Human Chronic Lymphocytic Leukemia Research Articles

Lessons learned from the Eµ-TCL1 mouse model of CLL

The Eµ-TCL1 mouse model has been used for over 20 years to study the pathobiology of chronic lymphocytic leukemia (CLL) and for preclinical testing of novel therapies. A CLL-like disease develops with increasing age in these mice due to a B cell specific overexpression of human TCL1. The reliability of this model to mirror human CLL is controversially discussed, as none of the known driver mutations identified in patients are found in Eµ-TCL1 mice. It has to be acknowledged that this mouse model was key to develop targeted therapies that aim at inhibiting the constitutive B cell receptor (BCR) signaling, a main driver of CLL. Inhibitors of BCR signaling became standard-of-care for a large proportion of patients with CLL as they are highly effective. The Eµ-TCL1 model further advanced our understanding of CLL biology owed to studies that crossed this mouse line with various transgenic mouse models and demonstrated the relevance of CLL-cell intrinsic and -extrinsic drivers of disease. These studies were instrumental in showing the relevance of the tumor microenvironment in the lymphoid tissues for disease progression and immune escape in CLL. It became clear that CLL cells shape and rely on stromal and immune cells, and that immune suppressive mechanisms and T cell exhaustion contribute to CLL progression. Based on this knowledge, new immunotherapy strategies were clinically tested for CLL, but so far with disappointing results. As some of these therapies were effective in the Eµ-TCL1 mouse model, the question arose concerning the translatability of preclinical studies in these mice. The aim of this review is to summarize lessons we have learnt over the last decades by studying CLL-like disease in the Eµ-TCL1 mouse model. The article focuses on pitfalls and limitations of the model, as well as the gained knowledge and potential of using this model for the development of novel treatment strategies to achieve the goal of curing patients with CLL.

Linc01588 deletion inhibits the malignant biological characteristics of hydroquinone-induced leukemic cells via miR-9-5p/SIRT1

Leukemia caused by environmental chemical pollutants has attracted great attention, the malignant leukemic transformation model of TK6 cells induced by hydroquinone (HQ) has been previously found in our team. However, the type of leukemia corresponding to this malignant transformed cell line model needs further study and interpretation. Furthermore, the molecular mechanism of malignant proliferation of leukemic cells induced by HQ remains unclear. This study is the first to reveal the expression of aberrant genes in leukemic cells of HQ-induced malignant transformation, which may correspond to chronic lymphocytic leukemia (CLL). The expression of Linc01588, a long non-coding RNA (lncRNA), was significantly up-regulated in CLL patients and leukemic cell line model which previously described. After gain-of-function assays and loss-of-function assays, feeble cell viability, severe apoptotic phenotype and the increased secretion of TNF-α were easily observed in malignant leukemic TK6 cells with Linc01588 deletion after HQ intervention. The tumors derived from malignant TK6 cells with Linc01588 deletion inoculated subcutaneously in nude mice were smaller than controls. In CLL and its cell line model, the expression of Linc01588 and miR-9–5p, miR-9–5p and SIRT1 were negative correlation respectively in CLL and cell line model, while the expression of Linc01588 and SIRT1 were positive correlation. The dual-luciferase reporter assay showed that Linc01588 & miR-9–5p, miR-9–5p & SIRT1 could bind directly, respectively. Furthermore, knockdown of miR-9–5p successfully rescued the severe apoptotic phenotype and the increased secretion of TNF-α caused by the Linc01588 deletion, the deletion of Linc01588 in human CLL cell line MEC-2 could also inhibit malignant biological characteristics, and the phenotype caused by the deletion of Linc01588 could also be rescued after overexpression of SIRT1. Moreover, the regulation of SIRT1 expression in HQ19 cells by Linc01588 and miR-9–5 P may be related to the Akt/NF-κB pathway. In brief, Linc01588 deletion inhibits the malignant biological characteristics of HQ-induced leukemic cells via miR-9–5p/SIRT1, and it is a novel and hopeful clue for the clinical targeted therapy of CLL.

