Pathogenesis Of Chronic Lymphocytic Leukemia Research Articles

Targeting the B Cell Receptor Signaling Pathway in Chronic Lymphocytic Leukemia

Aberrant signal transduction through the B cell receptor (BCR) plays a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). BCR-dependent signaling is necessary for the growth and survival of neoplastic cells, making inhibition of down-stream pathways a logical therapeutic strategy. Indeed, selective inhibitors against Bruton's tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) have been shown to induce high rates of response in CLL and other B cell lymphomas. In particular, the development of BTK inhibitors revolutionized the treatment approach to CLL, demonstrating long-term efficacy. While BTK inhibitors are widely used for multiple lines of treatment, PI3K inhibitors are much less commonly utilized, mainly due to toxicities. CLL remains an incurable disease and effective treatment options after relapse or development of TKI resistance are greatly needed. This review provides an overview of BCR signaling, a summary of the current therapeutic landscape, and a discussion of the ongoing trials targeting BCR-associated kinases.

Диагностический потенциал регуляторных не кодирующих белок РНК при хроническом лимфоцитарном лейкозе

This paper reviews current knowledge about regulatory non-coding protein RNAs (ncRNAs) involved in the pathogenesis of chronic lymphocytic leukemia (CLL) and their potential capabilities as diagnostic markers. Diversity of clinical course as well as absence of detectable chromosomal aberrations and somatic mutations in 20 % of patients increase the interest to study the epigenetic aspects of pathogenesis. In this context, ncRNAs are believed to be promising diagnostic markers since their expression is commonly tissue-specific and they are quite stable in body fluids. Among the regulatory ncRNAs involved in the CLL pathogenesis, microRNAs and long (lncRNAs) have been most studied, whereas ring-like, or circulatory, ncRNAs (circRNAs) require further analysis. Aberrant expression of ncRNAs may account for the resistance to treatment in CLL patients without detected genomic abnormalities. Bioinformatics analysis of RNA sequencing databases allows to isolate novel candidate ncRNA molecules, including those associated with RNA-mediated suppression of the Piwi protein-interacting transposons. This paper proposes new independent predictive models based on the expression of 2 (LNC-KIA1755-4, LNC-IRF2-32-LNCRNA), 4 (miR-125b, miR-15b, miR-181c, miR-412), and 6 (PRKCQ, TRG.AS1, LNC00467, LNC01096, PCAT6, SBF2.AS1) simultaneously assessed different ncRNAs. Since risk- and stage classification of hematological malignancies is performed not only on the basis of clinical but also molecular genetic markers, the monitoring of regulatory ncRNA expression can provide an additional tool for more effective stratification of patients. The present review is concerned with the methodology issues in analytical procedures which impede widespread use of laboratory ncRNA tests.

MiR-155 and miR-223 as markers of biological and clinical features of chronic lymphocytic leukemia

Introduction. Chronic lymphocytic leukemia (CLL) is a disease characterized by large individual differences both in the clinical course and in molecular patterns of expression of genes and regulatory RNAs, which can influence pathological changes. The involvement of regulatory microRNAs miR-155 and miR-223 in the pathogenesis of CLL is fairly well known, but there is insufficient information about possible fluctuations in the expression of miR-155 and miR-223 depending on the time course of pathology development and on parameters of medical treatment. Purpose – to investigate the expression of miR-155 and miR-223 in patients having CLL with different biological and clinical features and different characteristics of treatment in terms of peripheral-blood substrates (plasma, lymphocytes, and extracellular vesicles) and bone marrow. Material and Methods. This work involved samples of peripheral blood and bone marrow from 38 patients with a diagnosis of CLL from the City Hematology Center at the government-funded healthcare institution (Novosibirsk Oblast) City Clinical Hospital No. 2 from the years 2016 to 2017. Assessment of miR-155 and miR-223 expressions was carried out by reverse-transcription real-time PCR according to the TaqMan principle. Significance of differences between groups was evaluated either by the nonparametric Mann–Whitney test or by the nonparametric Kruskal–Wallis test with subsequent pairwise comparisons via the Mann–Whitney test. Results. High variation of the analyzed parameters was found. The expression levels of miR-155 and miR-223 in microvesicles of patients with unfavorable chromosomal anomalies were lower than those in patients with the chromosomal aberrations (or the normal karyotype) associated with a moderate effect on CLL prognosis. The expression level of miR-223 in peripheral blood lymphocytes of untreated patients with CLL was higher than that observed in treated patients. Conclusion. differences in the expression levels of miR-155 and miR-223 were identified depending on chromosomal aberrations and polychemotherapy. Our preliminary results will provide the basis for future larger studies on levels of microRNAs in CLL patients having specific features of the development, clinical course, and treatment of the disease.

B cell receptor signaling and associated pathways in the pathogenesis of chronic lymphocytic leukemia.

B cell antigen receptor (BCR) signaling is a key driver of growth and survival in both normal and malignant B cells. Several lines of evidence support an important pathogenic role of the BCR in chronic lymphocytic leukemia (CLL). The significant improvement of CLL patients' survival with the use of various BCR pathway targeting inhibitors, supports a crucial involvement of BCR signaling in the pathogenesis of CLL. Although the treatment landscape of CLL has significantly evolved in recent years, no agent has clearly demonstrated efficacy in patients with treatment-refractory CLL in the long run. To identify new drug targets and mechanisms of drug action in neoplastic B cells, a detailed understanding of the molecular mechanisms of leukemic transformation as well as CLL cell survival is required. In the last decades, studies of genetically modified CLL mouse models in line with CLL patient studies provided a variety of exciting data about BCR and BCR-associated kinases in their role in CLL pathogenesis as well as disease progression. BCR surface expression was identified as a particularly important factor regulating CLL cell survival. Also, BCR-associated kinases were shown to provide a crosstalk of the CLL cells with their tumor microenvironment, which highlights the significance of the cells' milieu in the assessment of disease progression and treatment. In this review, we summarize the major findings of recent CLL mouse as well as patient studies in regard to the BCR signalosome and discuss its relevance in the clinics.

