Chronic Lymphocytic Leukemia Progression Research Articles

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.

Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies

AbstractThe BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter’s syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

EBAG9 controls CD8+ T cell memory formation responding to tumor challenge in mice.

Insight into processes that determine CD8+ T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often use high-avidity CD8+ T cells in adoptive-transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon noninflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8+ T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen–mismatched leukocytes. CD8+ memory commitment was analyzed in Ebag9-deficient mice that exhibited enhanced tumor cell lysis. This property endowed Ebag9–/– mice with extended control of Tcl-1 oncogene–induced chronic lymphocytic leukemia progression. In Ebag9–/– mice, an expanded memory population was obtained for anti-HY and anti–SV-40 T antigen–specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8+ T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9+/+ and Ebag9–/– T cells. In Ebag9–/– cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation and anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8+ memory lineage.

Chronic Lymphocytic Leukemia Progression Diagnosis with Intrinsic Cellular Patterns via Unsupervised Clustering.

Simple SummaryDistinguishing between chronic lymphocytic leukemia (CLL), accelerated CLL (aCLL), and full-blown transformation to diffuse large B-cell lymphoma (Richter transformation; RT) has significant clinical implications. Identifying cellular phenotypes via unsupervised clustering provides the most robust analytic performance in analyzing digitized pathology slides. This study serves as a proof of concept that using an unsupervised machine learning scheme can enhance diagnostic accuracy. Identifying the progression of chronic lymphocytic leukemia (CLL) to accelerated CLL (aCLL) or transformation to diffuse large B-cell lymphoma (Richter transformation; RT) has significant clinical implications as it prompts a major change in patient management. However, the differentiation between these disease phases may be challenging in routine practice. Unsupervised learning has gained increased attention because of its substantial potential in data intrinsic pattern discovery. Here, we demonstrate that cellular feature engineering, identifying cellular phenotypes via unsupervised clustering, provides the most robust analytic performance in analyzing digitized pathology slides (accuracy = 0.925, AUC = 0.978) when compared to alternative approaches, such as mixed features, supervised features, unsupervised/mixed/supervised feature fusion and selection, as well as patch-based convolutional neural network (CNN) feature extraction. We further validate the reproducibility and robustness of unsupervised feature extraction via stability and repeated splitting analysis, supporting its utility as a diagnostic aid in identifying CLL patients with histologic evidence of disease progression. The outcome of this study serves as proof of principle using an unsupervised machine learning scheme to enhance the diagnostic accuracy of the heterogeneous histology patterns that pathologists might not easily see.

CircZNF91 Promotes the Malignant Phenotype of Chronic Lymphocytic Leukemia Cells by Targeting the miR-1283/WEE1 Axis.

Background Circular RNAs (circRNAs) are frequently dysregulated in cancers and are implicated in tumorigenesis and tumor progression. In this study, we investigated the role of circZNF91 in regulating the malignant phenotype of chronic lymphocytic leukemia (CLL) cells and the underlying molecular mechanism. Material/Methods The expression of circZNF91 was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding sequences between circZNF91/miR-1283 and miR-1283/WEE1 were predicted by the bioinformatic database. The functional interactions were confirmed by the dual-luciferase reporter, RT-qPCR, and Western blot assays. The functional roles of the circZNF91/miR-1283/WEE1 axis in CLL progression were examined by cell proliferation, apoptosis, and EdU incorporation assays. Results circZNF91 was upregulated in CLL samples. Silencing circZNF91 attenuated CLL cell proliferation and induced apoptosis and cell cycle arrest. circZNF91 could sponge miR-1283 to suppress its activity, which in turn upregulated WEE1 expression. Silencing circ-TTBK2 reduced WEE1 expression, while the inhibitor of miR-1283 enhanced WEE1 expression. The miR-1283/WEE1 axis mediated the effects of circZNF91 on cell proliferation and apoptosis, as well as induced cell cycle regulation. Conclusions The circZNF91/miR-1283/WEE1 axis is engaged in the pathological phenotypes of CLL cells, which could serve as potential targets for future therapy development.

TGF-β/SMAD Pathway Is Modulated by miR-26b-5p: Another Piece in the Puzzle of Chronic Lymphocytic Leukemia Progression.