B-1 derived anti-Thy-1 B cells in old aged mice develop lymphoma/leukemia with high expression of CD11b and Hamp2 that different from TCL1 transgenic mice

Human old aged unmutated chronic lymphocytic leukemia U-CLL are the TCL1+ZAP70+CD5+ B cells. Since CD5 makes the BCR signaling tolerance, ZAP70 increased in U-CLL not only TCL1+ alone. In mice, TCL1 (TCL1A) is the negative from neonate to old aged, as TC–. VH8-12/Vk21-5 is the anti-thymocyte/Thy-1 autoreactive ATA B cell. When ATA μκTg generation in mice, ATA B cells are the neonate generated CD5+ B cells in B-1, and in the middle age, CD5+ can be down or continuously CD5+, then, old aged CLL/lymphoma generation with increased CD11b in TC–ZAP70–CD5– or TC–ZAP70+CD5+. In this old aged TC–ATA B microarray analysis showed most similar to human CLL and U-CLL, and TC–ZAP70+CD5+ showed certain higher present as U-CLL. Original neonate ATA B cells showed with several genes down or further increase in old aged tumor, and old aged T-bet+CD11c+, CTNNB1hi, HMGBhi, CXCR4hi, DPP4hi and decreased miR181b. These old aged increased genes and down miR181b are similar to human CLL. Also, in old age ATA B cell tumor, high CD38++CD44++, increased Ki67+ AID+, and decreased CD180– miR15Olow are similar to U-CLL. In this old aged ATA B, increased TLR7,9 and Wnt10b. TC+Tg generated with ATAμκTg mice occurred middle age tumor as TC+ZAP70–CD5+ or TC+ZAP70+CD5+, with high NF-kB1, TLR4,6 and Wnt5b,6 without increased CD11b. Since neonatal state to age with TC+Tg continuously, middle age CLL/lymphoma generation is not similar to old aged generated, however, some increased in TC+ZAP70+ are similar to the old age TC– ATA B tumor. Then, TC– ATA B old age tumor showed some difference to human CLL. ATA B cells showed CD11b+CD22++, CD24 down, and hepcidin Hamp2++ with iron down. This mouse V8-12 similar to human V2-5, and V2-5 showed several cancers with macrophages/neutrophils generated hepcidin+ ironlow or some showed hepcidin– iron+ with tumor, and mouse V8-12 with different Vk19-17 generate MZ B cells strongly increased macrophage++ in old aged and generated intestine/colon tumor. Conclusion, neonate generated TC–ATA B1 cells in old aged tumor generation are CD11b+ in the leukemia CLL together with lymphoma cancer with hepcidin-related Hamp2++ in B-1 cell generation to control iron.

Mouse models of CLL: In vivo modeling of disease initiation, progression, and transformation to Richter transformation

Chronic lymphocytic leukemia (CLL) is a highly complex disease characterized by the proliferation of CD5+ B cells in lymphoid tissues. Current modern treatments have brought significant clinical benefits to CLL patients. However, there are still unmet needs. Patients relapse on Bruton's tyrosine kinase inhibitors and BCL2 inhibitors and often develop more aggressive diseases including Richter transformation (RT), an incurable complication of up to ∼10% patients. This evidence underscores the need for improved immunotherapies, combination treatment strategies, and predictive biomarkers. A mouse model that can recapitulate human CLL disease and certain components of the tumor immune microenvironment represents a promising preclinical tool for such purposes. In this review, we provide an overview of CRISPR-engineered and xenograft mouse models utilizing either cell lines, or primary CLL cells suitable for studies of key events driving the disease onset, progression and transformation of CLL. We also review how CRISPR/Cas9 established mouse models carrying loss-of-function lesions allow one to study key mutations driving disease progression. Finally, we discuss how next generation humanized mice might improve to generation of faithful xenograft mouse models of human CLL.