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia

The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eμ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adapter whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eμ-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eμ-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

Can Galectin-3 Be a Novel Biomarker in Chronic Lymphocytic Leukemia?

Galectin-3's (Gal-3) effect on the pathogenesis of chronic lymphocytic leukemia (CLL) has not yet been extensively studied. The present study aims to analyze the potential role of Gal-3 as a prognostic biomarker in CLL patients. The Gal-3 expression was evaluated in CLL cells with RT-qPCR and flow cytometry. Due to the unclear clinical significance of soluble Gal-3 in CLL, our goal was also to assess the prognostic value of Gal-3 plasma level. Because cell survival is significantly affected by the interaction between Gal-3 and proteins such as Bcl-2, the results of Gal-3 expression analysis were also compared with the expression of Bcl-2. The results were analyzed for known prognostic factors, clinical data, and endpoints such as time to first treatment and overall survival time. Our research confirmed that Gal-3 is detected in and on CLL cells. However, using Gal-3 as a potential biomarker in CLL is challenging due to the significant heterogeneity in its expression in CLL cells. Moreover, our results revealed that Gal-3 mRNA expression in leukemic B cells is associated with the expression of proliferation markers (Ki-67 and PCNA) as well as anti-apoptotic protein Bcl-2 and can play an important role in supporting CLL cells.

PD-1 and LAG-3 as early prognostic markers in the treatment of patients with chronic lymphocytic leukemia

Background. Recently, studies have been conducted all over the world to study the role of immune checkpoints in the pathogenesis of chronic lymphocytic leukemia (CLL) and the possibility of their use as prognostic markers. Of greatest interest are PD-1 (programmed cell death-1) and LAG-3 protein (lymphocyte-activation gene 3).Aim. To study the features of PD-1 (CD279) and LAG-3 (CD223) expression on blood B-cells of CLL patients and the possibility of their use as early markers for predicting the hematological response to therapy.Materials and methods. The blood of 30 patients with CLL in stage C according to Binet and 20 healthy individuals was studied by 10-color flow cytometry.Results. In patients with CLL, there were significant differences in the initial lymphocytes level, PD-1 and LAG-3 expression on B-lymphocytes, both with persons in the control group and among themselves with different hematological responses to therapy with rituximab according to the results of minimal residual disease monitoring.Conclusion. PD-1 and LAG-3 can be used as early markers for predicting the response of CLL patients to therapy. The combined use of initial lymphocytes level and PD-1 and LAG-3 expression on CD19+ blood cells has a greater prognostic value. New data obtained from the study of immune checkpoints PD-1 and LAG-3 may be useful in the development of targeted therapeutic agents.

Role of the tumor microenvironment in CLL pathogenesis

Chronic lymphocytic leukemia (CLL) cells extensively interact with and depend on their surrounding tumor microenvironment (TME). The TME encompasses a heterogeneous array of cell types, soluble signals, and extracellular vesicles, which contribute significantly to CLL pathogenesis. CLL cells and the TME cooperatively generate a chronic inflammatory milieu, which reciprocally reprograms the TME and activates a signaling network within CLL cells, promoting their survival and proliferation. Additionally, the inflammatory milieu exerts chemotactic effects, attracting CLL cells and other immune cells to the lymphoid tissues. The intricate CLL-TME interactions also facilitate immune evasion and compromise leukemic cell surveillance. We also review recent advances that have shed light on additional aspects that are substantially influenced by the CLL-TME interplay.

PRMT1 Promotes Chronic Lymphocytic Leukemia Progression Via Modulating Methylation of MAST1

