Chronic Lymphocytic Leukemia Microenvironment Research Articles

Cross-talk between leukemic and immune cells at the tumor microenvironment in chronic lymphocytic leukemia: An update review.

Chronic lymphocytic leukemia (CLL) is a mature-type B cell malignancy correlated with significant changes and defects in both the innate and adaptive arms of the immune system, together with a high dependency on the tumor microenvironment. Overall, the tumor microenvironment (TME) in CLL provides a supportive niche for leukemic cells to grow and survive, and interactions between CLL cells and the TME can contribute to disease progression and treatment resistance. Therefore, the increasing knowledge of the complicated interaction between immune cells and tumor cells, which is responsible for immune evasion and cancer progression, has provided an opportunity for the development of new therapeutic approaches. In this review, we outline tumor microenvironment-driven contributions to the licensing of immune escape mechanisms in CLL patients.

Unraveling the Bone Tissue Microenvironment in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in Western countries. Although characterized by the progressive expansion and accumulation of leukemic B cells in peripheral blood, CLL cells develop in protective niches mainly located within lymph nodes and bone marrow. Multiple interactions between CLL and microenvironmental cells may favor the expansion of a B cell clone, further driving immune cells toward an immunosuppressive phenotype. Here, we summarize the current understanding of bone tissue alterations in CLL patients, further addressing and suggesting how the multiple interactions between CLL cells and osteoblasts/osteoclasts can be involved in these processes. Recent findings proposing the disruption of the endosteal niche by the expansion of a leukemic B cell clone appear to be a novel field of research to be deeply investigated and potentially relevant to provide new therapeutic approaches.

Regulatory T cells contribute to the immunosuppressive phenotype of neutrophils in a mouse model of chronic lymphocytic leukemia

BackgroundImpaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL.MethodsIn this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg.ResultsThe obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the “healthy” phenotype of neutrophils.ConclusionsAltogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response.

Targeting conditioned media dependencies and FLT-3 in chronic lymphocytic leukemia

AbstractThe importance of the stromal microenvironment in chronic lymphocytic leukemia (CLL) pathogenesis and drug resistance is well established. Despite recent advances in CLL therapy, identifying novel ways to disrupt interactions between CLL and its microenvironment may identify new combination partners for the drugs currently in use. To understand the role of microenvironmental factors on primary CLL cells, we took advantage of an observation that conditioned media (CM) collected from stroma was protective of CLL cells from spontaneous cell death ex vivo. The cytokine in the CM-dependent cells that most supports CLL survival in short-term ex vivo culture was CCL2. Pretreatment of CLL cells with anti-CCL2 antibody enhanced venetoclax-mediated killing. Surprisingly, we found a group of CLL samples (9/23 cases) that are less likely to undergo cell death in the absence of CM support. Functional studies revealed that CM-independent (CMI) CLL cells are less sensitive to apoptosis than conventional stroma-dependent CLL. In addition, a majority of the CMI CLL samples (80%) harbored unmutated immunoglobulin heavy-chain variable (IGHV) region. Bulk-RNA sequence analysis revealed upregulation of the focal adhesion and RAS signaling pathways in this group, along with expression of fms-like tyrosine kinase 3 (FLT3) and CD135. Treatment with FLT3 inhibitors caused a significant reduction in cell viability among CMI samples. In summary, we were able to discriminate and target 2 biologically distinct subgroups of CLL based on CM dependence with distinct microenvironmental vulnerabilities.

A tumor microenvironment model of chronic lymphocytic leukemia enables drug sensitivity testing to guide precision medicine

The microenvironment of chronic lymphocytic leukemia (CLL) cells in lymph nodes, spleen, and bone marrow provides survival, proliferation, and drug resistance signals. Therapies need to be effective in these compartments, and pre-clinical models of CLL that are used to test drug sensitivity must mimic the tumor microenvironment to reflect clinical responses. Ex vivo models have been developed that capture individual or multiple aspects of the CLL microenvironment, but they are not necessarily compatible with high-throughput drug screens. Here, we report on a model that has reasonable associated costs, can be handled in a regularly equipped cell lab, and is compatible with ex vivo functional assays including drug sensitivity screens. The CLL cells are cultured with fibroblasts that express the ligands APRIL, BAFF and CD40L for 24 h. The transient co-culture was shown to support survival of primary CLL cells for at least 13 days, and mimic in vivo drug resistance signals. Ex vivo sensitivity and resistance to the Bcl-2 antagonist venetoclax correlated with in vivo responses. The assay was used to identify treatment vulnerabilities and guide precision medicine for a patient with relapsed CLL. Taken together, the presented CLL microenvironment model enables clinical implementation of functional precision medicine in CLL.