Abstract 2693: MicroRNA manipulation of macrophage polarization in DLBCL to augment antibody immunotherapy

Abstract Background: CD163 positive tumor associated macrophages (TAM), have been associated with poor response to immunochemotherapy in DLBCL. Targeting these TAMs to reverse immunosuppression is one approach for improving treatment response rates. MicroRNAs have the potential to modify hundreds of genes by interacting with multiple mRNA targets, thereby altering the global transcriptional profile of cells. Manipulation of microRNAs may prove an effective means of switching the transcriptomic profiles of TAMs to more activated, anti-tumor states resulting in the reversal of immune resistance and enhancement of treatment efficacy. Methods: MicroRNAs differentially expressed in DLBCL and associated with poor response were identified using whole gene correlated network analysis of DLBCL datasets. Peripheral blood mononuclear cells (PBMCs) were obtained from normal blood donors and differentiated into macrophages following cell culture with MCSF. Macrophages were polarized using immunosuppressive (IL4 + IL13) or immunostimulating (LPS + IFNγ) cytokines and microRNA expression measured using RT-qPCR. To assess antibody dependent cellular phagocytosis (ADCP), macrophages were generated and polarized as above and transfected with microRNA inhibitors (Thermofisher) prior to co-incubation with target cells. Human Chronic Lymphocytic Leukemia (CLL) cells were labelled with CSFE and used as target cells with Rituximab as the ADCP-inducing monoclonal antibody (mAb) and Herceptin as an isotype control. The percentage of Fc gamma receptor IIIA (FcγRIIIA)-expressing macrophages taking up target cells was determined using flow cytometry and analyzed using Flowjo software. Diagnostic biopsies from patients were then analyzed for microRNA 142-5p and CD206 expression using miRNAScope assays and immunohistochemistry. Results: There was significant upregulation of microRNA 142-5p in macrophages polarized with IL4 plus IL13 compared to macrophages polarized with LPS plus IFNγ and non-polarized macrophages. Inhibition of microRNA 142-5p in IL4 plus IL13 polarized macrophages led to a significant increase in FcγRIIIA, CD40 and CD47 expression resulting in enhanced phagocytosis of CLL cells opsonized with Rituximab plus anti-CD47 mAb when compared with controls. High microRNA 142-5p expression in combination with high CD206 expression predicted poor response to immunochemotherapy in 38 treatment naïve human DLBCL samples (median PFS 12 months v NR p=0.0096). Discussion: Transfection of a microRNA 142-5p inhibitor can repolarize immunosuppressive macrophages to a more anti-tumor phenotype leading to enhanced ADCP. CD206 positive TAM infiltration in combination with high microRNA 142-5p expression predicted poor outcome following treatment with immunochemotherapy in DLBCL. It may be possible to use microRNA directed therapies targeting the tumor microenvironment to enhance treatment efficacy. Citation Format: Jemma Longley, Russell Foxall, Steven Thirdborough, Steve Beers, Mark Cragg. MicroRNA manipulation of macrophage polarization in DLBCL to augment antibody immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2693.

A single bout of vigorous intensity exercise enhances the efficacy of rituximab against human chronic lymphocytic leukaemia B-cells ex vivo

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal proliferation and accumulation of mature B-cells and is often treated with rituximab, an anti-CD20 monoclonal antibody immunotherapy. Rituximab often fails to induce stringent disease eradication, due in part to failure of antibody-dependent cellular cytotoxicity (ADCC) which relies on natural killer (NK)-cells binding to rituximab-bound CD20 on B-cells. CLL cells are diffusely spread across lymphoid and other bodily tissues, and ADCC resistance in survival niches may be due to several factors including low NK-cell frequency and a suppressive stromal environment that promotes CLL cell survival. It is well established that exercise bouts induce a transient relocation of NK-cells and B-cells into peripheral blood, which could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab in blood. In this pilot study, n = 20 patients with treatment-naïve CLL completed a bout of cycling 15 % above anaerobic threshold for ∼ 30-minutes, with blood samples collected pre-, immediately post-, and 1-hour post-exercise. Flow cytometry revealed that exercise evoked a 254 % increase in effector (CD3−CD56+CD16+) NK-cells in blood, and a 67 % increase in CD5+CD19+CD20+ CLL cells in blood (all p < 0.005). NK-cells were isolated from blood samples pre-, and immediately post-exercise and incubated with primary isolated CLL cells with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab-mediated cell lysis increased by 129 % following exercise (p < 0.001). Direct NK-cell lysis of CLL cells – independent of rituximab – was unchanged following exercise (p = 0.25). We conclude that exercise improved the efficacy of rituximab-mediated ADCC against autologous CLL cells ex vivo and propose that exercise should be explored as a means of enhancing clinical responses in patients receiving anti-CD20 immunotherapy.