Introduction As a member of the protein arginine N-methyltransferase family, protein arginine methyltransferase 1 (PRMT1) plays a crucial regulatory role in the etiology of malignant neoplasms. PRMT1-mediated protein methylation promoted the maintenance of acute myeloid leukemia, which revealed the promising potency of PRMT1 inhibitors. Recent study suggested that dysregulation of PRMT5 promotes Richter's transformation in chronic lymphocytic leukemia (CLL). However, the effects of PRMT1 in the tumorigenesis and progression of CLL still remained ill-defined. Hence, the aim of this study was to investigate the clinical significance and mechanisms of PRMT1 underlying the development of CLL. Methods Peripheral blood samples were collected from 79 newly diagnosed CLL patients (47 males and 32 females; age range 39-85 years, median 63 years) in Shandong Provincial Hospital CLL (SPHCLL) cohort with informed consent. CRISPR-Cas9 technology was used to stably knockout PRMT1 in CLL cells. A label free quantitative proteomics analysis was implemented to reveal the protein methylation mediated by PRMT1. Assessment of cell viability, apoptosis and cell cycle were analyzed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital. Results This study examined the expression of PRMT1 in SPHCLL and GEO databases, and discovered the upregulation of PRMT1 mRNA in CLL cells. Aberrantly elevated expression of PRMT1 was observed in a cohort of newly diagnosed CLL patients than healthy donors in mRNA level (donors vs. CLL patients, 0.07±0.06 vs. 0.20 ±0.21, p=0.013). Furthermore, increased expression of PRMT1 was correlated with inferior prognosis in two long-term follow-up cohorts of CLL patients (HR=2.80, p=0.002, and HR=1.551, p=0.009). To elucidate the functional significance of PRMT1 in CLL, we established stable PRMT1 knockdown cells using lentiviral shRNAs and PRMT1 knockout cells using CRISPR/Cas9 technology. The suppression of PRMT1 remarkably inhibited cell proliferation, induced cell apoptosis and blocked cell cycle at G1/S phase in CLL cells. Furthermore, it was observed that C7280948, a selective inhibitor of PRMT1, resulted in the defective proliferation of CLL primary cells in a dose-dependent manner. We subsequently performed pre-clinical investigations of C7280948 in the orthotopic CLL xenograft murine model. Notably, the administration of C7280948 significantly diminished the leukemia burden and induced the lessened degree of splenomegaly in comparison to the control group (Fig. 1A). Moreover, a reduction in the population of CLL cells was detected in bone marrow and spleen of mice treated with C7280948. To decipher the role of PRMT1 in the pathogenesis of CLL, the label free quantitative proteomics analysis was conducted on PRMT1-deficent and control MEC1 cells. Knockdown of PRMT1 diminished asymmetric dimethylarginine of some proteins in CLL cells. In accordance with the results of proteomics analysis, microtubule associated serine/threonine kinase 1 (MAST1) was selected as a candidate target for further investigation. The methylation site of MAST1 protein was identified as R802 (Fig. 1B). Increased expression of MAST1 was correlated with inferior prognosis of CLL patients ( p<0.001), indicating the essential role of MAST1 in the progression of CLL. These results supported the hypothesis that PRMT1 interacted with MAST1 and enhanced the activity of MAST1 via methylating this oncoprotein in CLL cells. Conclusion The present study provides robust evidence for the oncogenic function of PRMT1 in the pathogenesis of CLL, highlighting the potential therapeutic efficacy of the selective PRMT1 inhibitor C7280948. Collectively, the targeted inhibition of PRMT1 exhibits promising prospects for the management of CLL patients.

CLL Cells within the Proliferation Centers of the Patient Lymph Nodes Are Enriched for Notch Signaling, Thus NOTCH a Viable Target for CLL Therapy

CLL cells within the Proliferation Centers of the Patient Lymph Nodes are enriched for Notch signaling, thus a Target for CLL Therapy. Shantaram S. Joshi, Michael Pitner, Matt Kling, Furqan Mehdi, Mamta Shukla, Ashima Shukla, Vipul Shukla, Nagendra Chaturvedi, Hamid Band , Javeed Iqbal The tissue microenvironment, particularly the lymph node (LN), plays a major role in regulating the leukemic progression and clinical heterogeneity of chronic lymphocytic leukemia (CLL). CLL cells proliferate in the patient lymph nodes, resulting in the formation of proliferation centers (PCs). The molecular basis of the CLL cell proliferation within PCs is not well known. Several reports have implicated the activated Notch signaling pathway in the pathogenesis of CLL. Using the IRF4 -/-Vh11 mouse model of CLL, we have previously shown that Notch signaling is indispensable for CLL development (Vipul Shukla et al, Oncotarget, 2016). Based on these studies, we hypothesized that CLL cell proliferation in the lymph node PCs involves activated NOTCH signaling. To this end, we have examined the expression levels of Notch signaling associated molecules namely IRF4, Notch 1, Notch 2, Nedd4, Fbxw7 and Itch1 on CLL cells within the patient lymph node proliferation centers (n=12) using immunohistochemistry and western blotting. Expression levels were scored based on the percentage of cells with positive staining: 5+ (>80%), 4+ (60-80%), 3+ (40-60%), 2+ (20-40%), 1+ (<20%) and 0 (No staining). IHC analyses showed that the expression levels of IRF4 to be 2+, Notch 1 was 4+, Notch 2 was -3 to 4+, Nedd4 was 1+, Fbxw7 was 3+ and ITCH1 was 1+. Western blotting of CLL cells from the lymph node proliferation centers (n=6) confirmed a similar pattern of expression of the Notch-associated genes. Together, these results validate the association of Notch signaling in CLL cells of the patient lymph node PCs similar to CLL cells from the IRF4 -/-Vh11 mouse model. Therefore, as a next logical step we treated the MEC-1 and OSU-CLL cells in culture and primary CLL cell from patients with NOTCH inhibitor RO4929097 (RO), a γ-secretase inhibitor for 24, 48 and 72 hours in vitro. There was a significant decrease in the expression NOTCH 1, NOTCH 2 in MEC-1, OSU-CLL and primary CLL cells determined using western blot technique. Also, we observed a significant decrease in proliferation of CLL cells from MEC-1 and OSU-CLL cell lines, following treatment with NOTCH inhibitor RO. In addition, NOTCH inhibitor combined with Bruton's Tyrosine Kinase (BTK) a member of the B-Cell Receptor signaling, showed a further significant decrease in the proliferation of CLL cells in culture. Together these experiments demonstrated that NOTCH targeting strategy is a viable therapy for aggressive CLL warranting further preclinical and clinical studies. This work was partially supported by bridge funding from the American Association of Hematology awarded to Dr. Runqing Lu, who passed away during the pursuit of this study.