Abstract 2516: Analysis of CLL Celllular Environment and Response (ACCER) is a novel method to understand the microenvironment in CLL

Abstract Microenvironments such as lymph nodes and bone marrow allow chronic lymphocytic leukemia (CLL) cells to survive after drug treatments. There are limited methods to study the to study the contribution of the microenvironment. We have adapted a solid tumour microenvironment cell culture system that provides elements of the CLL microenvironment called Analysis of CLL Cellular Environment and Response (ACCER). We optimized the cell number for patient’s primary CLL cells and HS-5 human bone marrow stromal cell line that will give sufficient cell number and viability with the ACCER. We then determined the amount of collagen type 1 to give the best extracellular matrix to seed CLL cells to the membrane. Finally, we determined that ACCER provide CLL cell protection against cell death following treatment with fludarabine and ibrutinib compared to co-culture conditions. This describes novel microenvironment model to investigate factors that promote drug resistance and cell survival in CLL. In the future, this will further validate new treatment strategies to overcome microenvironmental CLL cell survival signals. Citation Format: Spencer Gibson, Tricia Choquette, Elizabeth S. Henson, Xioyan Yang, James B. Johnston. Analysis of CLL Celllular Environment and Response (ACCER) is a novel method to understand the microenvironment in CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2516.

NF-kB and the CLL microenvironment.

Chronic lymphocytic leukemia (CLL) is the most prevalent type of leukemia in the western world. Despite the positive clinical effects of new targeted therapies, CLL still remains an incurable and refractory disease and resistance to treatments are commonly encountered. The Nuclear Factor-Kappa B (NF-κB) transcription factor has been implicated in the pathology of CLL, with high levels of NF-κB associated with disease progression and drug resistance. This aberrant NF-κB activation can be caused by genetic mutations in the tumor cells and microenvironmental factors, which promote NF-κB signaling. Activation can be induced via two distinct pathways, the canonical and non-canonical pathway, which result in tumor cell proliferation, survival and drug resistance. Therefore, understanding how the CLL microenvironment drives NF-κB activation is important for deciphering how CLL cells evade treatment and may aid the development of novel targeting therapeutics. The CLL microenvironment is comprised of various cells, including nurse like cells, mesenchymal stromal cells, follicular dendritic cells and CD4+ T cells. By activating different receptors, including the B cell receptor and CD40, these cells cause overactivity of the canonical and non-canonical NF-κB pathways. Within this review, we will explore the different components of the CLL microenvironment that drive the NF-κB pathway, investigating how this knowledge is being translated in the development of new therapeutics.

A novel method to investigate drug resistance in the chronic lymphocytic leukemia (CLL) microenvironment: Analysis of CLL Cellular Environment and Response (ACCER)

Microenvironments such as lymph nodes allow chronic lymphocytic leukemia (CLL) cells to survive and become drug resistant. There are limited methods to study the to study the contribution of the stromal microenvironment. We have adapted a solid tumor microenvironment cell culture system that provides elements of the CLL microenvironment called Analysis of CLL Cellular Environment and Response (ACCER). We optimized the cell number for patient’s primary CLL cells and HS-5 human bone marrow stromal cell line that will give sufficient cell number and viability with the ACCER. We then determined the amount of collagen type 1 to give the best extracellular matrix to seed CLL cells to the membrane. Finally, we determined that ACCER provide CLL cell protection against cell death following treatment with fludarabine and ibrutinib compared to co-culture conditions. This describes novel microenvironment model to investigate factors that promote drug resistance in CLL.