An unappreciated cell survival-independent role for BAFF initiating chronic lymphocytic leukemia.

Chronic Lymphocytic Leukemia (CLL) is characterized by the expansion of CD19+ CD5+ B cells but its origin remains debated. Mutated CLL may originate from post-germinal center B cells and unmutated CLL from CD5+ mature B cell precursors. Irrespective of precursor types, events initiating CLL remain unknown. The cytokines BAFF and APRIL each play a significant role in CLL cell survival and accumulation, but their involvement in disease initiation remains unclear. We generated novel CLL models lacking BAFF or APRIL. In vivo experiments were conducted to explore the impact of BAFF or APRIL loss on leukemia initiation, progression, and dissemination. Additionally, RNA-seq and quantitative real-time PCR were performed to unveil the transcriptomic signature influenced by BAFF in CLL. The direct role of BAFF in controlling the expression of tumor-promoting genes was further assessed in patient-derived primary CLL cells ex-vivo. Our findings demonstrate a crucial role for BAFF, but not APRIL, in the initiation and dissemination of CLL cells. In the absence of BAFF or its receptor BAFF-R, the TCL1 transgene only increases CLL cell numbers in the peritoneal cavity, without dissemination into the periphery. While BAFF binding to BAFF-R is dispensable for peritoneal CLL cell survival, it is necessary to activate a tumor-promoting gene program, potentially linked to CLL initiation and progression. This direct role of BAFF in controlling the expression of tumor-promoting genes was confirmed in patient-derived primary CLL cells ex-vivo. Our study, involving both mouse and human CLL cells, suggests that BAFF might initiate CLL through mechanisms independent of cell survival. Combining current CLL therapies with BAFF inhibition could offer a dual benefit by reducing peripheral tumor burden and suppressing transformed CLL cell output.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=&amp;lt;0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=&amp;lt;0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

Pre-Clinical Study on the Dual BCL2/BCL-XL Inhibitor AZD0466 for the Treatment of Chronic Lymphocytic Leukemia

