Supplemental Table S2 - Differential expression analysis of genes in leukemic or immune cells treated with sEV

Abstract A direct link between chronic inflammation and development of Acute Myeloid Leukemia (AML) has been highlighted in the past few years, demonstrating an interconnection between marked inflammatory phenotype and aberrant myeloproliferation in AML patients. Treating AML patients exhibiting a higher inflammatory signature with anti-inflammatory molecules resulted in significant increase of overall survival. Protein Arginine Methyltransferases (PRMTs) are epigenetic factors known to regulate gene expression through methylation of histone tails. It has been previously reported that PRMT1, 4, and 5 inhibition exhibit anti-proliferative effects on AML models. In this study, we investigated the role of another PRMT, called PRMT2, in the development of AML through its regulatory roles in inflammatory pathways. We first determined from an AML cohort (The Leucegene project, IRIC, Montréal, QC, Canada) that patients with a low PRMT2 expression display an enrichment of proinflammatory pathways compared to patients with a high PRMT2 expression. Therefore, we hypothesized that PRMT2 could be a key regulator of inflammatory processes in AML. We thus used a PRMT2 knockout mouse model (Prmt2 KO) and a PRMT2 knockout human AML cell line to validate our hypothesis. Although we demonstrated no difference in the bone marrow progenitors or mature cell populations of Prmt2 KO mice compared to control, we observed that Prmt2 KO Bone-Marrow Derived Macrophages (BMDMs) are more sensitive to LPS stimulation and express higher levels of pro-inflammatory cytokines, supporting our previous findings for a role of PRMT2 in the negative regulation of inflammatory processes. PRMT2 depleted human AML cells displayed an increased pro-inflammatory signature due to overactivation of STAT3, which is caused by an enhanced activation of the NFkB signaling pathway, leading to an overproduction of IL6. Together, these findings demonstrate that PRMT2 is a key regulator of the control of inflammation in AML. Recognition of PRMT2 as a biomarker of inflammation in AML would help to adapt treatment possibly through the synergistic use of anti-inflammatory molecules with other cytotoxic drugs. Citation Format: Camille Sauter, Thomas Morin, Fabien Guidez, John Simonet, Cyril Fournier, Céline Row, Denis Masnikov, Baptiste Pernon, Anne Largeot, Aziza Aznague, Yann Herault, Guy Sauvageau, Marc Maynadie, Mary Callanan, Jean-Noël Bastie, Romain Aucagne, Laurent Levadny Delva. Protein Arginine Methyltransferase 2 is involved in the control of inflammatory processes in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5588.

Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and is involved in various cellular processes, including cancer development. PRMT2 expression is increased in several cancer types although its role in acute myeloid leukemia (AML) remains unknown. Here, we investigate the role of PRMT2 in a cohort of patients with AML, PRMT2 knockout AML cell lines as well as a Prmt2 knockout mouse model. In patients, low PRMT2 expressors are enriched for inflammatory signatures, including the NF-κB pathway, and show inferior survival. In keeping with a role for PRMT2 in control of inflammatory signaling, bone marrow-derived macrophages from Prmt2 KO mice display increased pro-inflammatory cytokine signaling upon LPS treatment. In PRMT2-depleted AML cell lines, aberrant inflammatory signaling has been linked to overproduction of IL6, resulting from a deregulation of the NF-κB signaling pathway, therefore leading to hyperactivation of STAT3. Together, these findings identify PRMT2 as a key regulator of inflammation in AML.