Targeting Inflammatory Pathways to Reverse Immunosuppressive Tumor Microenvironment in Chronic Lymphocytic Leukemia

Targeting Inflammatory Pathways to Reverse Immunosuppressive Tumor Microenvironment in Chronic Lymphocytic Leukemia

Longitudinal Profiling of the T Cell Compartment in Patients with Chronic Lymphocytic Leukemia Treated with Venetoclax

Venetoclax (ven), an inhibitor of the BCL2 anti-apoptotic protein, has been proven remarkably effective in the treatment of patients with chronic lymphocytic leukemia (CLL), where the malignant B cells typically overexpress BCL2. BCL2 is also expressed by various bystander cells in the CLL microenvironment, including T cells. Hence, investigating the effects of ven treatment on T cells is highly relevant, especially considering that distinct T cell subpopulations exhibit different sensitivity to anti-apoptotic signaling blockade. Herein we interrogated the molecular and phenotypic profiles of the T cell compartment in 1 treatment-naïve and 15 relapsed/refractory CLL patients who received ven either as monotherapy (n=12) or in combination with an anti-CD20 antibody (obinutuzumab, n=1; rituximab, n=3). We studied peripheral blood samples collected prior to ven initiation and after 5 months of treatment, from which we isolated T cell subpopulations (CD3+ T cells n=8; CD4+ and CD8+ T cells, n=8). Longitudinal analysis of pre/post-treatment samples by flow cytometry (n=7) revealed a significant (p=0.01) decrease in the numbers of central memory (CD45RO+/CCR7+) CD4+ (21.8% vs 8.8%) and CD8+ cells (21.4% vs 9.7%) under ven; in contrast, a significant (p=0.01) increase of CD8+ effector memory cells (CD45RO+/CCR7-) was observed (27.8% vs 32.9%). Profiling of the T cell receptor beta (TRB) chain gene repertoire overtime (n=15) was performed by next generation sequencing (NGS). Clonotypes (i.e. TRBV-TRBD-TRBJ gene rearrangements with unique pairs of TRBV genes and identical CDR3 amino acid sequences) were calculated and clonality was estimated as the average cumulative frequency of the 10 most frequent clonotypes per sample (ACF-10). A significantly (p=0.01) more diverse repertoire was identified in CD4+ vs CD8+ cells at both pre- and post-treatment timepoints (average numbers of clonotypes pre-/post-treatment: CD4+ cells, 16,908/13,768; CD8+ cells, 4,437/3,633). Expanded clonotypes corresponding to oligoclonal expansions were documented in all examined subpopulations at both timepoints. However, significantly (p=0.01) more pronounced skewing was observed in CD8+ vs CD4+ cells (pre-/post-treatment ACF-10 values: 64.4%/70.6% vs 31.4%/42.2%, respectively). Repertoire comparisons overtime highlighted a fraction of T clonotypes that were retained after treatment in all examined subpopulations. Of note, repertoire conservation was significantly (p=0.01) greater in CD8+ vs CD4+ T cells [retained pre-treatment clonotypes: 7,102/31,063 (28%) vs 11,525/118,359 (12%), respectively]. Transcriptome profiling of CD4+ and CD8+ cells pre-/post-treatment was performed using the Oncomine Immune Response Research Assay (395 genes) in 5 cases attaining a complete response after treatment. While the transcriptomes of CD4+ cells remained essentially unaltered, significant differences were identified in CD8+ cells, where 19 genes were found to be downregulated (adj.p<0.05, fold change>2). These genes are implicated in functions such as immune response (e.g. TLR7, TLR9, CD40), regulation of immune system (e.g. HLA genes, CIITA, TNFAIP8) and lymphocyte activation pathways (e.g. ICOSLG, CEACAM1, CXCR5), with at least some of these having immunosuppressive functions (e.g. BTLA and CTLA4 encoding inhibitory receptors associated with T cell exhaustion). In conclusion, ven treatment led to increased numbers of CD8+ effector subpopulations along with prominent clonal expansions and transcriptional rewiring that could conceivably contribute to clinical response. Downregulation of immune-related genes implicated in core regulatory/activation pathways supports immune recovery by ven, offering a rationale for future combination strategies aiming to increase the depth of clinical response.