Targeting BCL2 with venetoclax has proven successful for treatment of chronic lymphocytic leukemia (CLL), but resistance develops in a subset of patients. Among other mechanisms, mutations in BCL2 and increased BCL-XL expression confer venetoclax refractoriness in patient subgroups. It is therefore important to identify alternative treatment options targeting BCL-XL and BCL2 for combating venetoclax resistance. AZD4320 was developed as a dual BCL-2/BCL-XL inhibitor. AZD0466 is a dendrimer-drug conjugate of AZD4320, designed to optimize drug release and improve drug tolerability. Hence, we used the nano drug AZD0466 for in vivo experiments and AZD4320 for in vitro and ex vivo experiments. Firstly, we studied the efficacy of AZD0466 in vivo in the Eµ-TCL1 murine CLL adoptive transfer model. Due to prevalence of primary resistance, murine tumors with the best ex vivo response to AZD4320 were selected and 10 million cells were transferred by i.v. in syngeneic recipient mice. Upon reaching a tumor load of 10% CD19+ CD5+ cells in peripheral blood, mice were randomized and treated with 30mg/kg ibrutinib in drinking water, 34mg/kg or 70mg/kg of AZD0466 once weekly (QW) by i.v, or the vehicle. Treatments were continued until reaching a predefined humane endpoint. AZD0466 at 70mg/kg led to a significant increase in survival (median 72 days vs. 38 days, P=0.0009) similar to ibrutinib (67 days, P=0.0026) when compared to vehicle (Fig. 1). Analysis of blood parameters at euthanasia did not reveal significant difference in platelet counts between AZD0466, ibrutinib and vehicle treatments. To evaluate if combination treatment of AZD0466 with BTK inhibitors would improve efficacy, we transplanted murine Eµ-TCL1 tumors into syngeneic recipient mice and randomized them for treatment with vehicle, ibrutinib (30mg/kg in drinking water), acalabrutinib (25mg/kg, p.o. QD), AZD0466 (70mg/kg, i.v., QW) and combination of AZD0466 with ibrutinib or acalabrutinib. AZD0466 and vehicle treatments showed similar platelet and RBC counts measured 2 or 3 days after the 2 nd and 4 th dosing but, a significant increase in platelet and RBC counts were observed with the combination treatments. A significant decrease in WBC count after 2 weeks, and in bone marrow tumor load after 4 weeks was observed with combined AZD0466 and acalabrutinib treatment compared to the single drugs. Though spleen weights did not differ after 4 weeks between vehicle and AZD0466 monotherapy, a strong decrease in spleen weight and tumor cell number in spleen was observed with the combination of AZD0466 with ibrutinib or acalabrutinib compared to vehicle. To validate the findings in human CLL, we performed dynamic BH3 profiling on primary CLL samples with mutated (N=5) and unmutated IGHV (N=7). Exposure of CLL cells to 1µM acalabrutinib for 24 hours significantly increased BCL2 dependency in all CLL samples with unmutated IGHV and in a subset of mutated IGHV samples. These tumors also showed an increased response to venetoclax and AZD4320 upon exposure to acalabrutinib. To study the efficacy of the dual BCL2/BCL-XL inhibitor in the context of venetoclax resistance, we stably expressed the BCL2 mutations A113G, D103E, D103V, D103Y, G101V, R129L, V156D and wildtype (WT) in the venetoclax sensitive mantle cell lymphoma cell line MAVER-1. Response to AZD4320 or venetoclax was analyzed using Annexin V/7AAD staining and flow cytometry after 48 hours of treatment. The BCL2 mutants showed varying degrees of resistance to venetoclax. MAVER-1 expressing WT had an IC50 of 17.7nM while D103E, D103V, D103Y, G101V showed the strongest resistance to venetoclax with 5, 13.8, 4.5 and 7.3 fold increase in IC50 compared to WT. Of note, compared to WT (IC50 43.9nM), D103E, D103V and D103Y were sensitive to AZD4320 with a fold change in IC50 of 0.6, 1.8 and 1.3, respectively. G101V mutant cells showed a 5 fold increase in IC50 to AZD4320 compared to WT (Fig. 2). Moreover, AZD4320 was highly efficacious in MAVER-1 and MINO cell line models where resistance to venetoclax mediated by BCL-XL upregulation was modelled by an in vitro dose escalation method. In summary, our pre-clinical study shows that the dual BCL2/BCL-XL inhibitor could represent an important treatment option for venetoclax resistance mediated by specific BCL2 mutations or BCL-XL upregulation and that its efficacy could be further improved upon combination treatment with BTKi.

Important Role for Glutamine in Human and Mouse TME Models of CLL ; Towards Development of a PET Tracer As a Novel Diagnostic Tool

