Development Of Chronic Lymphocytic Leukemia Research Articles

P598: EBI3/IL-27 DEPLETED MICROENVIRONMENT FAVOURS TUMOUR PROGRESSION IN CHRONIC LYMPHOCYTIC LEUKAEMIA

Background: Chronic Lymphocytic Leukaemia (CLL) is the most common adult leukaemia in western countries. Unfortunately, CLL remains an incurable disease, evidencing the urgency to develop novel effective treatments for this malignancy. CLL cells are known to be highly dependent on interactions with non-malignant cells in their tumour microenvironment (TME) for survival and proliferation. Such dependency highlights the potential of immunotherapy as a therapeutic approach in CLL. Recently, several pro-inflammatory cytokines have emerged as a powerful tool to reactivate the immune system in a wide range of malignancies. IL-27 is a member of the IL-12 family of heterodimeric cytokines reported to have pleiotropic functions during cancer development in different malignancies. Based on the existing literature, we wondered whether IL-27 affects the development and progression of CLL. Aims: The goal of this study is to elucidate the role of IL-27 in CLL development and progression. Methods: We used a constitutive knockout mouse model of the EBI3 subunit of IL-27 to abrogate IL-27 expression in vivo. We performed adoptive transfer (AT) of TCL1 derived CLL cells into Ebi3-/- and WT mice, as well as generated the transgenic mouse model Eµ-TCL1-Ebi3-/-.The splenic TME of these mice was characterized using flow cytometry. In addition, in vivo neutralization of IL-27 using a blocking antibody was conducted. Immunodeficient Rag2-/- mice were also injected with TCL1-derived CLL cells and either WT or Ebi3-/- CD3+ T cells to assess the role of T-cells in leukaemia control in the presence and absence of IL-27. Finally, the transcriptional differences among WT and Ebi3-/- T cells were assessed using bulk-RNA sequencing, and their impact on T cell cytotoxicity was investigated both in humans and mice using in vitro killing assays. The serum levels of IL-27 in CLL patients and sick mice were quantified via ELISA and compared to those of healthy controls. Results: A strikingly enhanced CLL development and survival reduction was observed when CLL was AT into Ebi3-/- mice, as well as in the transgenic Eµ-TCL1-Ebi3-/- mice when compared to controls (A). Consistently, flow cytometry analysis revealed an increasingly immunosuppressive splenic TME in the absence of EBI3, characterized by an enrichment of highly activated and immunosuppressive Tregs and a terminally exhausted CD8+ T cell subset. The aforementioned results were recapitulated in an IL-27 neutralization experiment (B). Using Rag2-/- mice, we demonstrated that T cells from EBI3-/- mice were less efficient in controlling CLL development (C). RNA sequencing revealed major changes in CD8+ T cells transcriptional program, in particular a decreased expression of crucial transporters (D). Finally, in vitro killing assays showed an increased cytotoxicity of both human and murine T cells in the presence of IL-27, while serum protein quantification showed a decrease in the levels of IL-27 in both CLL patients (E) and sick mice (F) when compared to their healthy counterparts. Image:Summary/Conclusion: Overall, our results demonstrate the antitumor role of IL-27 in CLL development and progression by promoting T-cell-mediated anti-tumor immunity, as well as establish this cytokine as a potential immunotherapeutic agent in CLL.