P603: GUT MICROBIOME IN CHRONIC LYMPHOCYTIC LEUKEMIA SHOWS DEPLETION OF SHORT-CHAIN FATTY ACIDS PRODUCING BACTERIA

Background: Recent studies have shown extensive crosstalk between our immune system and gut microbiome (GM). The host immune system plays a vital role in the maintenance of GM homeostasis by 1) establishing a balance between eliminating invading pathogens and promoting the growth of beneficial microbes, 2) producing short-chain fatty acids (SCFA), the main source of nutrition for the colon cells, and 3) modulating the immune system by cytokine production. Mounting evidence shows that the GM of patients with high rates of infection are characterized by an imbalance of bacteria, inducing proinflammatory states and reduced capacity for SCFA synthesis. As chronic lymphocytic leukemia (CLL) is, among others, also characterized by high rate of infectious complications and an altered immune system, it is warranted to explore composition of the CLL microbiome. Aims: We aim to investigate the hypothesis that deviation of the GM from homeostasis, i.e. loss of ‘health promoting’ gut microbes and/or overgrowth of pathogenic bacteria, distinguishes patients with CLL from the background population. Methods: Feces samples of patients with CLL were collected, immediately fixated and frozen within 72 h; total genomic DNA was sequenced. Feces samples of healthy controls were chosen to match the CLL population with respect to age, demographic data, sample collection method, and sequencing platform. Taxonomical profiling was done using an in-house bioinformatics pipeline. Results: A total of 61 CLL patients and 30 healthy individuals were included in the study. We observed reduced GM alpha diversity, and depletion of bacterial members of Lachnospiraceae and Ruminococcaceae families among the CLL patients when compared to healthy individuals. Our data show that members of the Lachnospiraceae family (Anaerostipes hadrus, Coprococcus comes, Blautia spp., Dorea spp.), and 3 members of Ruminococcaeae family (Ruminococcus torques, Ruminococcus bromii, Faecalibacterium prausnitzii) were among the most differentially abundant bacterial species between the microbiomes of healthy individuals and CLL. Their mean proportions were shown to be significantly higher in healthy microbiome samples. As further differentially abundant species we observed Bacteroides sp. and Alistipes finegoldii, which both demonstrated significantly higher mean proportions in CLL microbiomes (Fig 1). Image:Summary/Conclusion: To sum up, the CLL microbiomes in comparison to healthy controls demonstrated lower enrichment of Lachnospiraceae and Ruminococcaceae families, the major SCFAs-producing bacterial taxa reported to have a protective effect against inflammation. This supports the notion that proinflammatory risk factors identified in other cohorts with GM dysbiosis are also present within CLL patients. As CLL represents an antigen driven malignancy with immune dysfunction, we hypothesize that GM dysbiosis could both be implicated in the pathogenesis of CLL, through antigenic drive, and contribute to the distortion of the immune system in CLL. Notably, both immune dysfunction and treatment (e.g. antimicrobials) may influence the CLL microbiome itself and therefore confound cross-sectional clinical studies. We therefore plan to investigate the interaction between microbiome and CLL development in the TCL1 mouse model of CLL. Also, identification of potential mechanistic links between the GM and the microenvironment in CLL should be investigated, e.g. extravesicular vesicles or cytokines released from or impacted by the GM. In addition, modulation of the microbiome in animal models may help to establish causal connections between the GM and CLL development.

S137: SMALL EXTRACELLULAR VESICLES IN THE LEUKEMIA MICROENVIRONMENT SUSTAIN CLL PROGRESSION BY HAMPERING T CELL-MEDIATED ANTI-TUMOR IMMUNITY

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.