Abstract Background The epigenetic factors KAT6A (MOZ/MYST3) and KMT2A (MLL/MLL1) interact in normal hematopoiesis to regulate progenitors’ self-renewal. Both proteins are recurrently translocated in AML, leading to impairment of critical differentiation pathways in these malignant cells. We evaluated the potential of different KAT6A therapeutic targeting strategies to alter the growth of KAT6A and KMT2A rearranged AMLs. Methods We investigated the action and potential mechanisms of the first-in-class KAT6A inhibitor, WM-1119 in KAT6A and KMT2A rearranged (KAT6Ar and KMT2Ar) AML using cellular (flow cytometry, colony assays, cell growth) and molecular (shRNA knock-down, CRISPR knock-out, bulk and single-cell RNA-seq, ChIP-seq) assays. We also used two novel genetic murine KAT6A models combined with the most common KMT2Ar AML, KMT2A::MLLT3 AML. In these murine models, the catalytic activity of KAT6A, or the whole protein, can be conditionally abrogated or deleted. These models allowed us to compare the effects of specific KAT6A KAT activity inhibition with the complete deletion of the whole protein. Finally, we also tested these therapeutic approaches on human AML cell lines and primary patient AMLs. Results We found that WM-1119 completely abrogated the proliferative and clonogenic potential of KAT6Ar cells in vitro. WM-1119 treatment was associated with a dramatic increase in myeloid differentiation program. The treatment also decreased stemness and leukemia pathways at the transcriptome level and led to loss of binding of the fusion protein at critical regulators of these pathways. In contrast, our pharmacologic and genetic results indicate that the catalytic activity of KAT6A plays a more limited role in KMT2Ar leukemogenicity, while targeting the whole KAT6A protein dramatically affects leukemic potential in murine KMT2A::MLLT3 AML. Conclusion Our study indicates that inhibiting KAT6A KAT activity holds compelling promise for KAT6Ar AML patients. In contrast, targeted degradation of KAT6A, and not just its catalytic activity, may represent a more appropriate therapeutic approach for KMT2Ar AMLs.

Despite being tightly regulated, messenger RNA (mRNA) translation, a manner in which cells control expression of genes and rapidly respond to stimuli, is highly dysfunctional and plastic in pathologies including cancer. Conversely, the investigation of molecular mechanisms whereby mRNA translation becomes aberrant in cancer, as well as inhibition thereof, become critical in developing novel therapeutic approaches. More specifically, in malignancies such as chronic lymphocytic leukemia in which aberrant global and transcript specific translation has been linked with poorer patient outcomes, targeting translation is a relevant approach, with various translation inhibitors under development. Here we elaborate on a protein synthesis assay by flow cytometry, O-propargyl-puromycin, demonstrating global mRNA translation rate with a variety of different applications including cell lines, primary cells or co-culture systems in vitro. This method provides a comprehensive tool in quantifying the rate of global mRNA translation in cancer cells, as well as that of the tumor microenvironment cells, or in response to inhibitory therapeutic agents while offering the possibility to simultaneously assess other cellular markers.

Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 94 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 (C). Furthermore, inhibition of translation was associated with a block of proliferation (D) and a profound rewiring of MYC-driven metabolism. Interestingly, contrary to other models, in CLL, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation. We rather showed that PHBs are directly associated with the translation initiation complex (E). Knock-down of PHBs resembled FL3 treatment (F), confirming the direct involvement of PHBs in translation initiation. Importantly, inhibition of translation was efficient in controlling CLL development in vivo (G). Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in CLL patients (H). Summary/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. HemaSphere | 2023;7(S3) EHA2023 Hybrid Congress Copyright Information: (Online) ISSN: 2572-9241 © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) which allows third parties to download the articles and share them with others as long as they credit the author and the Abstract Book, but they cannot change the content in any way or use them commercially. Abstract Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx.Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 95

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.

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.