PB1857: NRF2 MODULATORS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: A POSSIBLE NEW THERAPEUTIC APPROACH

Background: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of resting clonal B lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. This disease has a notable heterogeneous clinical course, with different prognosis and distinct treatment needs. Oxidative stress (OS) is one of the mechanisms important in CLL development and progression. OS state consists in an unbalance between the production of reactive oxygen species (ROS) and the ability to neutralize them by antioxidant defenses. The nuclear factor erythroid 2–related factor 2, NRF2, is a transcription factor that regulates the expression of several antioxidants and detoxification systems and works as a protective mechanism in normal cells against malignant transformation. However, during the tumorigenic process, NRF2 protects tumor cells from OS effects compromising the therapeutic efficacy. Aims: The study aims to evaluate the therapeutic potential of two NRF2 modulators, Brusatol and ML385, in B cells from CLL patients. Methods: Peripheral blood mononuclear cells (PBMCs) from 13 CLL patients (stage initial, Binet A/B, n=8; advanced stage, Binet C, n=5) were isolated by density gradient using Ficoll-Paque Premium. PBMCs where immediately incubated in absence and presence of Brusatol and ML385. The cytotoxic effect was evaluated 24h and 48h after incubation by fluorometric microculture cytotoxicity assay. After 24h incubation with Brusatol (100nM) and ML385 (100μM) cells were stained with anti-CD5-PerCP, anti-CD19-APC and Annexin V, to assess cell death by flow cytometry. The statistical analysis was carried out in the total CLL patient group and in risk groups. The results were considered statistically significant when p<0.05. Results: CLL patients presented 93,3% (±3.7%) of neoplastic B cells (CD5+/CD19+), 3,7% (±3,0%) of normal B cells (CD5-/CD19+) and 2,7% (±1,2%) of normal T cells (CD5+/CD19-), with no statistical differences regarding staging groups. Brusatol induced a decreased survival rate in CLL cells comparing to ML385, being this effect dependent on the dose and disease stage. Brusatol induced a higher reduction of cell viability in PBMCs from advanced disease patients (Binet C). ML385 did not induced a significant decrease in cell survival, nevertheless its effect is more marked in advanced stage patients at 48h post incubation. IC50 was only achieved at 48h in advanced stage patients and corresponds to 65nM for Brusatol and to 114µM for ML385. NRF2 modulators induced a cytotoxic effect mainly mediated by apoptosis. Cytotoxicity induced by both drugs was more evident in neoplastic B cells, with no significant cytotoxicity in normal B and T cells. In PBMCs incubated with Brusatol an increase of neoplastic B cells in apoptosis about 68% was observed (Brusatol: 91,9% ± 7,1%; Control: 23,7% ± 4,3%), and of 34% in cells incubated with ML385 (ML385: 57,9% ± 10,2%; Control: 23,7% ± 4,3%). No differences of apoptotic cells were observed in advanced stage groups. Summary/Conclusion: Our results showed that neoplastic cells from CLL patients were sensitive to NRF2 modulators, suggesting that these drugs may constitute a new therapeutic approach in CLL patients, particularly in CLL patients in advanced stage. We also demonstrated the low cytotoxic effect of these modulators in normal lymphocytes and its higher efficacy in neoplastic cells from advanced stage patients. Altogether, these results show that these NRF2 modulators have an effect directed to the neoplastic cell and suggest a possible involvement of NRF2 on CLL development and progression. # equally contributing authors.

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.

S138: MICROENVIRONMENT-REGULATED TRANSCRIPTIONS FACTORS AHR AND HIF-1Α EXPRESSION IN TREGS PROMOTE CLL PROGRESSION BY IMPAIRING CD8+ T-CELL MEDIATED ANTI-TUMOR IMMUNITY

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. Aims: The aim of the present study is to investigate the role of AHR and HIF-1α in the suppressive ability of Tregs during CLL development. Methods: We generated conditional knock out mice (cKO) lacking Ahr or Hif1a genes exclusively in Tregs (Foxp3YFP-CreAhrfx/fx and Foxp3YFP-CreHif1afx/fx mice). We then performed adoptive transfer (AT) of CLL cells obtained from diseased Eµ-TCL1 mice into cKO and control mice. In order to decipher the mechanism by which AHR and HIF-1α pathways in Tregs affect CLL progression, we analyzed the splenic TME of recipient mice and evaluated immune checkpoint expression and cytokine production. Finally, we evaluated the suppression ability of the regulatory T cells with ex vivo suppression assays. Results: Generated cKO mice showed no sign of abnormalities or autoimmune phenotypes, and immune cells phenotyping revealed no major differences. However, we showed that CLL growth in cKO mice was drastically delayed compared to the control mice (A). Interestingly, this decrease was mitigated when CD8+ T cells were depleted. The analysis of the splenic TME in recipient cKO mice revealed an increase in IL-17 and TNF-α production, two major T-cell cytokines, in CD4+ T cells as compared to their WT counterparts. We also measured the expression of immune checkpoints and activation markers in the different T cell subpopulations and observed that Tregs lacking AHR or HIF-1α show decreased levels of the immune checkpoints CTLA-4 and TIGIT, two key proteins for the suppressive functions of Tregs. Ex vivo suppression assays demonstrated an increased proliferation of CD8+ T cells in the presence cKO Tregs (B-C). This result confirmed the decreased suppressive ability of Tregs in absence of the two transcription factors, explaining the observed delay in CLL progression in cKO mice. Image:Summary/Conclusion: Altogether, these results indicate that the TME-regulated transcription factors AHR and HIF-1α in Tregs are crucial for CLL development by promoting escape to anti-tumor immune response, and therefore represent potential therapeutic targets during CLL progression.