P607: A NOVEL LYMPH NODE-MIMICKING 3D CULTURE SYSTEM DISPLAYS LONG-TERM T CELL-DEPENDENT CLL PROLIFERATION AND SURVIVAL

Background: Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in the absence of microenvironmental survival signals1. No current system permits long-term expansion of CLL cells in vitro due to difficulties of mimicking a physiological microenvironment. The lymph node (LN) is the critical site of in vivo CLL proliferation and is also thought to play a key role in the development of resistance to targeted agents2,3. Developing an in vitro culture system for CLL with pathophysiological relevance will greatly benefit studies of CLL proliferation, microenvironment, clonal outgrowth and relevant drug screening. Aims: To design an in vitro CLL culture system that incorporates key aspects of the CLL LN; contribution of non-CLL cells and cytokines, and induction of drug resistance and prolonged proliferation of CLL cells. Methods: Primary CLL cells were cultured in ultra-low attachment (ULA) plates in parallel to standard 2D cultures. PBMCs were cultured with or without T cells (in a specific CLL:T cell ratio to mimic the LN composition), or PBMCs were co-cultured with primary lymph node fibroblasts. CLL cells were either stimulated directly with a B cell cocktail (BCC) consisting of IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28. Results: Compared to 2D cultures, 3D cultures showed several important CLL LN features. First, significantly increased CLL proliferation independent of IGHV mutation status, which was abrogated in the absence of T cells, or by JAK inhibitors. Notably, treatment with BTK inhibitors significantly inhibited CLL proliferation and resulted in disintegration of the 3D spheroid architecture. Second, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations. Third, 3D cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the 3D model. Fourth, when PBMCs were stimulated with BCC, spheroids developed proliferation center-like structures after 4 weeks of culture where T cells localized together with enrichment of Ki-67+ CLL cells. Finally, either B or T cell stimulation resulted in an induction of venetoclax resistance, showing that T cells are able to confer drug resistance to CLL cells in this model. Summary/Conclusion: We present a 3D culture system that underlines the role of T cells in sustained CLL proliferation, permitting investigation of CLL cells in the context of a protective niche consisting of multiple cell types, thereby opening up new avenues for clinically useful applications.

Role of the splenic microenvironment in chronic lymphocytic leukemia development in Eµ-TCL1 transgenic mice

The chronic lymphocytic leukemia (CLL) microenvironment has been receiving an increasing amount of attention, but there is currently limited data surrounding how the microenvironment affects initial development of CLL. We determined that the spleen is the initial site of CLL growth through monitoring of transgenic Eμ-TCL1 mice that develop CLL. Subsequently, we isolated stromal cells from the spleens of Eμ-TCL1 mice (EMST cells) that induce CLL cell division in vitro. Both cell–cell contact and soluble factors were involved in EMST-induced CLL cell division. These stromal cells are present in significantly larger numbers in the spleen than other lymphoid organs. We also noted that splenectomy delayed CLL development in Eμ-TCL1 mice and completely prevented CLL development in adoptive transfer mice. Our findings will allow future studies surrounding the CLL microenvironment to focus upon the splenic stromal cells.

Reduced Percentage of CD14dimCD16+SLAN+ Monocytes Producing TNF and IL-12 as an Immunological Sign of CLL Progression.

Monocytes are one of the least studied immune cells with a potentially important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Nevertheless, data regarding the role of subpopulations of monocytes in the CLL microenvironment are still limited. For the very first time, this study presents an assessment of monocyte subsets divided according to SLAN and CD16 expression in CLL patients. The study involved 70 freshly diagnosed CLL patients and 35 healthy donors. Using flow cytometry, monocyte subpopulations were assessed among PBMCs. CD14+ monocytes can be divided into: “classical” (CD14+CD16−SLAN−), “intermediate” (CD14+CD16+SLAN−) and “non-classical” (CD14dimCD16+SLAN+). In our study, we noted an increased percentage of non-classical monocytes with intracellular expression of TNF and IL-12. On the other hand, among the intermediate monocytes, a significantly higher percentage of cells synthesizing anti-inflammatory IL-10 was detected. The percentage of CD14dimCD16+SLAN+ monocytes producing TNF and IL-12 decreased with the stage of CLL and inversely correlated with the expression of the prognostic factors ZAP-70 and CD38. Moreover, the percentage of CD14dimCD16+SLAN+ monocytes producing TNF and IL-12 was lower in CLL patients requiring treatment. This may indicate the beneficial effect of non-classical monocytes on the anti-tumor response.

