Pathways In Chronic Lymphocytic Leukemia Research Articles

Genetic variability profiling of the p53 signaling pathway in chronic lymphocytic leukemia. Individual and combined analysis of TP53, MDM2 and NQO1 gene variants.

TP53 gene disruption, including 17p13 deletion [del(17p)] and/or TP53 mutations, is a negative prognostic biomarker in chronic lymphocytic leukemia (CLL) associated with disease progression, treatment failure and shorter survival. Germline variants in p53 signaling pathway genes could also lead to p53 dysfunction, but their involvement in CLL has not been thoroughly evaluated. The aim of this study was to determine the association of TP53, MDM2 and NQO1 gene variability with clinical and genetic data of CLL patients. Individual genotype and haplotype data of CLL patients were compared with clinical prognostic factors, cytogenetic and molecular cytogenetic findings as well as IGHV and TP53 mutational status. The study included 116 CLL patients and 161 healthy blood donors. TP53 (rs1042522, rs59758982, rs1625895), NQO1 (rs1800566) and MDM2 (rs2279744, rs150550023) variants were genotyped using different PCR approaches. Analysis of genotype frequencies revealed no association with the risk of CLL. TP53 rs1042522, rs1625895 and MDM2 rs2279744 variants were significantly associated with abnormal karyotype and the presence of del(17p). Similarly, these two TP53 variants were associated with TP53 disruption. Moreover, TP53 C-A-nondel and G-A-del haplotypes (rs1042522-rs1625895-rs59758982) were associated with an increased likelihood of carrying del(17p) and TP53 disruptions. MDM2 T-nondel haplotype (rs2279744-rs150550023) was found to be a low risk factor for del(17p) (OR = 0.32; CI: 0.12-0.82; p = 0.02) and TP53 disruptions (OR = 0.41; CI: 0.18-0.95; p = 0.04). Our findings suggest that TP53 and MDM2 variants may modulate the risk to have chromosome alterations and TP53 disruptions, particularly del(17p). To our knowledge this is the first study of several germline variants in p53 pathway genes in Argentine patients with CLL.

T-bet suppresses proliferation of malignant B cells in chronic lymphocytic leukemia

AbstractThe T-box transcription factor T-bet is known as a master regulator of the T-cell response but its role in malignant B cells has not been sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with a genetic knockout of Tbx21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity, induced by inflammatory signals provided by the microenvironment, triggered T-bet expression, which affected promoter-proximal and distal chromatin coaccessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling and negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of patients with CLL. Our study uncovered a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling, which has implications for the stratification and therapy of patients with CLL. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in the inflammatory signaling pathways in CLL.

IκBε deficiency accelerates disease development in chronic lymphocytic leukemia.

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.

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.

Targeting the NTSR2/TrkB oncogenic pathway in chronic lymphocytic leukemia

Current therapies that target the B-cell receptor pathway or the inhibition of anti-apoptotic proteins do not prevent the progressive forms of chronic lymphocytic leukemia (CLL), have low long-term efficacy and are subject to therapeutic resistance. Deciphering the mechanisms of leukemic cell survival and searching for new specific targets therefore remain major challenges to improve the management of this disease. It was evidenced that NTSR2 (neurotensin receptor 2), through the recruitment of TRKB (tropomyosin related kinase B), induces survival pathways in leukemic B cells. We have investigated the therapeutic potential of this protein complex as a new target. The binding domain of NTSR2 and TRKB was identified and a peptide targeting the latter was designed. The peptide binds TRKB and efficiently decreases the interaction of the two proteins. It is also effectively internalized by CLL-B cells in which it notably affects Src family kinase signaling and anti-apoptotic proteins levels. It demonstrated a cytotoxic effect both in vitro on the MEC-1 cell line and ex vivo on a cohort of 30 CLL patients. Altogether, these results underline the therapeutic potential of the NTSR2/TRKB protein complex as a target in CLL and open new perspectives for the development of targeted therapies.

ACOX1-mediated peroxisomal fatty acid oxidation contributes to metabolic reprogramming and survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.