mRNA translation dynamics enable tight regulation of protein expression and rapid stress adaptation; however, its contribution to oncogenesis is overlooked compared to genetic and epigenetic aberrations. Aberrations in mRNA translation are a common feature of cancer and are established as a therapeutic vulnerability. There is growing evidence of dysregulated protein synthesis in CLL. For instance, the Ribosomal Protein S15 (RPS15) coding gene, a part of the 40S ribosomal subunit, is frequently mutated in CLL, leading to altered translation fidelity and efficacy, and associated with poorer prognosis [1, 2] (Figure 1A). Reduced expression of Dyskerin, a modifier of ribosomal RNAs (rRNAs) critical for translation fidelity, is associated with translatome changes [3]. CLL subgroups with mutations/expression changes in ribosomal proteins and complexes could be further classified as "ribosomopathies". In these conditions, evolution from hypo- to hyperproliferative phenotypes is likely aided by a shift towards an oncogenic translatome and the acquisition of rescue mutations, explaining higher frequencies of cancer [4]. Whether the same mechanisms occur in RPS15-mutated CLL requires further investigation. Aberrations in mRNA translation in CLL. Different cellular mechanisms contribute to altered translation in CLL cells compared to non-leukemic B cells. mRNA translation could be targeted with different therapeutic approaches (e.g., targeting of the mutated ribosome, inhibition of translation initiation) as a novel treatment for CLL. (A) Defective translation caused by mutations in RPS15 and affected translation fidelity. (B) Activation of signaling pathways linked to the microenvironment (e.g., downstream of the BCR or TLR) alters gene expression and leads to increased translation rates in CLL cells. (C) Intrinsic factors such as ZAP-70 are involved in several pathways of abnormal translation in CLL. Figure created with Biorender. Abbreviations: BCR: B-Cell Receptor; CLL: Chronic Lymphocytic Leukemia; eIF4A: Eukaryotic translation Initiation Factor 4A; PDCD4: Programmed Cell Death 4; RPS15: Ribosomal Protein S15; TLR: Toll-Like Receptor; ZAP-70: Zeta chain of T cell receptor-Associated Protein kinase 70. Alongside translational program defects triggered by ribosomal protein mutations, increased protein synthesis is observed in CLL. The lymph node (LN) microenvironment drives intense proliferation through diverse pathways. Interestingly, CLL cells in patient LNs exhibit an enriched mRNA translation signature compared to circulating cells [5]. Stimulation of CLL cells markedly raises global translation rate via heightened Eukaryotic translation Initiation Factor 4A (eIF4A) and eIF4G1 expression, coupled with reduced Programmed Cell Death 4 (PDCD4) levels, an eIF4A inhibitor (Figure 1B). Furthermore, elevated expression of certain factors impacts translation rate. The Zeta chain of T cell receptor-associated protein kinase 70 (ZAP-70), highly expressed in aggressive CLL cases, interacts with ribosomal proteins and translation initiation factors, sustaining high translation of genes involved in cell survival [6] (Figure 1C). In their study [7], using pulsed Stable Isotope Labeling with Amino acids in Cell culture (SILAC) assay, Largeot et al. identified the mRNAs affected by the increased translation upon stimulation of CLL patient cells. Gene ontology analysis revealed a positive loop, with proteins involved in mRNA translation and also in apoptosis regulation and cytokine signaling. In addition, they revealed that both human and murine CLL cells display a high translation rate compared to B cells from healthy donors and also to B cells found in the blood of patients and in the murine splenic microenvironment. In addition, increased expression of translation-related genes is associated with increased disease progression and poor survival in a cohort of CLL patients [7], confirming the clinical significance of this process. These findings reveal potential therapeutic strategies targeting mRNA translation abnormalities in CLL. In RPS15-mutated CLL cases, strategies could be developed to expand on existing approaches for ribosomopathies. Structural variations between mutated and wild-type ribosomal proteins allow the design of specific small molecules targeting these mutations [4]. Moreover, since ribosomal protein mutations modify the translational program, inhibiting translation could also impede the oncogenic progression. Translation inhibitors have been developed to target elevated mRNA translation in cancers, some showing promising results in CLL treatment. These compounds primarily target the translation initiation machinery, globally inhibiting translation while affecting mRNA translational programs. Examples include DesMethyl DesAmino Pateamine A (DMDAPatA) targeting eIF4A; 4EGI-1 blocking eIF4E and eIF4G interaction; and flavagline family members (e.g., silvestrol, rocaglamide, FL3) targeting eIF4A or prohibitins (PHBs). Notably, these agents hinder various processes in the CLL pathogenesis, such as reducing translation of overexpressed anti-apoptotic proteins inducing apoptosis [8, 9], potentially decreasing elevated expression of oncogenes including MYeloCytomatosis oncogene (MYC) [10]. In their recent study, Largeot et al. reaffirmed the potential of targeting mRNA translation in CLL. Upon FL3 treatment, apoptosis primarily affected CLL cells, with a lesser impact on healthy B cells [7]. This could be linked to higher expression of translation initiation factors in CLL cells compared to healthy B cells. Alternatively, CLL cells may depend on a different translation initiation machinery readily targeted by FL3. Multi-omics analysis of FL3-treated CLL cells further confirmed reduced translation of several oncogenes, including MYC and Nuclear Factor Kappa light chain enhancer of activated B cells (NF-KB), as well as other oncogenes not previously linked to CLL such as E26 Transformation Specific 1 (ETS-1). Metabolomic profiling revealed that MYC loss is at least partially responsible for the observed metabolic changes upon mRNA translation inhibition. While limited evidence existed for translation inhibition's impact on CLL development in vivo, this study demonstrated FL3's efficacy in controlling CLL development, alone or combined with immune checkpoint blockade, using the Eμ-T-Cell Leukemia 1 (TCL1) adoptive transfer model. Subsequent profiling of splenic cell populations showed decreased translational activity in FL3-treated mice. Notably, treatment primarily reduced CLL cells and regulatory T cells (Treg) translation, highlighting varied susceptibilities of distinct immune populations to mRNA translation inhibition. A crucial breakthrough by Largeot et al. is the elucidation of the mechanism of action of FL3. In contrast to prior findings in other cancer models, FL3 did not inhibit translation via the RAt Sarcoma virus-Rapidly Accelerated Fibrosarcoma-Mitogen-Activated Protein Kinase (RAS-RAF-MAPK) signaling pathway. In the CLL context, PHBs interact directly with the translation initiation machinery. FL3, through its binding to PHBs, disrupts the translation initiation machinery, inhibiting mRNA translation [7]. This discovery holds significance in unraveling the complexity of mRNA translation initiation and uncovering novel players in this process. This new knowledge offers potential for developing targeted therapies that may alter the interaction between PHBs and the eIF4F complex. Additionally, it may shed light on potential resistance mechanisms to FL3 treatment. In summary, targeting mRNA translation may be a promising avenue to explore for CLL treatment. Although the development of translation inhibitors is advancing, their clinical application is limited and ongoing clinical trials will reveal their implementation in this setting. Combining therapies may yield better results, warranting further investigations for optimal combinations. Other explorations aimed at the role of mRNA translation inhibition in CLL transformation into Richter syndrome, which is in part driven by MYC and resistant to standard therapies, are anticipated. All authors wrote the main text, prepared the figure and revised the final manuscript. The authors thank Dr. Magretta Adiamah (Luxembourg Institute of Health) for proofreading the manuscript. The authors declare no conflict of interest. This work was supported by grants from the Luxembourg National Research Fund (FNR) and Fondation Cancer to Vanessa Klapp, Etienne Moussay, Jérôme Paggetti (PRIDE19/14254520/i2TRON, C20/BM/14582635, and C20/BM/14592342), from FNRS-Télévie to Anne Largeot (7.4502.17, 7.4503.19), and from the Plooschter Projet to Jérôme Paggetti and Etienne Moussay. Not applicable. Not applicable. Not applicable.