Aberrantly expressed Wnt5a in nurse-like cells drives resistance to Venetoclax in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of neoplastic B lymphocytes with high levels of Wnt5a in the plasma. Currently, the cell source of Wnt5a remains controversial. The receptor of Wnt5a is ROR1, whose expression is associated with disease progression and resistance to venetoclax, a BCL-2 inhibitor approved for the treatment of CLL. In this study, we found that the levels of Wnt5a in the plasma of CLL patients were positively correlated with absolute monocyte counts, but not lymphocyte counts. We cultured monocyte-derived nurse-like cells (NLCs) from patients with CLL, and detected Wnt5a expressed in NLCs. Flow cytometry and transwell assays showed that the antibody neutralizing Wnt5a inhibited the enhanced survival and migration in CLL cells co-cultured with NLCs. Furthermore, we performed a drug screening with CLL cells cultured with or without NLCs with a library containing 133 FDA-approved oncology drugs by using high-throughput flow cytometry. We observed a significant resistance to venetoclax in CLL cells co-cultured with NLCs. Immunoblot revealed the activation of NF-κB with enhanced expression of MCL-1 and BCL-XL in CLL cells co-cultured with NLCs. Neutralizing Wnt5a or blocking NF-κB pathway significantly decreased the expression of MCL-1 and BCL-XL, which leads to enhanced sensitivity to venetoclax in CLL cells co-cultured with NLCs. In conclusion, our data showed that NLCs could be one of the sources of Wnt5a detected in patients with CLL, and Wnt5a-induced NF-κB activation in the CLL microenvironment results in resistance to venetoclax in CLL cells.

Targeting Metabolic Alterations in CLL Microenvironment; Inhibition of Glutamine Import Attenuates Venetoclax Resistance

Introduction: For chronic lymphocytic leukemia (CLL), especially in the lymph node (LN) setting where cells receive proliferative and pro-survival signals, in-depth studies of altered metabolism and its relationship with therapeutic responses are still lacking. Venetoclax, a BCL-2 inhibitor currently in wide clinical use for CLL, has shown high efficiency yet emerging resistance is a growing clinical problem. In cell line models, induced resistance to Venetoclax was accompanied by profound metabolic changes 1. This is in accordance with our earlier findings on metabolic and apoptotic changes that CLL cells undergo within the LN environment 2. In the current study, we performed RNA sequencing and applied fluxomics with 13C 6-glucose and 13C 5-glutamine to investigate in detail the metabolic routes in LN CLL. This led to studies to manipulate glutamine metabolism in a venetoclax resistance model.Methods: Peripheral blood (PB) samples from CLL patients were in vitro stimulated for 24 hrs by CD40 or B cell receptor (BCR), which are two potential key signals in LN. RNAseq analysis was compared with microarray data of paired PB/LN patient samples 3. For fluxomics, CLL cells were cultured for 2 hrs in medium containing either 5 mM 13C 6-glucose or 1 mM 13C 5-glutamine. Incorporation of 13C in metabolic intermediates was analyzed by LC-MS. For glutamine blockade, CLL cells were stimulated in presence of specific inhibitors of glutamine/glutamate metabolism or amino acid transporters. Cells were then treated with venetoclax, and viability was measured.Results: Gene expression profiles demonstrated that CLL cells obtained from LN tissue as well as after in vitro CD40 or BCR stimulation showed increased expression of gene sets involved in glycolysis, oxidative phosphorylation / citric acid cycle (OXPHOS/TCA) and amino acid metabolism as well as Myc activation. This confirmed that in vitro stimulation can be used to model the CLL LN setting. For unstimulated PB CLL cells, fluxomics data demonstrated low uptake of either glucose or glutamine, with 13C labelling close to zero for most metabolites. In contrast, both CD40 or BCR stimulation increased the uptake and utilization of glucose and glutamine. 13C labelling from glucose was detected in all glycolytic intermediates analyzed in both CD40- and BCR-stimulated CLL cells. Glucose was catalyzed to lactate and also partly converted to acetyl-CoA, which entered the TCA cycle. Additionally, labelling from glucose was also increased in several metabolites of the pentose phosphate pathway (PPP) suggesting it entered nucleotide synthetic routes. Compared to glucose, the contribution of glutamine was much higher in the TCA cycle in both BCR and CD40-stimulated cells. All intermediates of the TCA cycle were highly enriched with 13C from glutamine (Figure 1A). Combined, these data revealed that glutamine is the key metabolite to fuel the TCA cycle in LN CLL cells, and prompted us to study effects of glutamine blockade in conditions of Venetoclax resistance. It was found that venetoclax resistance induced by CD40 or BCR stimulation was clearly attenuated by glutamine uptake inhibition. CLL cells became re-sensitized to Venetoclax in both CD40- or BCR-stimulated samples, with an approximate 100-fold shift in IC50 (Figure 1B).Conclusions: Our study highlights the role of glutamine, in addition to glucose, in the metabolic reprogramming that CLL cells undergo in the LN (Figure 1C). These processes show potential for therapeutic targeting. Inhibition of glutamine import could contribute to dampen tumor microenvironment-induced Venetoclax resistance.