Deciphering the Dynamics of the B-NHL Single-Cell Landscape during BTK Inhibition

Introduction: The inhibition of Bruton's tyrosine kinase (BTK) as a therapeutic strategy has dramatically improved the management of patients with chronic lymphocytic leukemia (CLL) and other B-cell-Non Hodgkin lymphomas (B-NHL). However, therapy resistance and treatment toxicity persist as significant clinical challenges. It has been previously reported that BTK inhibition not only targets the overactivated BCR pathway in CLL and other B-NHL, but also coincides with profound alterations in the tumor microenvironment including immune cells. Therefore, it is vital to understand these tumor-immune cell interactions during BTK inhibition to enhance response and reduce toxicity, advancing personalized care for CLL and B-NHL patients in the future. Methods: We developed a high-parametric analysis pipeline based on full spectrum cytometry, a novel technology allowing for the simultaneous assessment of 40 markers at single-cell level. In order to comprehensively assess the phosphoproteome relevant to BTK inhibition we established a signaling panel covering >30 proteins including pBTK, pSyk, pPLCg, pS6 and pAMPKa. Additionally, we designed 3 panels with up to 40 markers characterizing T cell activation and exhaustion, cellular trafficking, inhibitory immune checkpoints and cytokine production. Using our scalable and reproducible bioinformatics pipeline, we analyzed a cohort of 160 samples, longitudinally collected from 57 patients (CLL, MCL, MZL) before BTKi treatment, directly after treatment initiation, at leukocyte peak count and during therapy up to 6 months after therapy initiation. Results: Given that T cells are a crucial arm in governing anti-lymphoma immune response, we sought to elucidate the changes associated with BTK inhibition in the T cell compartment of the patients at high resolution (figure A). We observed certain changes in the fraction of T cell subtypes, notably a decrease in central memory stem T cells and an increase in γδ T cells at leukocyte peak and during later stages of therapy. Our analysis uncovered substantial changes in the T cell marker expression over the course of BTK inhibition. This includes a reversal of exhausted phenotype marked by downregulation of PD1, CD39 and CD38 and a skewed differentiation of CD4 T cells towards Th1 phenotype illustrated by a pronounced upregulation of the transcription factor T-bet. In addition, we detected restored proliferative and cytotoxic capacity in the CD8 and CD4 T cell compartments characterized by upregulation of GRZB, CD226, CD28, CD25 and Ki67. By leveraging our platform, we successfully captured the signalosome of malignant B cells at a high resolution. As anticipated, we detected significant heterogeneity in the malignant B cell signatures across individual patients. Interestingly, even at the initial sampling timepoint following treatment start, we observed substantially altered B cell signatures encompassing both the phosphoproteome and inhibitory immune checkpoints (figure B). Our preliminary data suggest that the dynamic changes induced by BTK inhibition extend beyond the signaling nodes associated with the BCR pathway and also involve the NFkB and p53 pathways. Conclusion: In this study, we established a platform for deep and longitudinal interrogation of the tumor and immune cell compartments to capture the dynamic and patient-specific changes occurring during BTK inhibition. Our preliminary data obtained from a large patient cohort of 57 B-NHL patients reveals a profound remodeling of not only the BCR signaling pathway in tumor cells, but also of the T cell compartment. Our data will be useful for a better understanding of treatment response and toxicity in the context of BTK inhibition.

Targeting BCL2 pathways in CLL: a story of resistance and ingenuity.

Chronic lymphocytic leukemia (CLL) is common amongst leukemic malignancies, prompting dedicated investigation throughout the years. Over the last decade, the treatment for CLL has significantly advanced with agents targeting B-cell lymphoma 2 (BCL2), Bruton's tyrosine kinase, and CD20. Single agents or combinations of these targets have proven efficacy. Unfortunately, resistance to one or multiple of the new treatment targets develops. Our review investigates various mechanisms of resistance to BCL2 inhibitors, including mutations in BCL2, alterations in the Bcl protein pathway, epigenetic modifications, genetic heterogeneity, Richter transformation, and alterations in oxidative phosphorylation. Additionally, the review will discuss potential avenues to overcome this resistance with novel agents such as bispecific antibodies, Bruton's tyrosine kinase (BTK) degraders, non-covalent BTK inhibitors, and chimeric antigen receptor T (CART).

NOTCH1-mutated chronic lymphocytic leukemia displays high endoplasmic reticulum stress response with druggable potential.