Abstract Small extracellular vesicle (sEV, or exosome) communication among cells in the tumor microenvironment has been modeled mainly in cell culture, whereas their relevance in cancer pathogenesis and progression in vivo is less characterized. Here we investigated cancer–microenvironment interactions in vivo using mouse models of chronic lymphocytic leukemia (CLL). sEVs isolated directly from CLL tissue were enriched in specific miRNA and immune-checkpoint ligands. Distinct molecular components of tumor-derived sEVs altered CD8+ T-cell transcriptome, proteome, and metabolome, leading to decreased functions and cell exhaustion ex vivo and in vivo. Using antagomiRs and blocking antibodies, we defined specific cargo-mediated alterations on CD8+ T cells. Abrogating sEV biogenesis by Rab27a/b knockout dramatically delayed CLL pathogenesis. This phenotype was rescued by exogenous leukemic sEV or CD8+ T-cell depletion. Finally, high expression of sEV-related genes correlated with poor outcomes in CLL patients, suggesting sEV profiling as a prognostic tool. In conclusion, sEVs shape the immune microenvironment during CLL progression. Significance: sEVs produced in the leukemia microenvironment impair CD8+ T-cell mediated antitumor immune response and are indispensable for leukemia progression in vivo in murine preclinical models. In addition, high expression of sEV-related genes correlated with poor survival and unfavorable clinical parameters in CLL patients. See related commentary by Zhong and Guo, p. 5. This article is highlighted in the In This Issue feature, p. 1