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.

Immunoglobulin Gene Sequence as an Inherited and Acquired Risk Factor for Chronic Lymphocytic Leukemia.

Simple SummaryChronic lymphocytic leukemia (CLL) is the most prevalent among adult leukemias. Over the years, several research efforts discovered a lot of intricate details about the cause of the disease, its mechanism, and the prognostic factors that help to understand the progression and outcome of the disease. Mutations in the immunoglobulin gene sequences in B cells are the most important prognostic factor for CLL. The cells having no to very less mutations show aggressive disease, while those having more mutations are either fairly indolent or non-aggressive. In this review, we discussed the current gain of knowledge about these mutations and their effects in the overall disease pathology.Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease characterized by the accumulation of CD5+ CD19+ malignant B cells. Autonomous ligand-independent B-cell signaling is a key process involved in the development of CLL pathogenesis. Together with other cytogenetic alterations, mutations in the immunoglobulin heavy chain variable (IGHV) gene act as a prognostic marker for CLL, with mutated CLL (M-CLL) being far more indolent than unmutated CLL (U-CLL). Recent studies highlight the role of a specific light chain mutation, namely, IGLV3-21R110G, in the development and prognosis of CLL. Such a mutation increases the propensity of homotypic BCR–BCR interaction, leading to cell autonomous signaling. In this article, we review the current findings on immunoglobulin gene sequence mutations as a potential risk factor for developing CLL.

Pre-diagnostic trajectories of lymphocytosis predict time to treatment and death in patients with chronic lymphocytic leukemia

BackgroundThe dynamics of pre-diagnostic lymphocytosis in patients with ensuing chronic lymphocytic leukemia (CLL) need to be explored as a better understanding of disease progression may improve treatment options and even lead to disease avoidance approaches. Our aim was to investigate the development of lymphocytosis prior to diagnosis in a population-based cohort of patients with CLL and to assess the prognostic information in these pre-diagnostic measurements.MethodsAll patients diagnosed with CLL in the Greater Copenhagen area between 2008 and 2016 were included in the study. Pre-diagnostic blood test results were obtained from the Copenhagen Primary Care Laboratory Database encompassing all blood tests requested by Copenhagen general practitioners. Using pre-diagnostic measurements, we developed a model to assess the prognosis following diagnosis. Our model accounts for known prognostic factors and corresponds to lymphocyte dynamics after diagnosis.ResultsWe explore trajectories of lymphocytosis, associated with known recurrent mutations. We show that the pre-diagnostic trajectories are an independent predictor of time to treatment. The implementation of pre-diagnostic lymphocytosis slope groups improved the model predictions (compared to CLL-IPI alone) for treatment throughout the period. The model can manage the heterogeneous data that are to be expected from the real-world setting and adds further prognostic information.ConclusionsOur findings further knowledge of the development of CLL and may eventually make prophylactic measures possible.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

Role of Interleukin-10 (1082G/A) and Splicing Factor 3B Subunit 1 (2098A/G) Gene Polymorphisms in Chronic Lymphocytic Leukemia

OBJECTIVE: Interleukin-10 (IL-10) gene polymorphisms might play a part in the development of some malignant tumors. It has been linked with high bcl-2 expression in some B-lymphocyte malignancies. Its relationship with chronic lymphocytic leukemia (CLL) development is still under investigation. Other studies have linked Splicing Factor 3B Subunit 1 (SF3B1) mutations to a poorer prognosis of CLL. From this context, we have great interest to investigate the effect of both IL-10 (1082G/A) and SF3B1 (2098A/G) gene polymorphisms on CLL in this study. MATERIALS AND METHODS: Peripheral blood mononuclear cells were analyzed for IL-10 (1082G/A) and SF3B1 (2098A/G) gene polymorphisms by real-time quantitative polymerase chain reaction in 80 newly diagnosed CLL patients and 80 controls. RESULTS: Our results showed that the IL-10 (G/A) genotype, IL-10 (A/A) genotype and IL-10 A allele and SF3B1 (A/G) genotype and SF3B1 G allele were increased significantly in the patients group compared with the control group. CONCLUSION: IL-10 gene polymorphisms (1082 G/A and A/A) and A alleles might be associated with increased risk of CLL development compared with G/G genotypes and G alleles and are a probable risk factor for the disease. Also, our study demonstrated that SF3B1 (2098A/G) polymorphisms and G allele are related to and might be a causative factor for CLL.