Constitutive activation of NOTCH1-wild-type (NT1-WT) signaling is associated with poor outcomes in chronic lymphocytic leukemia (CLL), and NOTCH1 mutation (c.7541_7542delCT), which potentiates NOTCH1 signaling, worsens the prognosis. However, the specific mechanisms of NOTCH1 deregulation are still poorly understood. Accumulative evidence mentioned endoplasmic reticulum (ER) stress/unfolded protein response (UPR) as a key targetable pathway in CLL. In this study, we investigated the impact of NOTCH1 deregulation on CLL cell response to ER stress induction, with the aim of identifying new therapeutic opportunities for CLL. We performed a bioinformatics analysis of NOTCH1-mutated (NT1-M) and NT1-WT CLL to identify differentially expressed genes (DEGs) using the rank product test. Quantitative real-time polymerase chain reaction (qPCR), Western blotting, cytosolic Ca2+, and annexin V/propidium iodide (PI) assay were used to detect curcumin ER stress induction effects. A median-effect equation was used for drug combination tests. The experimental mouse model Eμ-TCL1 was used to evaluate the impact of ER stress exacerbation by curcumin treatment on the progression of leukemic cells and NOTCH1 signaling. Bioinformatics analysis revealed gene enrichment of the components of the ER stress/UPR pathway in NT1-M compared to those in NT1-WT CLL. Ectopic expression of NOTCH1 mutation upregulated the levels of ER stress response markers in the PGA1 CLL cell line. Primary NT1-M CLL was more sensitive to curcumin as documented by a significant perturbation in Ca2+ homeostasis and higher expression of ER stress/UPR markers compared to NT1-WT cells. It was also accompanied by a significantly higher apoptotic response mediated by C/EBP homologous protein (CHOP) expression, caspase 4 cleavage, and downregulation of NOTCH1 signaling in NT1-M CLL cells. Curcumin potentiated the apoptotic effect of venetoclax in NT1-M CLL cells. In Eμ-TCL1 leukemic mice, the administration of curcumin activated ER stress in splenic B cells ex vivo and significantly reduced the percentage of CD19+/CD5+ cells infiltrating the spleen, liver, and bone marrow (BM). These cellular effects were associated with reduced NOTCH1 activity in leukemic cells and resulted in prolonged survival of curcumin-treated mice. Overall, our results indicate that ER stress induction in NT1-M CLL might represent a new therapeutic opportunity for these high-risk CLL patients and improve the therapeutic effect of drugs currently used in CLL.

Dimethyl itaconate selectively targets inflammatory and metabolic pathways in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) co-evolves with its own microenvironment where inflammatory stimuli including toll-like receptors (TLR) signaling can protect CLL cells from spontaneous and drug-induced apoptosis by upregulating IκBζ, an atypical co-transcription factor. To dissect IκBζ-centered signaling pathways, we performed a gene expression profile of primary leukemic cells expressing either high or low levels of IκBζ after stimulation, highlighting that IκBζ is not only an inflammatory gene but it may control metabolic rewiring of malignant cells thus pointing to a novel potential opportunity for therapy. We exploited the capacity of the dimethyl itaconate (DI), an anti-inflammatory electrophilic synthetic derivative of the metabolite Itaconate, to target IκBζ. CLL cells, murine leukemic splenocytes, and leukocytes from healthy donors were treated in vitro with DI that abolished metabolic activation and reduced cell viability of leukemic cells only, even in the presence of robust TLR prestimulation. RNA sequencing highlighted that in addition to the expected electrophilic stress signature observed after DI treatment, novel pathways emerged including the downregulation of distinct MHC class II complex genes. In conclusion, DI not only abrogated the proinflammatory effects of TLR stimulation but also targeted a specific metabolic vulnerability in CLL cells.