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 Myc (Yeomans et al., 2016, Blood). Aims: Here, we tested the targeting of translation initiation in CLL as a novel therapeutic strategy and identified prohibitin as partner of the translation initiation machinery. Methods: In order to target translation, we used the FL3 molecule, a synthetic flavagline, which is a known inhibitor of translation initiation in other types of cancers (Boussemart et al., 2014, Nature). This drug binds to the scaffold proteins prohibitins (PHBs), but the mechanism for translation inhibition is still unclear. PHBs are crucial partners for the activation of the Ras-Raf-MEK-ERK pathway, ultimately leading to eIF4E (a factor of the eIF4F translation initiation machinery) phosphorylation and to activation of translation. Results: We confirmed the increase in translation in human primary CLL cells upon stimulation (A). In addition, we showed that in the Eµ-TCL1 murine model, CLL cells have a higher translation rate compared to normal B cells and T cells. In vitro treatment of human CLL cells (either cell lines or primary patient samples) with FL3 leads to a decrease in translation rate. It is associated with a strong decrease of cell viability (B) and apoptosis induction, even at a low nanomolar dose. By western blot, we showed that in CLL, contrary to other cancer types, FL3 does not prevent RAF1 and ERK phosphorylation. However, eIF4e phosphorylation is impaired. This observation strongly suggests a direct impact of the drug through its molecular target PHBs on the translation initiation machinery. By co-immunoprecipitation, proximity-ligation assay (C) and Nanoluciferase experiments, we demonstrated the interaction between PHBs and the members of the eIF4F complex. In addition, gene silencing of PHB by shRNA leads to a decrease in translation. This indicates for the first time a direct role of PHBs in translation, and allows a better understanding of FL3’s mechanism of action. We thus propose that PHB is necessary for the correct assembly of the eIF4F complex. Interestingly, we showed that the loss of eIF4e phosphorylation is neither responsible for the impairment of translation nor for the decrease in cell proliferation. By pulse SILAC experiments, we determined the proteins that are subjected to increased translation upon TLR stimulation, and to decreased translation upon FL3 treatment. Among others, oncogenes such as c-MYC or ETS-1 (D) have been identified. In vivo treatment of mice with FL3 after TCL1 adoptive transfer leads to a significant delay in CLL progression and an increased survival (E-F), demonstrating the relevance and possible impact of this molecule. Finally, high expression of translation initiation-related genes correlated with poor survival and unfavorable clinical parameters in CLL patients (G). Image:Summary/Conclusion: To conclude, we identified translation initiation as a potential therapeutic target in CLL. The use of the inhibitor of translation FL3, efficiently impairs proliferation of CLL cells in vitro and controls CLL development in vivo. Moreover, we start to unveil the mechanism of action of this molecule, by the identification of a novel interactor of the translation initiation machinery, namely prohibitins.