Progressive Gut Microbiome Dysbiosis Is Associated with Chronic Lymphocytic Leukemia Pathogenesis and Development in the Eµ-TCL1 Mouse Model

Introduction: The gut microbiome is home to a community of microorganisms such as bacteria, fungi, viruses, archaea, and protozoa. Maintaining a balance between “favorable” and “unfavorable” bacteria within the gut microbiome is essential for homeostatic physiological processes including proper immune system development, regulation of metabolic pathways, protection against pathogens, and gut epithelium integrity. Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by the accumulation of CD5 + B-cells in the blood, bone marrow, and secondary lymphoid tissues. The pathogenesis of CLL is related to the activation of critical survival pathways such as B-cell and toll-like receptor signaling pathways, where microbial antigens are known to be key stimulators driving CLL B-cell proliferation. In this study, we investigated how the gut microbiota composition contributes and correlates to CLL disease progression. Methods: Using a well-established murine model representative of CLL development in humans, we profiled the gut microbiota of transgenic Eµ-TCL1 mice and age-matched wild-type B6 (WT B6) mice over the course of 12 months (n = 7-10/group). Fecal pellets were collected aseptically from study mice at four time points (4, 7, 10, and 12 months) for DNA extraction using the QIAamp® PowerFecal® Pro DNA Kit. To characterize the gut microflora, we utilized 16S rRNA sequencing using the Illumina 16S metagenomics protocol. Bacterial taxonomy was assigned and analyzed using the QIIME2™ platform. Linear Discriminant Analysis (LDA) Effect Size (LEfSe) analysis was employed to identify differentially expressed bacterial taxa between cohorts. Beta diversity indices were used to evaluate intersample differences in the fecal microbiota. Mice were monitored for peripheral expansion of leukemia cells (%CD45 +/CD19 +/CD5 + cells) via flow cytometry. Next, we evaluated the microbiome in an aggressive model of CLL. Utilizing the Eµ-TCL1 adoptive transfer murine model, similar methods were adopted to profile the gut microbiome over the course of 7 weeks. Lastly, to evaluate the contribution of the gut microbiota to CLL development, we employed an antibiotic-mediated gut microflora ablation to the adoptive transfer Eµ-TCL1 model, with leukemia expansion monitored post-engraftment on a weekly basis. Results: A unique microbiome signature was identified in both the indolent and aggressive model of CLL. Evaluation of beta diversity, using Bray-Curtis distance metrics, confirmed distinct differences in the gut microbial communities between Eµ-TCL1 and WT B6 cohorts. At the phylum level, the gut microbiota of the Eµ-TCL1 and WT B6 cohorts were similarly dominated by Firmicutes and Bacteroidota. However, Proteobacteria Deferribacterota and Desulfobacterota were more abundant in the Eµ-TCL1 mice over 12 months as compared to age-matched WT B6. Abundance of Actinobacteriota increased specifically at 12 months in the Eµ-TCL1 mice. Additionally, LEfSe analysis showed enrichment of certain phyla in the Eµ-TCL1 mice at 4 months, 10 months, and 12 months. Presence of these bacteria paralleled with expansion of disease. In the aggressive model of CLL, beta diversity confirmed distinct differences in gut microbial communities as CLL disease progressed. Moreover, certain phyla were enriched in leukemic mice at 7 weeks post-engraftment including Proteobacteria and Actinobacteriota. Lastly, in the antibiotic-mediated gut flora ablation model, a delay of disease onset was reflected in mice with ablated gut microbiomes compared to mice with intact gut microbiomes, resulting in prolonged overall survival. Conclusion: We provide novel results associating the gut microbiome with the initiation and progression of CLL disease. In both the indolent and aggressive CLL disease models, we showed that there is a distinct gut microbiome as evident by beta diversity. A dysbiotic signature represented by increasing Proteobacteria abundance was similarly identified in both models. Proteobacteria are known to be evident in diseases characterized by inflammation. These findings support future studies to determine a potential causal mechanism between gut microbiota changes and CLL pathogenesis.

Evidence of Defective Cell-Autonomous Signalling By B Cell Receptors in CLL Stereotyped Subset #1