Abstract 4725: The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia

Abstract Introduction: The introduction of Bruton’s tyrosine kinase (BTK) inhibitors (BTKi) to target B cell receptor (BCR) pathways in chronic lymphocytic leukemia (CLL) has significantly improved clinical responses. However, the effectiveness of BTKi is limited by the development of adaptive drug resistance. Therefore, there is an urgent need to identify critical biologic features that determine BTKi efficacy in order to develop more effective therapies. Epigenetic aberrations play an important role in tumor initiation, progression, and drug resistance. However, it is currently unknown if BCR signaling is also dependent on epigenetic mechanisms to support oncogenic gene expression and survival in CLL B cells. Methods and Results: To gain new insights into the role of epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in other cohorts at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies show that suppressing BCR signaling by BTKi treatment leads to an alteration of a pro-survival epigenetic signature (defined by histone modifications and chromatin accessibility landscapes) in CLL and that the genes regulated by these chromatin changes are enriched in pathways associated with malignant B cell survival (i.e., BCR signaling and apoptosis pathways). Importantly, disruption of this pro-survival epigenetic signature is necessary for effective BTKi treatment. We further find that the transcription factor (TF) Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) couples the BCR signaling to control this pro-survival epigenetic program that supports CLL B cell survival. In specific, we show that nuclear depletion of NFATc1 is required for BTKi induced epigenetic alteration and effective BTKi treatment. Depletion of NFATc1 disrupts the pro-survival epigenetic signature and inhibits CLL B cells survival, which improves the efficacy of BTKi treatment in CLL. Conclusion: Here using leukemic cells from CLL patients with BTKi treatment, we demonstrate that epigenetic regulators are exploited by the oncogenic signaling pathway to support malignant cell survival. Specifically, NFATc1 is utilized by the BCR signaling pathway to control an epigenetic signature that supports CLL cell survival. This BCR regulated epigenetic machinery can be targeted to maintain and enhance BTKi treatment efficacy in CLL. Citation Format: Zhiquan Wang, Huihuang Yan, Justin C. Boysen, Esteban Braggio, Sameer A. Parikh, Neil E. Kay. The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4725.

SOHO State of the Art Updates and Next Questions: New Targetable Pathways in Chronic Lymphocytic Leukemia.

Regulatory approvals of Bruton tyrosine kinase (BTK) inhibitors and BCL2 inhibitors have transformed the therapeutic paradigm in chronic lymphocytic leukemia (CLL). However, despite significant improvement, treatment discontinuations due to an acquired resistance mutation or intolerance to these agents are common. Those who are refractory and/or intolerant to both these classes of drugs - the "double exposed/refractory" patients - pose a real challenge in clinical practice and are in dire need of novel therapeutic approaches. In this manuscript, we review the ongoing efforts addressing this unmet clinical need including the ongoing development of non-covalent BTK inhibitors, BTK degraders, novel BH3-mimetics, therapeutic antibodies targeting novel antigens and immune cell enabling therapies.

Investigation of Lipid Metabolism Dysregulation in Chronic Lymphocytic Leukemia and the Role of ENPP2 in Tumorigenesis of Chronic Lymphocytic Leukemia

Introduction Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world, and although the advent of novel targeted drugs has greatly improved the prognosis of CLL patients, CLL remains incurable at present. Improving the risk stratification of CLL patients and finding new CLL therapeutic targets are urgent issues to be addressed. Methods We performed untargeted metabolomic analysis using LC-MS on sera from CLL patients and healthy controls (HC). Statistical analysis was used to screen for differential metabolites (DMs). Differentially expressed genes (DEGs) were also screened for enrichment pathway enrichment relying on transcriptome data analysis through the GEO database. Finally, DMs and DEGs were analyzed together to explore CLL metabolic alteration pathways. In this study, we targeted the gene ENPP2. Peripheral blood samples from 82 CLL patients were collected at the Department of Hematology in Shandong Provincial Hospital. Expressions levels of ENPP2 in CLL patients were determined by quantitative RT-PCR. In addition, cell viability and apoptosis were assessed by cell counting kit-8 and annexin V-PE/7AAD staining. Results A total of 52 differential metabolites were annotated, 40 of them were up-regulated and 12 were down-regulated. There were significant differences in sphingolipids (SP), glycerolipids (GL), glycerophospholipids (GP), and fatty acids (FA) levels in CLL patients compared to health controls. Fifty-two differential metabolites between samples from CLL patients and HC were analyzed by pathway analysis using metaboanalyst v5.0. KEGG enrichment showed that differential metabolites in CLL patients and HC were mainly annotated and enriched in human diseases, metabolism, and organismal systems (Figure 1A). Furthermore, we obtained differential expression information from the GEO database for CLL patients with HC. A total of 539 DEGs were screened, of which 279 were up-regulated and 260 were down-regulated. A total of 257 KEGG pathways were enriched. In the current work, a joint analysis of the two histological data was performed to validate each other. A total of 20 KEGG pathways were repeatedly obtained from metabolomics and transcriptomics data-based pathway analysis, including pathways in cancer, insulin resistance, long-term depression, regulation of lipolysis in adipocytes metabolic pathways, glycerophospholipid metabolism, glycerolipid metabolism, sphingolipid metabolism, arachidonic acid metabolism, ether lipid metabolism, inositol phosphate metabolism, choline metabolism in cancer, phospholipase D signaling pathway. A PPI network was constructed for the identified significantly different genes, from which we also screened for the gene ENPP2, a secreted lysophospholipase D, that we desired to investigate further (Figure 1B). We validated the difference in ENPP2 expression between CLL patients and healthy donors in the GEO database and its guiding effect on CLL prognosis. The results showed that elevated expression of ENPP2 in CLL patients and relatively high expression in CLL patients implied a worse prognosis. In addition, to validate the role of ENPP2 in CLL, we did the further investigation in CLL patients in our own center. Aberrantly increased expression of ENPP2 were detected in CLL primary cells and cell line (MEC1) at mRNA compared with normal B cells. To investigate the biological processes of ENPP2 involving in CLL progression, functional assays were performed. The survival rate of CLL cells treated with ENPP2 inhibitor PF-8380 was significantly decreased, while apoptosis was increased. In addition, we performed a combination of ibrutinib with PF8380, which showed a further decrease in cell viability and showed better anti-CLL effects. Conclusion Taken together, the present work provides evidence that the identified metabolic biomarkers can be used for CLL diagnosis and screening. The combined analysis of metabolomics and transcriptomics data provides a comprehensive understanding of the metabolic pathways in CLL. Besides, this study was the first investigation on the role of ENPP2 in CLL. ENPP2 expression was increased in CLL cells, and the ENPP2 inhibitor PF-8380 inhibited cell proliferation and induced apoptosis, with therapeutic potential. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ezrin Is a Potential Druggable Target in Chronic Lymphocytic Leukemia