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.

B-cell antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

Clonal Evolution of High-Risk Chronic Lymphocytic Leukemia: A Contemporary Perspective.

Clonal evolution represents the natural process through which cancer cells continuously search for phenotypic advantages that enable them to develop and expand within microenvironmental constraints. In chronic lymphocytic leukemia (CLL), clonal evolution underpins leukemic progression and therapeutic resistance, with differences in clonal evolutionary dynamics accounting for its characteristically diverse clinical course. The past few years have witnessed profound changes in our understanding of CLL clonal evolution, facilitated by a maturing definition of high-risk CLL and an increasing sophistication of next-generation sequencing technology. In this review, we offer a modern perspective on clonal evolution of high-risk CLL, highlighting recent discoveries, paradigm shifts and unresolved questions. We appraise recent advances in our understanding of the molecular basis of CLL clonal evolution, focusing on the genetic and non-genetic sources of intratumoral heterogeneity, as well as tumor-immune dynamics. We review the technological innovations, particularly in single-cell technology, which have fostered these advances and represent essential tools for future discoveries. In addition, we discuss clonal evolution within several contexts of particular relevance to contemporary clinical practice, including the settings of therapeutic resistance to CLL targeted therapy and immunotherapy, as well as Richter transformation of CLL to high-grade lymphoma.

R-Loop Associated Mitotic Stress Confers Vulnerabilities in Splicing Factor Mutant Leukemia

