational efficiency.