The amino acid transporter ASCT2, encoded by the gene SLC1A5, is the main glutamine importer in proliferating human and mouse cells. We recently showed that glutamine is the main fuel of the tricarboxylic acid (TCA) cycle in cells from chronic lymphocytic leukemia (CLL) patients, and that blocking its import attenuated CD40/BCR-induced venetoclax (VEN) resistance 1. In the current study, we have investigated the role of ASCT2 and the consequences of its inhibition in human and mouse CLL cells and in healthy lymphocytes by flow cytometry, gene expression analysis, extracellular flux analysis, metabolomics, CRISPR/Cas9 gene editing and adoptive-cell transfer experiments. To enable in vivo assessment of ASCT2 in the future with Positron Emission Tomography (PET), we have explored the potential use of 18F-labelelled ( 2S,2R)-4-fluoroglutamine (4- 18F]FGln) 2 as PET tracer. Increased expression of ASCT2 upon PMA/ionomycin stimulation was observed in healthy B and T cells. In BCR/CD40-stimulated CLL cells, the increased expression was irrespective of IGHV status. We dissected the main routes of glutamine usage in CD40- and BCR-stimulated CLL cells and found that in addition to its role in the TCA cycle, glutamine import is linked to mTOR signaling. The ASCT2 inhibitor V9302 impaired mTOR signaling and decreased translation of new proteins, including the anti-apoptotic proteins Bcl-XL and Mcl-1. Of relevance for therapeutic approaches, the effector and proliferative capacity of human T cells was preserved upon V9302 treatment. This suggests that blocking ASCT2 will not affect immune responses. The SLC1A5 gene was deleted by CRISPR/Cas9 technique in murine TCL1 cells 3. Adoptive cell transfer experiments of SLC1A5-KO and WT TCL1 cells injected at 50-50% ratio in recipient mice showed selective outgrowth of SLC1A5-WT cells, with no detection of the SLC1A5-KO in the spleen of overt leukemic mice. This indicates that TCL1 leukemic cells rely on SLC1A5/ASCT2 for growth. Finally, we explored the high expression of ASCT2 and glutamine addiction in CLL from the diagnostic perspective. With the aim of improving the currently suboptimal [ 18F]FDG PET for imaging of CLL LN sites 4, we successfully synthesized 4-[ 18F]FGln. We observed uptake in CD40-stimulated CLL cells, which was decreased in the presence of V9302. Experiments to perform PET with 4-[ 18F]FGln in TCL1 mice to demonstrate an in vivo proof of concept are ongoing. Overall, these data support a crucial role for ASCT2 and glutamine in leukemia development and VEN resistance in CLL. This provides a basis for targeting the transporter for therapeutic options and/or taking advantage of its function for diagnostic approaches using 4-[ 18F]FGln as PET tracer. 1. Chen, Z. et al. Characterization of metabolic alterations of chronic lymphocytic leukemia in the lymph node microenvironment. Blood 140, 630-643, doi:10.1182/blood.2021013990 (2022). 2. Qu, W. et al. Synthesis of optically pure 4-fluoro-glutamines as potential metabolic imaging agents for tumors. J Am Chem Soc 133, 1122-1133, doi:10.1021/ja109203d (2011). 3. Nardi, F. et al. Assessing gene function in human B cells: CRISPR/Cas9-based gene editing and mRNA-based gene expression in healthy and tumor cells. Eur J Immunol 52, 1362-1365, doi:10.1002/eji.202149784 (2022). 4. Rhodes, J. M. &amp; Mato, A. R. PET/Computed Tomography in Chronic Lymphocytic Leukemia and Richter Transformation. PET Clin 14, 405-410, doi:10.1016/j.cpet.2019.03.007 (2019).

Live Lymphoma and CLL Cells inside the Bone Marrow Fibroblasts: Implication in Residual Disease Persistence