Background: Chronic Lymphocytic Leukemia (CLL), the most common type of leukemia in adults, is characterized by the clonal expansion of CD5+ CD19+ B cells. CLL progression is highly dependent on complex interactions with non-malignant cells of the tumor microenvironment. Despite great advance in the standard of care in the last decades, there are still unmet medical needs (long-life treatment, resistance…). Altered cellular metabolism has emerged as a hallmark of cancer by sustaining the uncontrolled growth of cancer cells. The one-carbon pathway is a major driver for tumor proliferation, providing building blocks for biosynthesis of nucleotides through pyrimidines (dTTP) and purines synthesis, redox metabolism (GSH), and energy balance by suppling ATP and NADPH to cells (A). Understanding the metabolic reprogramming occurring in cancer, notably in CLL, may provide insights to support the development of novel therapies. Aims: We aim to get insights into one-carbon metabolism regulation in CLL, identify new therapeutic targets and find new treatment options for CLL patients. Methods: Through a collaboration with a biotech company that develops inhibitors of the one-carbon metabolism, we tested new MTHFD1/2 inhibitors, which we believe, are promising in the context of CLL. The cytotoxic activity of the inhibitors was evaluated on a panel of murine and human CLL cell lines and primary cells, and also on others B cell malignancies such as Mantle Cell Lymphoma (MCL), Diffuse Large B-Cell Lymphoma (DLBCL), and Multiple Myeloma (MM). The molecular mechanisms sustaining the cytotoxic activity were evaluated by performing metabolic tracing, rescue experiments and CRISPR/Cas9 KO. Results: We showed a strong expression of both MTHFD1 and 2 in all the cancer cell lines tested and in stimulated primary human CLL cells. A higher expression of both enzymes was also observed in primary CLL cells, isolated from the Eµ-TCL1 murine model of CLL compared to normal B cells. In vitro treatment with the inhibitors efficiently reduced cell viability of CLL cell lines and primary cells, as for MCL and DLBCL cell lines, at low nanomolar dose while no effect was observed on MM cell lines (B). This effect is associated with a blockade of cell proliferation. Using 13C-serine isotope tracing, we could showed that the inhibitors significantly reduced ATP production from serine in CLL cells, probably resulting in altered nucleotides biosynthesis. By performing rescue experiments with thymidine or hypoxanthine, we confirmed that the cytotoxic effect of the inhibitors on CLL cells is mediated through a defect in pyrimidine synthesis (C). Interestingly, we demonstrated that the resistant MM cell lines overexpressed the SHMT1 enzyme, and could be sensitized to MTHFD1/2 inhibition by combination therapy with SHMT1/2 inhibitor (D). Taken together these results suggest that SHMT1 may be a key player in the response to MTHFD1/2 inhibition. Furthermore, we have previously reported that CLL induces a dramatic remodeling of tumor microenvironment, notably leading to the accumulation of highly immunosuppressive Tregs (Wierz et al., Blood, 2018). We showed that both enzymes are overexpressed in those highly immunosuppressive Tregs, and that MTHD1/2 inhibitors are able to reduce the viability of ex vivo polarized Tregs. Image:Summary/Conclusion: MTHFD1/2 inhibitors display a high cytotoxic activity in CLL and other B-cell malignancies by impairing cell proliferation through a defect in pyrimidine synthesis. These inhibitors also exhibit cytotoxic effect on activated Treg cells ex vivo, reinforcing its therapeutic potential for CLL treatment.

Background: Small extracellular vesicles (sEV) are nano-sized particles released by every cell and found in all biofluids. Given their composition and abundance, sEV are commonly involved in cell-to-cell communication through the transfer of genetic material and proteins. Furthermore, sEV possess direct functions carried out by sEV-ligands capable to affect the biological functions of targeted cells. In cancer, tumor-derived sEV are involved in the re-education of microenvironment (ME) cells promoting tumor proliferation, immune escape and metastasis. We previously demonstrated that leukemia-derived sEV are involved in the re-education of surrounding cells and increased immune escape. Indeed, chronic lymphocytic leukemia (CLL)-derived sEV induce stromal cell conversion into cancer-associated fibroblasts (Paggetti et al., Blood, 2015), and modulate PD-L1 expression in monocytes (Haderk et al. Science Immunology, 2017). Aims: The goal of the present work was to characterize leukemia ME-derived sEV (LME-sEV) and to evaluate their role in the disease development and progression in vivo. Methods: To obtain a biological representation of sEV in CLL microenvironment, we isolated LME-sEV directly from spleens of leukemic mice, obtaining a complex mix of sEV released by both CLL and ME cells alike. Small EV characterization was performed using a wide range of techniques, including qPCR, mass spectrometry and single sEV flow cytometry (FC). The effect on target cells was evaluated both ex vivo and in vivo using high-throughput techniques, FC, qPCR and cytotoxic assay. Small EV impact on CLL development in vivo was evaluated by generating a novel preclinical mouse model in which sEV release is genetically impaired due to Rab27a/b knock-out. Finally, we analyzed the expression of sEV-related genes in a cohort of 144 CLL patients using qPCR followed by regression analysis. Results: LME-sEV showed a distinct proteome (A) and RNA contents compared to healthy counterparts (HCME-sEV), including miRNA enriched in the plasma of CLL patients. Furthermore, FC-based immune checkpoint (ICP) screening showed the presence of multiple ICP ligands anchored on CLL-derived sEV (CD20+ subset of LME-sEV) (B), while high expression of the corresponding ICP receptors was found on T cells from matching LME. We also found that LME-sEV are internalized by different T cell subsets, thus we performed in vivo and ex vivo functional studies to assess sEV impact on T cells. High-throughput analysis of cells isolated from spleens of control mice treated with LME-sEV revealed considerable physiological changes mainly in CD8+ T cells. Indeed, CD8+ T cells showed alterations in their transcriptome, proteome and metabolome leading to cell exhaustion, decreased functions and survival. In line with this, absence of sEV dramatically delayed CLL progression in vivo. This effect was due to CLL inability to escape immune surveillance in absence of sEV and this was rescued by LME-sEV treatment (C). Finally, we identified a consistent sEV gene signatures in CLL patients correlating with treatment-free survival, overall survival, and with unfavorable clinical parameters routinely used in CLL diagnosis and prognosis (D). Image:Summary/Conclusion: By using different preclinical murine models and strategies, our results demonstrated for the first time that sEV in CLL ME play a key pro-tumoral role in leukemia development by negatively affecting the anti-tumor immune response. Furthermore, high expression of sEV-related genes correlated with poor survival and clinical parameters in CLL patients, suggesting sEV profiling as prognostic tool in CLL.