B cell receptor (BCR)-mediated signalling is central in the pathogenesis of chronic lymphocytic leukaemia (CLL), and represents a target for pharmacological intervention. Notably, so far characterized CLL-derived BCRs are capable of triggering cell-autonomous intracellular signalling through homotypic interactions without requirement for exogenous antigens. Approximately 41% of all CLL cases can be classified in subsets based on homologous, otherwise known as stereotyped, BCRs expressed by the malignant clones, sharing remarkably similar sequences in the VH CDR3 region and highly restricted immunoglobulin heavy and/or light variable genes. Since cases within a subset display clinicobiological consistency in contrast with the pronounced heterogeneity of CLL at large, analysis of the mechanism of BCR-mediated signalling can unveil correlations between molecular details of BCR activation and the course of the disease. Our present study focused on BCRs of stereotyped subset #1, the second largest stereotyped subset, known for a particularly aggressive clinical course. Cases belonging to subset #1 express unmutated IGHV genes of phylogenetic clan I (namely, IGHV1, IGHV5 and IGHV7 subgroup genes) partnered by light chains encoded by the IGKV1-39/1D-39 gene. First, we used X-ray crystallography to determine the high-resolution crystal structures of the BCR Fab fragments from three subset #1 cases utilizing the IGHV1-2, IGHV1-3, and IGHV5-10 genes partnered with IGKV1-39/1D-39 to obtain information on the combining sites' structures and possible modes of intermolecular interactions that may mediate cell-autonomous signalling. Analysis of the experimental structures revealed that, despite the high sequence homology of the three proteins, the heavy and light chain CDR loops in the three BCR Fabs are arranged differently to shape binding cavities with distinct dimensions and polarity, thus suggesting that distinct antigens may be the physiological target of each subgroup. Unlike BCRs from other subsets previously characterised, the intermolecular contacts between the subset #1 Fab molecules in the three crystals involve diverse residues that are not indicative of a shared homotypic interaction. In addition, the surfaces involved in the contacts are small and the computed energies for the protein-protein contacts observed are suggestive at best of very weak interactions. In accordance to this, when we used analytical ultracentrifugation along with chromatographic and calorimetric methods to detect BCR Fab oligomerization in solution, no evidence of self-association was obtained, thus indicating that the receptors in solution either do not interact, or are characterized by self-dissociation constants that exceed the sensitivity limit of the techniques employed. To assess whether the cellular environment could promote self-association and signalling activity of the full-length BCR, we expressed subset #1 BCRs in the model triple knockout (TKO) pre-B-cell line, designed to measure Ca 2+ ion influx using cytofluorimetry. All subset #1 BCRs tested were unable to mobilize autonomously the Ca 2+ ions, despite being fully expressed on the cell surface and capable of signalling upon stimulation with anti-BCR antibody as a proxy for exogenous ligand. This result is in sharp contrast with the behaviour of all other CLL-derived BCRs so far characterised in this system that could activate a cell-autonomous signal as evidenced from a persistent Ca 2+ ion influx in the transduced cells without requiring external stimuli. Hence, subset #1 BCRs differ from the so far characterized BCRs from CLL cases belonging to other stereotyped subsets, including clinically aggressive subset #2 and its satellite subset #169 as well as the clinically indolent stereotyped subset #4. In conclusion, our combined biochemical and cellular analysis demonstrates that a constitutively active cell-autonomous signalling may not be a unifying feature of all CLL clones as previously proposed, and that other mechanisms may be at the roots of the BCR-mediated signal required for leukemic cell proliferation and development of CLL.

The Unique BCR Inhibiting Properties of BMS-986205 in Chronic Lymphocytic Cells

B cell receptor (BCR) signaling is recognised as a central pathway in the pathogenesis of chronic lymphocytic leukaemia (CLL), the most common leukemia in the western world. In CLL, the BCR pathway is activated by antigens in the tissue microenvironment to promote the maintenance and expansion of leukemic cells. The impressive inhibition of clonal expansion of CLL cells and the disease control achieved with tyrosine kinase inhibitors that block BCR signaling provided further evidence of the role of BCR in CLL. Indoleamine 2,3-dioxygenase 1 (IDO1) is the inducible and rate-limiting enzyme involved in the catabolism of tryptophan to kynurenine. It was discovered as a modulator of the innate immune response during infection. Subsequent discoveries have implicated IDO1 as a player in acquired immune tolerance. IDO1 activity has been identified in several types of cancer cells as well as in their tumor-surrounding microenvironment. We have previously investigated and characterized the impact of IDO1 expression in CLL. Our results show that CLL microenvironmental stimuli positively modulate IDO1 expression in leukemic cells and that increased IDO1 activity leads to sustained survival of malignant lymphocytes and impaired drug sensitivity through the autocrine/paracrine activation of the aryl hydrocarbon receptor by kynurenine (Atene CG et al. 2022). To assess the functional and clinical significance of IDO1 enzymatic activity in CLL, we used several inhibitors that are currently in late-stage clinical trials. Using BMS-986205 (BMS), an irreversible inhibitor of IDO1 with the best cell-based potency, in primary patient cells, we observed not only a reduction in kynurenine production due to enzymatic inhibition of IDO1, but also lower levels of IDO1 RNA and protein following BCR stimulation with α-IgM. As we have previously shown that BCR activation can induce IDO1 expression, we treated CLL cells with increasing doses of BMS for 3 hours and then added α-IgM to the culture. BMS is able to counteract the pro-survival effect of BCR triggering, leading to significant apoptosis of CLL cells, while the viability of CLL cells in the absence of external stimuli was not significantly modulated. Focusing on the complex signaling cascade activated by BCR engagement, we observed that BMS impaired the phosphorylation of upstream kinases such as SYK and BTK, but also CD79a, CD19 and BLNK, in addition to downstream effectors such as AKT. We therefore investigated which factors that negatively regulate the BCR pathway are modulated by BMS treatment. By analyzing both mRNA and protein levels, we measured a significant induction of the phosphatases PTPN6, PTPN22 and SHIP1. Since BMS also showed a downregulation of total SYK expression, we are analyzing the expression of the proto-oncogene CBL, an E3 ubiquitin ligase known to be involved in the negative regulation of SYK. Further studies should be undertaken to determine whether, in addition to its role as an IDO1 inhibitor, BMS may also have the ability to indirectly modulate and disrupt BCR signaling. Efforts are underway to identify the proteome-wide direct target of BMS in CLL cells. In conclusion, our limited and preliminary data suggest that BMS could be considered as a potential therapeutic strategy to target CLL cells with a double hit: by blocking the pro-survival effect of BCR signaling, one of the driving forces for CLL cell survival and proliferation, and by inhibiting IDO1/kynurenine action to restore sensitivity to key leukemia treatments.

