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 antibodybased 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 setup , quality control, and analysis. Additionally, we discuss different advantages and pitfalls of this technique.

Dysregulation of mRNA translation, including preferential translation of mRNA with complex 5'-UTRs such as the MYC oncogene, is recognized as an important mechanism in cancer. In this study, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which can be inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis consisting of pulsed SILAC, RNA sequencing and polysome profiling performed in CLL patient samples and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibition of translation was associated with a block of proliferation and a profound 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 translation initiation complex and can be targeted by FL3. Knock-down of PHBs resembled FL3 treatment. Importantly, inhibition of translation was efficient in controlling 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 CLL patients. In conclusion, 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 CLL patients.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in the elderly and is characterized by the accumulation of mature B lymphocytes in peripheral blood and primary lymphoid organs. In order to proliferate, leukemic cells are highly dependent on complex interactions with their microenvironment in proliferative niches. Not only soluble factors and BCR stimulation are important for their survival and proliferation, but also the activation of transcription factors through different signaling pathways. The aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF)-1α are two transcription factors crucial for cancer development, whose activities are dependent on tumor microenvironment conditions, such as the presence of metabolites from the tryptophan pathway and hypoxia, respectively. In this study, we addressed the potential role of AHR and HIF-1α in chronic lymphocytic leukemia (CLL) development in vivo. To this end, we crossed the CLL mouse model Eµ-TCL1 with the corresponding transcription factor-conditional knock-out mice to delete one or both transcription factors in CD19+ B cells only. Despite AHR and HIF-1α being activated in CLL cells, deletion of either or both of them had no impact on CLL progression or survival in vivo, suggesting that these transcription factors are not crucial for leukemogenesis in CLL.

Failure of immunotherapy after applying checkpoint inhibitors or CAR-T cells is linked to T cell exhaustion. Here, we explored the T cell landscape in chronic lymphocytic leukemia (CLL) by single-cell omics analyses of blood, bone marrow and lymph node samples of patients and spleen samples of a CLL mouse model. By single-cell RNA-sequencing, mass cytometry (CyTOF), and multiplex image analysis of tissue microarrays, we defined the spectrum of phenotypes and transcriptional programs of T cells and and their differentiation state trajectories. We identified disease-specific accumulation of distinct regulatory T cell subsets and T cells harboring an exhausted phenotype exclusively in the CLL lymph node tissue. Integration of TCR data revealed a clonal expansion of CD8+ precursor exhausted T cells, suggesting their reactivity for CLL cells. Interactome analyses identified the TIM3 ligand Galectin-9 as novel immunoregulatory molecule in CLL. Blocking of Galectin-9 in CLL-bearing mice slowed down disease development and reduced the number of TIM3 expressing T cells. Galectin-9 expression correlated with shorter survival of CLL patients. Thus, Galectin-9 contributes to immune escape in CLL and represents a novel target for immunotherapy.

Background:CLL is the most common type of leukemia in adult, and despite great advance in the standard of care in the last decades, there is still no cure available. CLL cells are dependent on their microenvironment for proliferation and survival. Microenvironmental stimuli are associated with an increase in translation globally but also at the level of specific transcripts, including the MYC oncogene. Interestingly, translation was recognized as one "Achilles' heel" of cancer cells and increased translation seems to be a common feature of a large variety of tumors. Several inhibitors of translation are available. We used FL3, a synthetic flavagline that was shown to bind prohibitins (PHBs). These proteins are found in several cellular localizations that dictate their activity. At the membrane, they are required for the RAF activation by RAS in a large variety of cancers, leading to the phosphorylation of eukaryotic initiation factor 4E (eIF4E) through the MAPK pathway, and ultimately resulting in increased translation. By binding to PHBs, FL3 was shown to prevent the activation of RAF and therefore decreases the translation.

Background:Background: Chronic Lymphocytic Leukemia (CLL) progression is highly dependent on complex interactions between tumor cells and the tumor microenvironment (TME). Indeed, CLL cells can modify stromal cells and immune cells to promote the survival of the leukemic clone and to escape from the immune system surveillance. Within the TME, regulatory T cells (Tregs) represent a subtype of CD4+ T cells with immunosuppressive abilities, causing the evasion of cancer cells from the immune system. We previously characterized extensively the immune microenvironment of pre-clinical CLL mouse models using mass cytometry, and we described a significant increase in the Tregs subsets with an enhanced immunosuppressive and activated phenotype compared to non-leukemic animals (Wierz et al., Blood, 2018). Interestingly, TIGIT+ Tregs are more immunosuppressive than their TIGIT-Treg counterparts and express higher levels of several transcription factors, including Ahr and Hif1α (Joller et al., Immunity, 2014), both involved in the cellular response to microenvironment-mediated stimuli.

BM-MSC, immune checkpoint blockade, NK cells, TIGITAccounting for 10% of all blood cancers, multiple myeloma (MM) is an incurable haematological malignancy characterized by the accumulation of clonal plasma cells in the bone marrow (BM) of patients. This malignancy often develops in elderly patients through the accumulation of a heterogeneous range of cytogenetic abnormalities that promote tumour growth and survival in the BM microenvironment. 1 The gold standard for the treatment of newly diagnosed patients without co-morbidities remains high-dose chemotherapy plus autologous stem cell transplantation. Unfortunately, MM patients relapse after each treatment regimen, leading to a continuous and dynamic search for new non-refractory therapeutic options. 2 Novel agents including immunomodulatory drugs, proteasome inhibitors, dexamethasone and more recently monoclonal antibodies have transformed the treatment paradigm of MM. Nowadays, these agents are extensively used in the clinic to target relapsed and refractory diseases. Of particular interest, immune checkpoint inhibitors have emerged as powerful tools to disrupt the deeply immunosuppressive interactions in the BM MM microenvironment,This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Background: Background:Abnormal distribution and impaired function of T cells are key features of chronic lymphocytic leukemia (CLL), a malignancy of mature B lymphocytes that develops in secondary lymphoid tissue and blood. These defects have been linked to failure of immune control and resistance to immunotherapy including immune checkpoint or CAR T-cell therapy. A better characterization of T cells and their loss of function in CLL will help to improve such treatment approaches.