The RNA splicing factor SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL). Co-expression of mutant Sf3b1 and Atm deletionin murine B cellsresults in CLL development, in which CLL cells exhibit RNA splicing dysregulation, increased DNA damage and recurrent chromosomal amplification. These findings suggest a strong link between splicing factormutation, chromosomal instability (CIN), and oncogenesis, although the mechanism has yet to be elucidated.CIN in cells with defective RNA processing is triggered, at least in part, by excessive R-loop (RNA:DNA hybrid with displaced single strand DNA). Using dotblot assay, we observed R-loop augmentation in SF3B1 mutant (MT) compared to wild-type (WT) cells in both human cell lines and murine CLL cells. Along with R-loop accumulation, SF3B1 MT increased DNA double strand breaks (DSB) (measured by comet assay) and mis-segregated chromosomes during mitosis (quantified by immunofluorescence using anti-phospho-histone H3 and anti-αtubulin antibodies). Overexpression of R-loop resolving enzyme RNaseH1 in SF3B1 MT cell lines reduced DSB (t-test p-value <0.0001) and mitotic stress (t-test p-value <0.0001), confirming the causative role of R-loop in CIN.To elucidate the mechanisms underlying SF3B1 MT-associated CIN, we used DNA:RNA hybrid immunoprecipitation (DRIP) coupled with sequencing to map R-loop in SF3B1 MT and WT Nalm6 cells. Although SF3B1 MT caused minimal changes in overall R-loop distribution, it led to increased R-loop formation at centromeric regions. Proper centromeric R-loop (cen-R-loop) formation is essential for accurate chromosome segregation. Those R-loop are coated by phosphorylated replication protein A (p-RPA). p-RPA and centromere immunofluorescence (IF) co-staining revealed increased cen-R-loop in SF3B1 MT cells. Intriguingly, overexpression of RNaseH1 reduced both levels of centromeric p-RPA and percentage of cells with defective mitosis, suggesting SF3B1 MT associated cen-R-loop impacts CIN.To further discern the molecular underpinnings of R-loop mediated CIN, we investigated the effect of their accumulation on spindle geometry in metaphase cells. By analyzing the total body of chromosomes two-dimensional area, we observed one fold greater magnitude of chromosome oscillations in SF3B1 Nalm6 MT cells. Accordingly, compared to WT cells, SF3B1 MT cells displayed longer and wider spindles to accommodate an aneuploid chromosomal content. Removal of R-loops rescued mitotic spindle architecture and CIN in SF3B1 MT cells, highlighting a direct contribution of R-loop to kinetochore-microtubule organization and stability.As there is no evidence of strong binding of SF3B1 to centromeres, we tested the possibility that SF3B1 MT contributes to cen-R-loop formation through alternative splice variants. Through overlapping conserved SF3B1 MT-associated splice variants across isogenic cell lines and murine model with available R-loop interactome data, we identified SERBP1, which encodes an RNA-binding protein, as a target candidate . SF3B1 MT induces loss-of-function in SERBP1 through alternative splicing. Knockdown of SERBP1 triggered R-loop accumulation, increased centromeric p-RPA coating, and aberrant mitosis, which recapitulated the SF3B1 MT phenotype. Importantly, overexpression of SERBP1 WT alleviated both DSB and R-loop accumulation in SF3B1 MT cells. These observations strongly suggest that SF3B1 MT augments R-loop formation through dysregulated splicing of SERBP1 and possibly other genes involved in R-loop metabolism.We also investigated the possibility that the impact of RNA splicing defect on R-loop biology is a generalizable phenomenon. Examing a panel of leukemia-associated splicing factors (U2AF1, SRSF2, ZRSR2, RNU1), we found that these mutations all display R-loop dependent mitotic stress. Thus, mitotic stress caused by aberrant R-loop formation could be a convergent mechanism in splicing factor mutated leukemia cells.Altogether, we demonstrated that SF3B1 mutation promotes CIN by destabilizing mitotic spindles through aberrant R-loop accumulation at the centromere, via dysregulated splicing in genes such as SERBP1. Our study highlights an unrecognized role of cen-R-loop as a critical link between RNA splicing dysregulation and CIN, providing an opportunity for therapeutic targeting of R-loop in splicing factor mutant leukemias. DisclosuresWu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Lack of Bmf Facilitates the Selection of Highly Responsive B-Cell Receptor Clones in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is a disease of inhibited cell death, increased proliferation and high importance of interactions with the microenvironment e.g. with T-cells or stromal cells. A central effector in this concert is the activation of B-cell receptor (BCR) signalling pathway, associated with selection of specific BCR qualities (either autonomous signalling or reactivity to chronic (auto)-antigenic stimuli) and these signals play an essential role in CLL disease development and progression, also evidenced by the clinical success of kinase inbibitors directed at this signal. Mechanisms modulating selection of specific BCR types are ill understood, but since checkpoints during BCR selection in B cells are guarded by the Bcl2 family of proteins it is likely that cell death proteins are also influencing these decisions in CLL. To study the importance of microenvironmental interactions and antigen stimulation Tcl1 tg mice, which develop a murine CLL highly similar to the human disease 1,2, were frequently applied in the past, aiming to overcome the limitations of more or less artificial CLL in vitro culture systems. During clonal evolution of CLL, predominantly unmutated and stereotyped IgVH-11 and IgVH-12 BCRs are selected in Tcl1 tg mice which were shown to be specific for the autoantigen phosphatidylcholine (PtC) 3, however, the detailed mechanisms of clonal selection in CLL are still unclear. Here we propose a role of the BH3-only and pro-apoptotic protein Bmf in clonal selection by eliminating CLL cells expressing highly responsive BCRs.Methods: Tcl1 tg mice were crossed to Bmf knockout (KO) mice and CLL development monitored over time. After establishment of overt leukemia, mice were sacrificed and the overall survival time analysed. Additionally, we performed BCR sequencing, RNA-Seq and phosphoproteomics after 10µg/ml aIgM treatment using mass cytometry (Helios, CyTOF) and flow cytometry.Results: We found that Tcl1 tg Bmf KO mice developed phenotypically unchanged murine CLL but had an earlier onset of disease (Figure 1A) and an altered usage of BCR IgVH genes. While Tcl1 tg mice usually use PtC specific IgVH genes from the VH11 and V12 family, absence of Bmf led to a skewing to non PtC specific IgVH genes, mainly form the VH1 family (Figure 1B). We thus speculated that lack of Bmf favoured the selection of CLL clones with hyper responsive BCRs, when compared to Tcl1 tg mice with functional Bmf. Indeed we could show that after aIgM stimulation Tcl1 tg BmfKO CLL cells showed increased BCR signalling as measured by mass cytometry (Figure 1C), which was also confirmed by conventional flow cytometry (Figure 1D). Importantly, strong BCR stimulation induces cell death in B cells. Indeed, this can be observed in CLL cells from Tcl1 mice. In Tcl1 tumors that are deficient for BMF this cell death induction is not observed, suggesting that loss of Bmf protects from excessive cell death due to strong BCR stimulation.Conclusions: Here we report a novel role of Bmf in the selection of BCR clones in murine CLL. The higher BCR signalling capacity and the skewing of IgVH gene usage in the absence of Bmf indicates that highly responsive BCR clones are more often selected instead of eliminated during clonal selection.. Interestingly, a SNP in the Bmf gene has been reported to be susceptibility locud for CLL, suggesting that low expression of BMF may have similar effects in huma CLL. Indeed, in preliminary analyses, we observed a similar contribution of Bmf in clonal selection and BCR responsiveness in human CLL and decreased resistance to the BCR signalling inhibitor ibrutinib. Therefore our findings might also be important in a clinical context.