TRAF3-Inactivated Chronic Lymphocytic Leukemia Cells Show an Enhanced Metabolic Plasticity That Can be Attenuated By Glutaminolysis and Mitochondrial Pyruvate Import Inhibition

I ntroduction: TRAF3 gene is located in 14q32.32 and has been identified as a tumor suppressor gene involved in B-cell survival, immune response, and cellular metabolism. TRAF3 is frequently encompassed within 14q deletion and affected by inactivating mutations in 4% of chronic lymphocytic leukemia (CLL) patients. Recently, our group found that CLL patients harboring biallelic TRAF3 inactivation (deletion and mutation) showed a dismal prognosis, with a short time to first treatment (Pérez Carretero, AJH 2022). However, the biological role of TRAF3 alterations in CLL pathogenesis remains to be elucidated. Thus, the aims of this study wereto assess the functional consequences of these alterations and to determine their impact on therapy response. Methods: Previously reported TRAF3 mutations were mimicked in a CRISPR/Cas9-edited CLL model by using the PGA1 cell line (PGA1- TRAF3 mut). Metabolomic (liquid chromatography-mass spectrometry), transcriptomic (RNAseq) and functional analyses were carried out. To evaluate the metabolic specificities of PGA1- TRAF3 mut cells, oxidative phosphorylation (OXPHOS) and glycolytic capacity were studied by assessing the mitochondrial oxygen consumption rate (OCR) and extracellular acidification (ECAR), respectively. Additionally, we assessed the metabolite profile of 25 primary CLL cells (n=3 TRAF3 mut cells, n=22 TRAF3 wt cells) and their response to a library of drugs (CLL drugs and metabolic inhibitors), in the presence and absence of microenvironment (with and without HS-5 co-culture and IL-2 and CpG stimulation). Results: Metabolomic analysis of CRISPR/Cas9-edited CLL models showed an enrichment of metabolites involved in Warburg effect (pyruvate, phosphoenolpyruvate, and lactate), citric acid cycle (TCA- acetyl-CoA, α-ketoglutarate, succinate) and glutamate metabolism (α-ketoglutarate) in PGA1- TRAF3 mut cells. Moreover, PGA1- TRAF3 mut cells exhibited higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis was also slightly enhanced in the TRAF3 mut cells. Remarkably, primary CLL cells with TRAF3 mutations presented a higher concentration of phosphoenolpyruvate, glutamate and the TCA intermediates fumarate and malate, as well as higher levels of NADH and ATP, proving an increased energy production in these cells. Altogether, these results reveal that TRAF3 mut cells showed an enhanced mitochondrial glycolytic metabolism. Given the metabolic changes identified in TRAF3 mut cells, we subsequently assessed their OCR and ECAR after treatment with metabolic inhibitors. Unlike in WT cells, UK5099 administration (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) induced an increase in the maximal respiration of TRAF3 mut cells, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. Therefore, we tested the effect of C968 (glutaminolysis inhibitor), finding the same effect on TRAF3 mut cells, which reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis). Notably, the transcriptomic analysis revealed an upregulation of the glutamine transporter SLC1A4 (p-adj=0.007), what could be favoring glutamine uptake for an enhanced glutaminolysis. Interestingly, by blocking pyruvate import and glutaminolysis simultaneously, a decrease in TRAF3 mut CLL cells proliferation was observed (p=0.03), with equal levels of the previously dysregulated metabolites to those of the WT, which may indicate that this treatment sensitizes TRAF3 mut cells through its relieving effect on metabolic reprogramming. Moreover, primary CLL cells with TRAF3 mutations were also sensitive to the combination, in the presence and absence of microenvironment (p=0.01, p=0.04 respectively), demonstrating the potential of the combination of metabolic inhibitors as a new therapeutic approach in TRAF3 mut patients. Conclusion: Our work identifies a novel role of TRAF3 alterations in the CLL cellular metabolism. Specifically, TRAF3 inactivation induced an increased mitochondrial glycolytic activity and distinct metabolic dependencies in CLL cells, contributing to an enhanced metabolic plasticity (mitochondrial glycolysis/glutaminolysis) potentially targetable in CLL. Funding: PI21/00983 FEHH-GELLC(CPC), JYCL(ARS)

Comparison of Dietary Intake and Dietary Inflammatory Index in Patients with Chronic Lymphocytic Leukemia By Treatment Status to Cohabitating Controls