Despite development in therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or are refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors, but its role in hematological neoplasms is still poorly explored. There is evidence that EZR plays a key function in cell survival and activation of BCR-mediated signaling in B-cell lymphomas. Here, we uncover the expression of EZR in samples from CLL and healthy donors, and the cellular and molecular effects of the NSC305787, a pharmacological EZR inhibitor, in CLL cellular models. For gene expression studies, EZR mRNA expression data from healthy donors (normal lymphocytes, n = 11) and CLL patients (n = 103) samples were derived from the publicly accessible AmaZonia! database 2008 (discovery cohort). Purified CD19+ cells from peripheral blood samples were collected from 56 CLL patients and 10 age-matched healthy donors from the University Hospital of the Medical School of Ribeirão Preto (validation cohort). For functional and molecular assays, peripheral blood mononuclear cells of 24 CLL patients from the Instituto do Câncer do Estado de São Paulo were used. All samples were obtained after informed consent and protocol approved by the Ethical Committee of the Institution. For functional transcriptomics, RNA-seq data published by Landau et al. were obtained from cBioPortal, and all transcripts were pre-ranked according to their differential expression by comparing samples with high versus low EZR expression using limma-voom package in Galaxy and used for gene set enrichment analysis (GSEA). The MEC-1 cell line was cultured following the recommendations of DSMZ. PBMCs from CLL patients were cultured in RPMI Media 1640 and 10% autologous serum. NSC305787 was used as an EZR inhibitor. Cell viability was determined by MTT assay, apoptosis rates were determined by annexin V/PI labeling and flow cytometry, DNA content was performed by PI labeling and flow cytometry, protein expression was determined by Western Blot assay, and gene expression was evaluated by quantitative PCR. Statistical analyses were performed using GraphPad Prism 8; Mann-Whitney test, Student's t-test, or ANOVA and Bonferroni post-test were used for measurable factors, as appropriate. A value of p < 0.05 was considered statistically significant. EZR was highly expressed in lymphocytes derived from CLL patients (discovery cohort: p = 0.003; validation cohort: p = 0.005). GSEA indicated that high EZR expression was positively associated with relevant signaling pathways associated with CLL development and progression, including p53, PI3K/AKT/mTOR, NFkB, and MAPK (all p < 0.05 and FDR q < 0.05). Of note, pharmacological inhibition of EZR had remarkable antineoplastic effects in primary cells derived from CLL patients, corroborating the potential relevance of this target in disease maintenance. In a CLL cell line, EZR inhibition reduced viability, clonogenicity, cell cycle progression, and induced apoptosis (all p < 0.05). Pharmacological EZR inhibition also reduced activation of the ERK, S6RP, and NFkB, indicating that EZR not only associates, but participates in the activation of these signaling pathways in CLL. In conclusion, our results indicate that EZR is high expressed in CLL and associated with molecular signatures relevant to the development and maintenance of the disease. Pharmacological EZR inhibitions attenuate various cellular and molecular behaviors associated with high risk in CLL and may present a new class of drugs to expand the treatment repertoire of this disease. Supported by: FAPESP, CNPQ, and CAPES.