Targeting PAK1 Overcomes Resistance to Ibrutinib in Chronic Lymphocytic Leukemia

Objective:Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance.Methods:An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining.Results:Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G).To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I).IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI<1) of this combinatorial therapy (Figure 1K).Conclusions:In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Distinctive Signaling Profiles With Distinct Biological and Clinical Implications in Aggressive CLL Subsets With Stereotyped B-Cell Receptor Immunoglobulin.

The ontogeny and evolution of chronic lymphocytic leukemia (CLL) are critically dependent on interactions between leukemic cells and their microenvironment, including antigens, the latter recognized through the clonotypic B-cell receptor immunoglobulin (BcR IG). Antigen selection is key to the pathogenesis of CLL, as evidenced by the remarkable skewing of the BcR IG gene repertoire, culminating in BcR IG stereotypy, referring to the existence of subsets of patients with (quasi)identical BcR IG. Notably, certain of these subsets have been found to display distinct, subset-biased biological background, clinical presentation, and outcome, including the response to treatment. This points to BcR IG centrality while also emphasizing the need to dissect the signaling pathways triggered by the distinctive BcR IG expressed by different subsets, particularly those with aggressive clinical behavior. In this mini-review, we discuss the current knowledge on the implicated signaling pathways as well as the recurrent gene mutations in these pathways that characterize major aggressive stereotyped subsets. Special emphasis is given on the intertwining of BcR IG and Toll-like receptor (TLR) signaling and the molecular characterization of signaling activation, which has revealed novel players implicated in shaping clinical aggressiveness in CLL, e.g., the histone methyltransferase EZH2 and the transcription factor p63.

Treatment with ibrutinib does not induce a TP53 clonal evolution in chronic lymphocytic leukemia.

Not available.

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia.

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.

Longitudinal analyses of CLL in mice identify leukemia-related clonal changes including a Myc gain predicting poor outcome in patients

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy mainly occurring at an advanced age with no single major genetic driver. Transgenic expression of TCL1 in B cells leads after a long latency to a CLL-like disease in aged Eµ-TCL1 mice suggesting that TCL1 overexpression is not sufficient for full leukemic transformation. In search for secondary genetic events and to elucidate the clonal evolution of CLL, we performed whole exome and B-cell receptor sequencing of longitudinal leukemia samples of Eµ-TCL1 mice. We observed a B-cell receptor stereotypy, as described in patients, confirming that CLL is an antigen-driven disease. Deep sequencing showed that leukemia in Eµ-TCL1 mice is mostly monoclonal. Rare oligoclonality was associated with inability of tumors to develop disease upon adoptive transfer in mice. In addition, we identified clonal changes and a sequential acquisition of mutations with known relevance in CLL, which highlights the genetic similarities and therefore, suitability of the Eµ-TCL1 mouse model for progressive CLL. Among them, a recurrent gain of chromosome 15, where Myc is located, was identified in almost all tumors in Eµ-TCL1 mice. Interestingly, amplification of 8q24, the chromosomal region containing MYC in humans, was associated with worse outcome of patients with CLL.

Statins in mature B-cell lymphomas and leukaemias.

Statins in mature B-cell lymphomas and leukaemias.