Chronic lymphocytic leukemia (CLL) is an incurable disease with a varied presentation ranging from indolent and asymptomatic to morbid and life-limiting. As tumor biology insufficiently explains this variability, we hypothesized that behavioral and environmental factors including diet and the gut microbiome, where diet is a key influence, may influence CLL pathogenesis and response to therapy. We performed a cross-sectional sample and survey collection study of the gut microbiome, blood, and diet in patients with CLL treated at the University of Nebraska Medical Center between 2021-2023. Eligible subjects included 1) untreated CLL with Rai stage 2-4 disease (untreated CLL), 2) CLL treated with a Bruton's tyrosine kinase inhibitor (BTKi), and 3) individuals without cancer cohabitating with enrolled CLL subjects (control). All subjects provided stool samples for microbial analysis, and subjects with CLL provided blood for serum cytokine analysis. All subjects completed the Diet History Questionnaire version 3.0 (DHQ-3). The DHQ-3 is a self-administered food frequency questionnaire, where participants complete 135 questions on food/beverages and 26 on dietary supplements consumed in the past month. A total and component healthy eating index (HEI) score was derived from the DHQ-3 and represents how well diet intake corresponds to the dietary guidelines recommended by the United States Department of Agriculture and Health and Human Services. The HEI is based on 13 individual components (Figure 1) and scored from 0-100, where higher scores indicate healthier diets. Inflammatory capacity of the diet was assessed by the Dietary Inflammatory Index (DII) (Shivappa et al, Public Health Nutrition, 2013), a measure of dietary components associated with serum markers of inflammation. The DII has a range of scores up to 7.98 suggestive of greater systemic inflammation (median DII score 0.23), and a minimum of -8.87, suggestive of consumption of anti-inflammatory foods. The DII was calculated and compared between each cohort. Statistical analysis was performed using ANOVA. A total of 58 subjects enrolled on the study, 42 with CLL and 16 cohabitating controls. A total of 27 subjects were included in the analysis after data cleaning. Fifteen subjects (56%) were treated with a BTKi, 5 (19%) had untreated Rai stage 2 disease, and 7 (26%) were cohabitating controls. Median ages (72 vs 67 vs 71 for BTKi, untreated, control, respectively) were similar. Median absolute lymphocyte count was 3.2 cells x10^3/µL (1.3 - 131 range) in the BTKi and 55 cells x10^3/µL (13-134 range) in the untreated cohort. There were 5/15 (33%) with 17p deletion and/or p53 mutation in the BTKi cohort and 2/5 (40%) in the untreated cohort. Median time from diagnosis to study entry was 30 months (range 3-151 months) in the untreated cohort, and median time on BTKi therapy was 30 months (range 2-92 months). Within the BTKi cohort, 33% received ibrutinib, 20% received zanubrutinib, and 47% received acalabrutinib. Overall dietary habits were largely similar between the 3 cohorts (Table 1). The mean total HEI score was higher in the untreated (66) and control (66) populations compared to the BTKi cohort (59) ( p = 0.23), although not significantly different. These scores are similar to the 2020 median score of 61 for Americans 60 years and above. Added sugar intake was highest in the untreated cohort (HEI component score of 9.6, 6.3, 8.6 untreated, BTKi, control p = 0.005, respectively) (Figure 1). After calculating the DII for each cohort, no statistically significant differences were observed (Table 1). The range of DII scores across subjects was -5.2 - 4.0, within the expected range for DII calculations. DII scores were 0.44, 0.2, 0.82 p = 0.89 for BTKI, untreated CLL, and control cohorts respectively. To our knowledge, this study is the first to analyze dietary patterns in well-defined homogenous CLL populations (BTKi vs Rai stage 2+ CLL cohort) and compare them to cohabitating individuals. Although overall dietary patterns and DII were similar across cohorts, slight differences in added sugar intake was observed between the cohorts, suggestive that subtle yet important dietary differences may be observed. These data provide a baseline to study personalize dietary approaches to patients with CLL, who increasingly ask clinicians for nutritional interventions as part of their comprehensive cancer care.

Normal B cells express ZAP70 in chronic lymphocytic leukemia: A link between autoimmunity and lymphoproliferation?

ZAP70 has a prognostic value in chronic lymphocytic leukemia (CLL), through altered B-cell receptor signaling, which is important in CLL pathogenesis. A good correlation between ZAP70 expression in CLL cells and the occurrence of autoimmune phenomena has been reported. Yet, the great majority of CLL-associated autoimmune cytopenia is due to polyclonal immunoglobulin (Ig) G synthesized by nonmalignant B cells, and this phenomenon is poorly understood. Here, we show, using flow cytometry, that a substantial percentage of CD5- nonmalignant B cells from CLL patients expresses ZAP70 compared with CD5- B cells from healthy subjects. This ZAP70 expression in normal B cells from CLL patients was also evidenced by the detection of ZAP70 mRNA at single-cell level with polyclonal Ig heavy- and light-chain gene transcripts. ZAP70+ normal B cells belong to various B-cell subsets and their presence in the naïve B-cell subset suggests that ZAP70 expression may occur during early B-cell development in CLL patients and potentially before malignant transformation. The presence of ZAP70+ normal B cells is associated with autoimmune cytopenia in CLL patients in our cohort of patients, and recombinant antibodies produced from these ZAP70+ nonmalignant B cells were frequently autoreactive including anti-platelet reactivity. These results provide a better understanding of the implication of ZAP70 in CLL leukemogenesis and the mechanisms of autoimmune complications of CLL.