Natural killer (NK) cells are innate effector lymphocytes with strong antitumor effects against hematologic malignancies such as chronic lymphocytic leukemia (CLL). However, NK cells fail to control CLL progression on the long term. For effective lysis of their targets, NK cells use a specific cell-cell interface, known as the immunological synapse (IS), whose assembly and effector function critically rely on dynamic cytoskeletal changes in NK cells. Here we explored the role of CLL cell actin cytoskeleton during NK cell attack. We found that CLL cells can undergo fast actin cytoskeleton remodeling which is characterized by a NK cell contact-induced accumulation of actin filaments at the IS. Such polarization of the actin cytoskeleton was strongly associated with resistance against NK cell-mediated cytotoxicity and reduced amounts of the cell-death inducing molecule granzyme B in target CLL cells. Selective pharmacological targeting of the key actin regulator Cdc42 abrogated the capacity of CLL cells to reorganize their actin cytoskeleton during NK cell attack, increased levels of transferred granzyme B and restored CLL cell susceptibility to NK cell cytotoxicity. This resistance mechanism was confirmed in primary CLL cells from patients. In addition, pharmacological inhibition of actin dynamics in combination with blocking antibodies increased conjugation frequency and improved CLL cell elimination by NK cells. Together our results highlight the critical role of CLL cell actin cytoskeleton in driving resistance against NK cell cytotoxicity and provide new potential therapeutic point of intervention to target CLL immune escape.

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.

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.

Extracellular vesicles (EV), comprising microvesicles and exosomes, are particles released by every cell of an organism, found in all biological fluids, and commonly involved in cell-to-cell communication through the transfer of cargo materials such as miRNA, proteins, and immune-related ligands (e.g., FasL and PD-L1). An important characteristic of EV is that their composition, abundance, and roles are tightly related to the parental cells. This translates into a higher release of characteristic pro-tumor EV by cancer cells that leads to harming signals toward healthy microenvironment cells. In line with this, the key role of tumor-derived EV in cancer progression was demonstrated in multiple studies and is considered a hot topic in the field of oncology. Given their characteristics, tumor-derived EV carry important information concerning the state of tumor cells. This can be used to follow the outset, development, and progression of the neoplasia and to evaluate the design of appropriate therapeutic strategies. In keeping with this, the present brief review will focus on B-cell malignancies and how EV can be used as potential biomarkers to follow disease progression and stage. Furthermore, we will explore several proposed strategies aimed at using biologically engineered EV for treatment (e.g., drug delivery mechanisms) as well as for impairing the biogenesis, release, and internalization of cancer-derived EV, with the final objective to disrupt tumor–microenvironment communication.