Ezrin is highly expressed and a druggable target in chronic lymphocytic leukemia

AimsDespite the development of therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors and plays a key role in cell survival and BCR-mediated signaling activation in B-cell lymphomas. However, its role in hematological neoplasms remains poorly explored. Main methodsThe present study assessed EZR expression in samples from CLL patients and healthy donors and evaluated the cellular and molecular effects of a pharmacological EZR inhibitor, NSC305787, in CLL cellular models. Key findingsEZR was highly expressed and positively associated with relevant signaling pathways related to CLL development and progression, including TP53, PI3K/AKT/mTOR, NF-κB, and MAPK. NSC305787 reduced viability, clonogenicity, and cell cycle progression and induced apoptosis in CLL cells. Pharmacological EZR inhibition also attenuated ERK, S6RP, and NF-κB activation, indicating that EZR not only associates with but also activates these signaling pathways in CLL. Ex vivo assays revealed that the EZR inhibition-induced cell viability reduction was independent of molecular risk and the Binet stage. SignificanceOur study provides insights into EZR as a pharmacological target in CLL, shedding light on a novel strategy for treating this disease.

Hes1 Controls Proliferation and Apoptosis in Chronic Lymphoblastic Leukemia Cells by Modulating PTEN Expression.

Hairy and enhancer of split homolog-1 (HES1), regulated by the Notch, has been reported to play important roles in the immune response and cancers, such as leukemia. In this study, we aim to explore the effect of HES1-mediated Notch1 signaling pathway in chronic lymphocytic leukemia (CLL). Reverse transcription quantitative polymerase chain reaction and Western blot assay were conducted to determine the expression of HES1, Notch1, and PTEN in B lymphocytes of peripheral blood samples of 60 CLL patients. We used lentivirus-mediated overexpression or silencing of HES1 and the Notch1 signaling pathway inhibitor, MW167, to detect the interaction among HES1, Notch1, and PTEN in CLL MEC1 and HG3 cells. MTT assay and flow cytometry were employed for detection of biological behaviors of CLL cells. HES1 and Notch1 showed high expression, but PTEN displayed low expression in B lymphocytes of peripheral blood samples of patients with CLL in association with poor prognosis. HES1 bound to the promoter region of PTEN and reduced PTEN expression. Overexpression of HES1 activated the Notch1 signaling pathway, thus promoting the proliferation of CLL cells, increasing the proportion of cells arrested at the S phase and limiting the apoptosis of CLL cells. Collectively, HES1 can promote activation of the Notch1 signaling pathway to cause PTEN transcription inhibition and the subsequent expression reduction, thereby promoting the proliferation and inhibiting the apoptosis of CLL cells.

Cellular Therapy in High-Risk Relapsed/Refractory Chronic Lymphocytic Leukemia and Richter Syndrome.

Despite the development of highly effective, targeted inhibitors of B-cell proliferation and anti-apoptotic pathways in chronic lymphocytic leukemia (CLL), these treatments are not curative, and many patients will develop either intolerance or resistance to these treatments. Transformation of CLL to high-grade lymphoma—the so-called Richter syndrome (RS)—remains a highly chemoimmunotherapy-resistant disease, with the transformation occurring following targeted inhibitors for CLL treatment being particularly adverse. In light of this, cellular therapy in the form of allogenic stem cell transplantation and chimeric antigen receptor T-cell therapy continues to be explored in these entities. We reviewed the current literature assessing these treatment modalities in both high-risk CLL and RS. We also discussed their current limitations and place in treatment algorithms.

Targeting the Non-Canonical NF-κB Pathway in Chronic Lymphocytic Leukemia and Multiple Myeloma.