РОЛЬ ИЗМЕНЕНИЙ РЕДОКС-СТАТУСА В ПАТОГЕНЕЗЕ ХРОНИЧЕСКОГО ЛИМФОЛЕЙКОЗА

Introduction. Chronic lymphocytic leukemia is a hemoblastosis of CD5+ B lymphocytes characterized by lymphocytosis and damages to lymphatic organs and occurring in the older age group. Its etiology and pathogenesis have not been fully understood as of today. Oxidative stress is an important factor in the regulating the hemostasis of hemopoietic stem cells and in activating intracellular survival signaling pathways in chronic lymphocytic leukemia cells. Aim. This study is aimed at analyzing the current data on the role of redox status changes in the pathogenesis of chronic lymphocytic leukemia. Materials and Methods. We reviewed relevant studies published in 2018-2023 and research articles collected in scientific electronic bibliographic databases PubMed and Social Sciences Citation Index, dealing with the pathogenesis of chronic lymphocytic leukemia and the role of free-radical oxidation processes in it. Results and Discussion. In chronic lymphocytic leukemia, oxidative stress with a systemic excess of reactive oxygen species, an imbalance in the effectiveness of antioxidant defense is caused mainly by activation of oxidative phosphorylation in mitochondria, low levels of NADPH-oxidase type 2, increased expression of hemoxygenase-1, glutathione peroxidase and glutathione recycling enzymes, superoxide dismutase-2, thioredoxins, and decreased expression of catalase. Oxidative stress subsequences in the chronic lymphocytic leukemia cells include the activation of transcription factors related to erythroid nuclear factor, nuclear kappa B factor, and FOXO family transcription factors. One of the mechanisms of resistance to drug therapy and oxidative stress of chronic lymphocytic leukemia cells is the intracellular signaling pathway dependent on erythroid nuclear factor-2, due to the activation of expression in cells of superoxide dismutase-2, catalase, glutathione peroxidase, peroxiredoxin-3 and -5, heme oxygenase-1, thioredoxin-1 and -2, reduced glutathione, natural killer cell activity, which is associated with lifespan, chemotaxis, proliferation and survival. FOXO family proteins are believed to suppress carcinogenesis. FOXO3a increases the expression of superoxide dismutase-2, catalase, glutathione peroxidase, peroxiredoxin-3 and -5, and the activity of natural killer cells, which promotes the survival of tumor cells. Conclusion. The development of new targeted pharmacological agents that are capable of accumulating reactive oxygen species and reducing antioxidant protection due to the degradation of erythroid nuclear factor-2 and activation of NADPH-quinone oxidoreductase-1 is underway, which modernizes the therapy of chronic lymphocytic leukemia.

Genetic markers of chronic lymphocytic leukemia: a retrospective study of 312 patients from a reference center in Lebanon.

Aim: Chronic lymphocytic leukemia (CLL) is a highly heterogenous hemopathy. Genetic stratification of CLL patients has important prognostic and therapeutic values -mainly immunoglobulin heavy chain variable regiongene (IGHV) mutational status and the presence of cytogenetic abnormalities. The genetics of CLL in Lebanon is scarcely described in the literature. Patients & methods: In this work, we studied the genetic biomarkers of 312 Lebanese CLL patients. Results: Prominent IGHV genes were IGHV4-34, IGHV1-69 and IGHV3-30; and CLL #1 and #5 presented major subsets. Some similarities as well as major differences were highlighted when comparing our data with previously published data. Conclusion: The distribution of IGHV alleles in our series differed from previously described distributions, suggesting involvement of antigenic selection and regional variables in CLL pathogenesis.

Focal adhesion kinase activation by calcium-dependent calpain is involved in chronic lymphocytic leukaemia cell aggressiveness.

Signalling events downstream the B-cell receptor (BCR) are central for the survival and progression of chronic lymphocytic leukaemia (CLL) cells. Focal adhesion kinase (FAK), regulated through calpain, interacts with molecules of BCR signalling, cytoskeletal modelling and disease progression, such as Src/Lyn, cortactin and HS1. Hypothesizing that FAK might play a key role in CLL pathogenesis, we observed a down-modulation of FAK whole form, associated with FAK cleavage due to calpain activity upon BCR stimulation. Patients, whose cells were able to release Ca++ after BCR stimulation, had less amount of full-length FAK, which translated into a higher presence of cleaved/activated form of the protein phosphorylated at Y397, these features being mostly shown by immunoglobulin heavy chain (IGHV)-unmutated poor-prognosis patients. Moreover, we found that cortactin and HS1 proteins were overexpressed in those cells, suggesting a possible interplay with FAK. Treatment with the FAK inhibitor Defactinib was able to induce apoptosis in CLL cells. In conclusion, the malignant phenotype in unfavourable-prognosis patients seems to be encouraged by the overexpression of cortactin and HS1, that, together with FAK, may be involved in a druggable pathogenetic pathway in CLL.

Autonomous B-cell receptor signaling and genetic aberrations in chronic lymphocytic leukemia-phenotype monoclonal B lymphocytosis in siblings of patients with chronic lymphocytic leukemia.

Clonal expansion of CD5-expressing B cells, commonly designated as monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL). The mechanisms driving subclinical MBL B-cell expansion and progression to CLL, occurring in approximately 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for the pathogenesis of CLL. The objectives of this study were functional characterization of the BCR of MBL in siblings of CLL patients and a comparison of genetic variants in MBL-CLL sibling pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per microliter (median: 37/μL) in 34 of 191 (17.8%) siblings of CLL patients. Clonal BCR isolated from highly purified CLL-phenotype cells induced robust calcium mobilization in BCR-deficient murine pre-B cells in the absence of external antigen and without experimental crosslinking. This autonomous BCR signal was less intense than the signal originating from the CLL BCR of their CLL siblings. According to genotyping by single nucleotide polymorphism array, whole exome, and targeted panel sequencing, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, respectively. Likewise, the prevalence of recurrent CLL-associated genetic variants was similar between CLL and matched MBL samples. However, copy number variations and small variants were frequently subclonal in MBL cells, suggesting their acquisition during subclinical clonal expansion. These findings support a stepwise model of CLL pathogenesis, in which autonomous BCR signaling leads to a non-malignant (oligo)clonal expansion of CD5+ B cells, followed by malignant progression to CLL after acquisition of pathogenic genetic variants.