BTK Inhibitors in Chronic Lymphocytic Leukemia: Biological Activity and Immune Effects

Bruton´s tyrosine kinase (BTK) inhibitor (BTKi)s block the B-cell receptor (BCR) signaling cascade by binding to the BTK enzyme preventing the proliferation and survival of malignant and normal B cells. During the past decade, the clinical use of BTKis for the treatment of B-cell malignancies has exponentially grown, changing the treatment landscape for chronic lymphocytic leukemia (CLL) in particular. At present, three different covalent BTKis, ibrutinib, acalabrutinib and zanubrutinib, are FDA-approved and many new inhibitors are under development. Despite having remarkable selectivity for BTK, the first-in-class BTKi ibrutinib can also bind, with various affinities, to other kinases. The combined inhibition of BTK (“on-target” effect) and other kinases (“off-target” effect) can have additive or synergistic anti-tumor effects but also induce undesired side effects which might be treatment-limiting. Such “off-target” effects are expected to be more limited for second-generation BTKis. Moreover, the blockade of BCR signaling also indirectly affects the tumor microenvironment in CLL. Treatment with BTKis potentially impacts on both innate and adaptive immunity. Whether this affects infection susceptibility and vaccination efficacy requires further investigation. Here, we summarize the available knowledge on the impact of BTKis on the immune system and discuss the possible clinical implications. Indeed, a deeper knowledge on this topic could guide clinicians in the management and prevention of infections in patients with CLL treated with BTKis.

T Cell Defects and Immunotherapy in Chronic Lymphocytic Leukemia.

Simple SummaryThe treatment of chronic lymphocytic leukemia (CLL) is a rapidly evolving field; however, despite recent progress, CLL remains incurable. Different types of immunotherapy have emerged as therapeutic options for CLL, aiming to boost anti-tumor immune responses; that said, despite initial promising results, not all patients benefit from immunotherapy. Relevant to this, the tumor microenvironment (TME) in CLL has been proposed to suppress effective immune responses while also promoting tumor growth, hence impacting on the response to immunotherapy as well. T cells, in particular, are severely dysfunctional in CLL and cannot mount effective immune responses against the malignant cells. However, reinvigoration of their effector function is still a possibility under the proper manipulation and has been associated with tumor regression. In this contribution, we examine the current immunotherapeutic options for CLL in relation to well-characterized T cell defects, focusing on possible counteracts that enhance anti-tumor immunity through the available treatment modalities.In the past few years, independent studies have highlighted the relevance of the tumor microenvironment (TME) in cancer, revealing a great variety of TME-related predictive markers, as well as identifying novel therapeutic targets in the TME. Cancer immunotherapy targets different components of the immune system and the TME at large in order to reinforce effector mechanisms or relieve inhibitory and suppressive signaling. Currently, it constitutes a clinically validated treatment for many cancers, including chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes with great dependency on microenvironmental signals. Although immunotherapy represents a promising therapeutic option with encouraging results in CLL, the dysfunctional T cell compartment remains a major obstacle in such approaches. In the scope of this review, we outline the current immunotherapeutic treatment options in CLL in the light of recent immunogenetic and functional evidence of T cell impairment. We also highlight possible approaches for overcoming T cell defects and invigorating potent anti-tumor immune responses that would enhance the efficacy of immunotherapy.