Simple SummaryThis study was designed to investigate the potential for targeting the NF-κB-inducing kinase, NIK, in two common B-cell malignancies, chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). Using a selective NIK inhibitor, CW15337, we were able to demonstrate that cell lines and primary tumor cells were sensitive to the effects of NIK inhibition, whilst normal lymphocytes were significantly more resistant to its cytotoxic effects. Sensitivity to CW15337 was associated with the nuclear expression of the NF-κB subunit, p52. Importantly, tumor samples from a subset of poor prognosis CLL patients, with mutations in a gene called BIRC3, showed elevated p52 expression and were particularly sensitive to NIK inhibition. Furthermore, the combination of CW15337 and ABT-199 (venetoclax) reversed the drug resistance observed when treating tumor cells with ABT-199 alone. Our study shows the potential for targeting NIK in both CLL and MM.In this study, we evaluated an NF-κB inducing kinase (NIK) inhibitor, CW15337, in primary chronic lymphocytic leukemia (CLL) cells, CLL and multiple myeloma (MM) cell lines and normal B- and T-lymphocytes. Basal NF-κB subunit activity was characterized using an enzyme linked immunosorbent assay (ELISA), and the effects of NIK inhibition were then assessed in terms of cytotoxicity and the expression of nuclear NF-κB subunits following monoculture and co-culture with CD40L-expressing fibroblasts, as a model of the lymphoid niche. CW15337 induced a dose-dependent increase in apoptosis, and nuclear expression of the non-canonical NF-κB subunit, p52, was correlated with sensitivity to CW15337 (p = 0.01; r2 = 0.39). Co-culture on CD40L-expressing cells induced both canonical and non-canonical subunit expression in nuclear extracts, which promoted in vitro resistance against fludarabine and ABT-199 (venetoclax) but not CW15337. Furthermore, the combination of CW15337 with fludarabine or ABT-199 showed cytotoxic synergy. Mechanistically, CW15337 caused the selective inhibition of non-canonical NF-κB subunits and the transcriptional repression of BCL2L1, BCL2A1 and MCL1 gene transcription. Taken together, these data suggest that the NIK inhibitor, CW15337, exerts its effects via suppression of the non-canonical NF-κB signaling pathway, which reverses BCL2 family-mediated resistance in the context of CD40L stimulation.

Druggable Molecular Pathways in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in adults, is characterized by a high degree of clinical heterogeneity that is influenced by the disease’s molecular complexity. The genes most frequently affected in CLL cluster into specific biological pathways, including B-cell receptor (BCR) signaling, apoptosis, NF-κB, and NOTCH1 signaling. BCR signaling and the apoptosis pathway have been exploited to design targeted medicines for CLL therapy. Consistently, molecules that selectively inhibit specific BCR components, namely Bruton tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) as well as inhibitors of BCL2, have revolutionized the therapeutic management of CLL patients. Several BTK inhibitors and PI3K inhibitors with different modes of action are currently used or are in development in advanced stage clinical trials. Moreover, the restoration of apoptosis by the BCL2 inhibitor venetoclax offers meaningful clinical activity with a fixed-duration scheme. Inhibitors of the BCR and of BCL2 are able to overcome the chemorefractoriness associated with high-risk genetic features, including TP53 disruption. Other signaling cascades involved in CLL pathogenesis, in particular NOTCH signaling and NF-kB signaling, already provide biomarkers for a precision medicine approach to CLL and may represent potential druggable targets for the future. The aim of the present review is to discuss the druggable pathways of CLL and to provide the biological background of the high efficacy of targeted biological drugs in CLL.

The TKI Era in Chronic Leukemias.

Tyrosine kinases are proteins involved in physiological cell functions including proliferation, differentiation, and survival. However, the dysregulation of tyrosine kinase pathways occurs in malignancy, including hematological leukemias such as chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Particularly, the fusion oncoprotein BCR-ABL1 in CML and the B-cell receptor (BCR) signaling pathway in CLL are critical for leukemogenesis. Therapeutic management of these two hematological conditions was fundamentally changed in recent years, making the role of conventional chemotherapy nearly obsolete. The first, second, and third generation inhibitors (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) of BCR-ABL1 and the allosteric inhibitor asciminib showed deep genetic and molecular remission rates in CML, leading to the evaluation of treatment discontinuation in prospective trials. The irreversible BTK inhibitors (ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, and spebrutinib) covalently bind to the C481 amino acid of BTK. The reversible BTK inhibitor pirtobrutinib has a different binding site, overcoming resistance associated with mutations at C481. The PI3K inhibitors (idelalisib and duvelisib) are also effective in CLL but are currently less used because of their toxicity profiles. These tyrosine kinase inhibitors are well-tolerated, do have some associated in-class side effects that are manageable, and have remarkably improved outcomes for patients with hematologic malignancies.