Simple SummaryTGF-β is a key immunoregulatory pathway that can limit the proliferation of B-lymphocytes. Chronic lymphocytic leukemia (CLL) has been historically conceptualized as a neoplasm characterized by accumulation of mature B cells escaping programmed cell death and undergoing cell-cycle arrest in the G0/G1 phase. However, new evidence indicates that tumor expansion is in fact a dynamic process in which cell proliferation also plays an important role. In general, cancers progress by the emergence of subclones with genomic aberrations distinct from the initial tumor. Often, these subclones are selected for advantages in cell survival and/or growth. Here, we provide novel evidence to explain, at least in part, the origins of CLL progression in a subgroup of patients with a poor clinical outcome. In this cohort, the immunoregulatory pathway TGF-β/SMAD is modulated by miR-26b-5p and the impairment of this axis bypasses cell cycle arrest in CLL cells facilitating disease progression.Clinical and molecular heterogeneity are hallmarks of chronic lymphocytic leukemia (CLL), a neoplasm characterized by accumulation of mature and clonal long-lived CD5 + B-lymphocytes. Mutational status of the IgHV gene of leukemic clones is a powerful prognostic tool in CLL, and it is well established that unmutated CLLs (U-CLLs) have worse evolution than mutated cases. Nevertheless, progression and treatment requirement of patients can evolve independently from the mutational status. Microenvironment signaling or epigenetic changes partially explain this different behavior. Thus, we think that detailed characterization of the miRNAs landscape from patients with different clinical evolution could facilitate the understanding of this heterogeneity. Since miRNAs are key players in leukemia pathogenesis and evolution, we aim to better characterize different CLL behaviors by comparing the miRNome of clinically progressive U-CLLs vs. stable U-CLLs. Our data show up-regulation of miR-26b-5p, miR-106b-5p, and miR-142-5p in progressive cases and indicate a key role for miR-26b-5p during CLL progression. Specifically, up-regulation of miR-26b-5p in CLL cells blocks TGF-β/SMAD pathway by down-modulation of SMAD-4, resulting in lower expression of p21−Cip1 kinase inhibitor and higher expression of c-Myc oncogene. This work describes a new molecular mechanism linking CLL progression with TGF-β modulation and proposes an alternative strategy to explore in CLL therapy.

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

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

A Specific CD44lo CD25lo Subpopulation of Regulatory T Cells Inhibits Anti-Leukemic Immune Response and Promotes the Progression in a Mouse Model of Chronic Lymphocytic Leukemia.

Regulatory T cells (Tregs) are capable of inhibiting the proliferation, activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study, using the Eµ-TCL1 mouse model of CLL, we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly, we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation, the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment, supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far, however, above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific, described in this study Tregs subpopulation. In addition, functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover, inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit, both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether, activation of Tregs appears to be crucial for CLL progression.

Quantitative features to assist in the diagnostic assessment of chronic lymphocytic leukemia progression†.

The use of artificial intelligence methods in the image-based diagnostic assessment of hematological diseases is a growing trend in recent years. In these methods, the selection of quantitative features that describe cytological characteristics plays a key role. They are expected to add objectivity and consistency among observers to the geometric, color, or texture variables that pathologists usually interpret from visual inspection. In a recent paper in The Journal of Pathology, El Hussein, Chen et al proposed an algorithmic procedure to assist pathologists in the diagnostic evaluation of chronic lymphocytic leukemia (CLL) progression using whole-slide image analysis of tissue samples. The core of the procedure was a set of quantitative descriptors (biomarkers) calculated from the segmentation of cell nuclei, which was performed using a convolutional neural network. These biomarkers were based on clinical practice and easily calculated with reproducible tools. They were used as input to a machine learning algorithm that provided classification in one of the stages of CLL progression. Works like this can contribute to the integration into the workflow of clinical laboratories of automated diagnostic systems based on the morphological analysis of histological slides and blood smears. © 2021 The Pathological Society of Great Britain and Ireland.

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.

Inside the chronic lymphocytic leukemia cell: miRNA and chromosomal aberrations.

Alterations in microRNA (miRNA/miRs) expression are associated with the occurrence and course of human diseases, including chronic lymphocytic leukemia (CLL). Expression of miRNAs may vary among patients with CLL in different cytogenetic risk groups. The present study assessed the expression levels of the following miRNAs in 35 patients with CLL: hsa-miR-15a, −16-1, −29a, −29c, −34a, −34b, −155, −181a, −181b, −221, −222 and −223. Fluorescent in situ hybridization (FISH) analysis was performed for 13q14d, 17p13 and 11q22 deletions and chromosome 12 trisomy. Significantly higher expression levels of miR-181a, −221 and −223 were observed in the group at low risk of disease progression (stage 0) compared with the group with high risk of CLL progression (P=0.036, P=0.019 and P=0.038, respectively). The present study revealed that the expression levels of miRNA-181b and miRNA-223 were significantly higher in the group of patients without D13S319 deletion (P=0.039 and P=0.037, respectively). Moreover, the expression levels of miR-15a and miRNA-29c were demonstrated to be significantly higher in the group of patients with CLL who had a tumor protein p53 deletion, identified by FISH, compared with patients without this lesion (P=0.047, P=0.03 respectively). Based on receiver operating characteristic curve analysis, the present study revealed that miR-181a, −221 and −223 expression was able to distinguish low and high risk of CLL progression in patients. Among the tested miRNAs, miRNA-181a, −221 and −223 were indicated to have the greatest diagnostic potential in CLL.