PI3K-δ Inhibition Influences T-Cell Populations and Anti-Tumoral Immune Function in Preclinical Models

Chronic lymphocytic leukemia (CLL) is characterized by continuously activated B-cell receptor (BCR) signaling. Phosphoinositide 3-kinase delta (PI3K-δ) is an important mediator of BCR signaling, which controls cell survival and proliferation through activation of AKT. The PI3K-δ inhibitor idelalisib (IDELA) is a highly effective treatment option for relapsed/refractory CLL patients. In the frontline setting, IDELA in treatment-naïve patients was associated with frequent adverse effects (AEs), such as an increased infection rate, colitis, and pneumonitis. Therefore, understanding the impact of PI3K-δ inhibition on the immune cells in the CLL microenvironment is of importance to identify novel treatment strategies to overcome these AEs.Methods and Results: In this study, we specifically focused on the effect of PI3K-δ inhibition on T-cell function in CLL using the Eµ-TCL1 adoptive transfer (TCL1 AT) model. In brief, Eµ-TCL1 tumor cells were transferred intravenously into syngeneic littermates, followed by continuous treatment after reaching a tumor load of 50% in peripheral blood. GS-649443, a tool compound with more favorable murine pharmacokinetic properties than IDELA, was used for PI3K-δ inhibition in vivo . Tumor load, as well as the immune microenvironment, was analyzed after 2 weeks of continuous treatment of the drug.Since PI3K-δ signaling has been previously demonstrated to be important for CD4+ regulatory T-cell (Treg)-mediated suppression of antitumor immunity, we studied the impact of PI3K-δ inhibition on differentiation and activation of Tregs. GS-649443 treatment resulted in a dramatic reduction in numbers of Foxp3+ CD25+ CD4+ Tregs, as well as a decrease in Treg maturation and activation markers (CD103, GITR, KLRG1, and CD69) compared to vehicle treatment.We further assessed the impact of PI3K-δ inhibition on TCL1 AT-induced changes in CD8+ T-cell differentiation and subset distribution. Notably, treatment with GS-649443 resulted in a dose-dependent decrease in effector T-cell numbers, accompanied by a decrease in activation markers such as CD69 and CD137. In line with these findings, effector cell proliferation, as measured by Ki-67, significantly decreased in GS-649443-treated mice. In accordance with that, degranulation capacity, granzyme B, and IFNγ production by CD8+ T cells was substantially reduced by GS-649443 treatment. These results were further confirmed in a treatment study with PI-3065, another PI3K-δ-specific inhibitor.To investigate a direct impact of PI3K-δ inhibition on CD8+ T cells, we performed in vitro studies with GS-649443. For that purpose, we used splenocytes from Nur77-GFP mice, a reporter mouse model for T-cell receptor (TCR) activity, and stimulated these with aCD3 antibody after pre-treatment with GS-649443 or vehicle. PI3K-δ inhibition led to a profound reduction of TCR signaling, which was accompanied by decreased expression of activation markers like CD69, CD44, CD25, and CD137 on CD8+ T-cells. Furthermore, GS-649443 pre-treatment resulted in significant dose-dependent decrease in CD8+ T-cell proliferation rates, as measured by CFSE dilution. These findings were confirmed using aCD3-stimulated human peripheral blood mononuclear cells that were pretreated with IDELA in vitro, which resulted in an impairment of CD8+ T-cell activation measured by CD69 and CD25 expression, as well as proliferation measured by CFSE dilution.Conclusion: This study clearly shows that PI3K-δ inhibition abrogates the immunosuppressive activity of Tregs in an in vivo model of CLL. Additionally, PI3K-δ inhibition of CD8+ T cells leads to decreased activation and proliferation of these cells, which might contribute to the immune-mediated AEs observed with IDELA when used as frontline treatment. DisclosuresStilgenbauer:Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.