The rise of the lymphocyte clones: targeted agents strike back

Chronic lymphocytic leukemia (CLL) is an indolent lymphoproliferative disorder commonly seen in the elderly. Its diagnosis is often incidental. Treatment is not always required, and a clinical and biological follow-up may be sufficient. However, progressive CLL may result in shortened life expectancy and significant complications. It is therefore critical to precisely stratify CLL prognosis to guide therapeutic choices accordingly. Therapeutic options have largely evolved in the past years and current molecular biomarkers allow a better risk stratification, leading to a significant improvement of survival. We summarize herein modern diagnostic and therapeutic management of CLL.

Single-cell sequencing demonstrates complex resistance landscape in CLL and MCL treated with BTK and BCL2 inhibitors

AbstractThe genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to Bruton tyrosine kinase (BTK) inhibitors and B-cell lymphoma 2 (BCL2) inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of 8 patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.

Anti-telomerase immune response predicts disease progression in chronic lymphocytic leukemia

Human telomerase reverse transcriptase (hTERT) is broadly expressed in many cancers. High hTERT expression have been described in chronic lymphocytic leukemia (CLL). Here we investigated the relationship between anti-hTERT immunity and disease progression in 49 CLL patients. Anti-hTERT T cell responses were evaluated by IFNγ-ELISpot. Complementary flow cytometry analyses were performed, and data were analyzed in regards of the treatment received by CLL patients afterward and disease progression. Anti-hTERT responses were more frequently observed in non-progressive watch and wait patients, and in progressive patients scheduled to receive ibrutinib, as compared to patients scheduled to receive other types of treatment. In vitro , addition of the anti-PD-1 antibody nivolumab increased anti-hTERT responses. Importantly, Kaplan Meier analyses showed significantly longer progression-free survival in patients with anti-hTERT immune responses at diagnosis as compared to non-responder patients. Our results show that anti-hTERT T cell responses represent a new potential biomarker predictive of CLL clinical outcome.

How can we discover the most valuable types of big data and artificial intelligence-based solutions? A methodology for the efficient development of the underlying analytics that improve care

BackgroundMuch has been invested in big data and artificial intelligence-based solutions for healthcare. However, few applications have been implemented in clinical practice. Early economic evaluations can help to improve decision-making by developers of analytics underlying these solutions aiming to increase the likelihood of successful implementation, but recommendations about their use are lacking. The aim of this study was to develop and apply a framework that positions best practice methods for economic evaluations alongside development of analytics, thereby enabling developers to identify barriers to success and to select analytics worth further investments.MethodsThe framework was developed using literature, recommendations for economic evaluations and by applying the framework to use cases (chronic lymphocytic leukaemia (CLL), intensive care, diabetes). First, the feasibility of developing clinically relevant analytics was assessed and critical barriers to successful development and implementation identified. Economic evaluations were then used to determine critical thresholds and guide investment decisions.ResultsWhen using the framework to assist decision-making of developers of analytics, continuing development was not always feasible or worthwhile. Developing analytics for progressive CLL and diabetes was clinically relevant but not feasible with the data available. Alternatively, developing analytics for newly diagnosed CLL patients was feasible but continuing development was not considered worthwhile because the high drug costs made it economically unattractive for potential users. Alternatively, in the intensive care unit, analytics reduced mortality and per-patient costs when used to identify infections (− 0.5%, − €886) and to improve patient-ventilator interaction (− 3%, − €264). Both analytics have the potential to save money but the potential benefits of analytics that identify infections strongly depend on infection rate; a higher rate implies greater cost-savings.ConclusionsWe present a framework that stimulates efficiency of development of analytics for big data and artificial intelligence-based solutions by selecting those applications of analytics for which development is feasible and worthwhile. For these applications, results from early economic evaluations can be used to guide investment decisions and identify critical requirements.

Combined Ibrutinib and Venetoclax for First-Line Treatment of Patients with Chronic Lymphocytic Leukemia (CLL): Focus on Long-Term MRD Results

Combined Ibrutinib and Venetoclax for First-Line Treatment of Patients with Chronic Lymphocytic Leukemia (CLL): Focus on Long-Term MRD Results

B Cell-Restricted Depletion of Dnmt3a Activates Notch Signaling and Causes Chronic Lymphocytic Leukemia

B Cell-Restricted Depletion of Dnmt3a Activates Notch Signaling and Causes Chronic Lymphocytic Leukemia

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.

Addressing a New Challenge in Chronic Lymphocytic Leukemia: Outcomes of Therapies after Exposure to Both a Covalent Bruton's Tyrosine Kinase Inhibitor and Venetoclax

Addressing a New Challenge in Chronic Lymphocytic Leukemia: Outcomes of Therapies after Exposure to Both a Covalent Bruton's Tyrosine Kinase Inhibitor and Venetoclax

Long-Term Outcome of Treatment-Naïve and Relapsed/Refractory Patients with CLL and TP53 Aberrations Treated with Ibrutinib, with or without Rituximab

Long-Term Outcome of Treatment-Naïve and Relapsed/Refractory Patients with CLL and TP53 Aberrations Treated with Ibrutinib, with or without Rituximab