Bruton tyrosine kinase inhibitors (BTKi) have generated remarkable clinical responses in patients with chronic lymphocytic leukemia (CLL) and other mature B-cell malignancies. However, deep, and durable response to BTK inhibition is uncommon. A significant number of patients with measurable residual disease relapse with resistance to therapies. Although mutational mechanisms responsible for resistance have been well described, it is not known what mechanism led to persistence of the minimal residual disease (MRD) in the first place. The tumor microenvironment (TME) contributes to drug resistance. Various roles for stroma participation have been described, however, none of these include the physical intracellular sheltering of tumor cells. Cell-in-cell (CIC) describes the microscopic observations of the embodiment of an intact cell by another whole cell ( Fig. 1A). Pathologists have reported this phenomenon sporadically in human fixed tissues for over a century. However, its biological, pathological, and clinical significance remains obscure. In the absence of systematic studies, understanding the significance of live CIC remains challenging. In this investigation, we studied primary human CLL samples using an ex vivo co-culture model system which mimics the CLL TME. In the TME system, CLL cells were cocultured with a primary bone marrow fibroblast line (BMF) in the presence of B-cell growth factors. The tumor cells survive for weeks, become larger, downregulate surface CXCR4. Phenotypically, tumor cells proliferate and divide into daughter cells. Collectively, these features recapitulate the behavior of lymph node-resident CLL cells. Using the TME system, we have demonstrated that modeled drug response correlates well with patient clinical response to BTK inhibitor ibrutinib and BCL2 inhibitor venetoclax. Interestingly, with confocal microscopy and 3D reconstruction, we observed that CLL cells placed in the model system were actively internalized by BMF in all 41 CLL cases studied. CLL cells were live and mobile. Moreover, one CLL cell was captured halfway in ( Fig. 1B). The number of internalized CLL cells was quantified and correlated with patients' clinical and pathological features. No correlations were found between CIC and widely used CLL prognostic indicators, including cytogenetic features and IGHV mutational status. However, we have found that patients who were on treatment for CLL at the time of sample collection had higher levels of CIC than those not treated. We hypothesized that live CIC may be an important mechanism contributing to residual disease persistence and drug resistance. To test the hypothesis, we exposed CLL cells in the TME model system to covalent and noncovalent BTKi including ibrutinib, zanubrutinib, and pirtobrutinib at clinically achievable concentrations. Our results showed that drug exposure drove CLL cells into BMF. This happened specifically to the BTK inhibitors as BCL2 inhibitor venetoclax was not observed to cause such an increase in CIC. These laboratory findings are consistent with the clinical observations that the rate of MRD negativity is much higher with venetoclax treatment than with BTKi. Additionally, CIC was observed in both follicular lymphoma (FL) cell line and primary FL cells suggesting the phenomenon is not limited to CLL. Further, we found that CIC was stimulated by several B-cell growth factors. The chemokine CXCL12-CXCR4 axis played a critical role in this process. Specifically, conditioned medium containing CXCL12 ligand promoted cell internalization, while plerixafor, a CXCR4 antagonist, largely prevented CIC from occurring. Furthermore, plerixafor blocks ibrutinib-driven CIC in cell lines as well as in primary CLL cells. Our study represents the first report implicating the CIC phenomenon in residual disease persistence using a cohort of patient-derived CLL cells assessed with 3D imaging and quantitative approach. These findings suggest that live CIC may be an important mechanism tumor cells use to avoid harmful drug insults, survive, persist, and later proliferate, eventually leading to clinical relapse. Targeting the CXCL12-CXCR4 axis may become a potential therapeutic strategy to minimize residual disease and future relapse. Fig.1. A. Images are reconstructed in 3D using Imaris v10.0. BMF cells are visualized as transparent red surfaces and CLL cells as green surfaces. B. One CLL cell was captured halfway in.

The effect of HYPE knock-out on the AMPylome of human OSU-CLL leukemia cells*

In humans, AMPylation of cellular proteins is carried out by Huntingtin yeast-interacting protein E (HYPE), activated under conditions of endoplasmic reticulum stress, such as in cancer cells. Extracts of the human chronic lymphocytic leukemia cell line, OSU-CLL, were fractionated using immuno-precipitation with antibodies against adenosine-phosphate and then AMP-Tyr. The proteins isolated were modified with AMP, the ‘AMPylome.’ AMP-labelled peptides isolated from HYPE wild-type (WT) and HYPE knock-out (KO) cells were identified using tandem mass spectrometry. A total of 213 proteins were identified from WT cell extracts, while only 23 of these were pulled down from KO cells, consistent with the presence of another AMPylator, besides HYPE. The KO cells were more sensitive to fludarabine nucleoside (2-FaraA) than WT cells. Ingenuity Pathway Analysis of the AMPylated proteins identified in WT cells clustered actin binding proteins of the cytoskeleton, and proteins of the RHO GTPase pathway that would jointly stimulate cell proliferation.

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

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

The inhibitory receptor Siglec-G controls the severity of chronic lymphocytic leukemia.

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5+ B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5+ B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eμ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist.

Lymphocyte migration and retention properties affected by ibrutinib in chronic lymphocytic leukemia.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is widely used for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). A prominent effect of ibrutinib is to disrupt the retention of CLL cells from supportive lymphoid tissues, by altering BTK-dependent adhesion and migration. To further explore the mechanism of action of ibrutinib and its potential impact on non-leukemic cells, we quantified multiple motility and adhesion parameters of human primary CLL cells and non-leukemic lymphoid cells. In vitro, ibrutinib affected CCL19-, CXCL12- and CXCL13-evoked migration behavior of CLL cells and non-neoplastic lymphocytes, by reducing both motility speed and directionality. De-phosphorylation of BTK induced by ibrutinib in CLL cells was associated with defective polarization over fibronectin and inability to assemble the immunological synapse upon B-cell receptor engagement. In patients' samples collected during a 6-month monitoring of therapy, chemokine-evoked migration was repressed in CLL cells and marginally reduced in T cells. This was accompanied by profound modulation of the expression of chemokine receptors and adhesion molecules. Remarkably, the relative expression of the receptors governing lymph node entry (CCR7) versus exit (S1PR1) stood out as a reliable predictive marker of the clinically relevant treatment-induced lymphocytosis. Together, our data reveal a multifaceted modulation of motility and adhesive properties of ibrutinib on both CLL leukemic cell and T-cell populations and point to intrinsic differences in CLL recirculation properties as an underlying cause for variability in treatment response.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL

AbstractDysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5′ untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation–related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

Oncogenic role and target properties of the lysine-specific demethylase KDM1A in chronic lymphocytic leukemia

AbstractIn chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eμ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eμ-TCL1A vs iKdm1aKD;Eμ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti–B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

New insights into the Lck-NF-κB signaling pathway.

Lck is essential for the development, activity, and proliferation of T cells, which may contribute to pathological progression and development of human diseases, such as autoimmune disorders and cancers when functioning aberrantly. Nuclear factor-κB (NF-κB) was initially discovered as a factor bound to the κ light-chain immunoglobulin enhancer in the nuclei of activated B lymphocytes. Activation of the nuclear factor-κB pathway controls expression of several genes that are related to cell survival, apoptosis, and inflammation. Abnormal expression of Lck and nuclear factor-κB has been found in autoimmune diseases and malignancies, including rheumatoid arthritis, systemic lupus erythematosus, acute T cell lymphocytic leukemia, and human chronic lymphocytic leukemia, etc. Nuclear factor-κB inhibition is effective against autoimmune diseases and malignancies through blocking inflammatory responses, although it may lead to serious adverse reactions that are unexpected and unwanted. Further investigation of the biochemical and functional interactions between nuclear factor-κB and other signaling pathways may be helpful to prevent side-effects. This review aims to clarify the Lck-nuclear factor-κB signaling pathway, and provide a basis for identification of new targets and therapeutic approaches against autoimmune diseases and malignancies.

Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles.

In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.

Establishment and Characterization of Mouse Models with Primary and Acquired BTK Inhibitor Resistance

Background: The second-generation Bruton's tyrosine kinase (BTK) inhibitor acalabrutinib is effective for treatment of patients with chronic lymphocytic leukemia (CLL), including high-risk and relapsed patients. Resistance to BTK inhibitors can develop not only due to on-target mutations, but also by bypassing the BTK signaling pathway. Mouse models represent useful tools in studying mechanisms of resistance as well as testing the efficacy of alternative therapy options to overcome resistance. Eµ-TCL1-transgenic (TCL1-tg) mice have the advantage of immunocompetence and resemble the human CD49d-high CLL cohort (Szenes et al., 2020). Transplantation of tumors from these mice to wild-type recipients allows acceleration of pathophysiology, selection of specific tumor clones, and more efficient testing of drug combinations. Here, we developed acalabrutinib resistance models by serial transplantation and long-term administration of the drug in drinking water, characterized these models, and compared molecular resistance signatures with those of human patient samples. Methods: Models were developed starting from four primary TCL1-tg mice at overt disease with tumor burden in spleen ranging up to 95% of lymphocytes. Serial retransplantation to wild-type C57BL/6J recipients for two to four rounds and acalabrutinib therapy pressure resulted in several drug resistant pedigrees. Mice were sacrificed at end-stage disease and tumor burden in spleen, bone marrow and blood was analyzed by flow cytometry using anti-CD5/CD19 antibodies. Purified leukemic cells were subjected to RNA-seq, real-time quantitative PCR and mutational analyses. Results: Three response patterns were identified in recipient mice: a) sustained sensitivity (models were stopped after 23 weeks of treatment), b) acquired resistance (with initial response followed by development of resistance under continuous therapy pressure and c) retransplantation-induced resistance in a case of an aggressive tumor clone that was responding to acalabrutinib in the parental mouse. The sensitive models displayed a 63% reduction in spleen weight upon treatment and a concomitant 65% reduction in blood tumor burden. In acquired or primary resistant mice, spleen weight and tumor burden was not significantly different from that of untreated mice, with blood tumor burden in the magnitude of 100,000 leukemic cells/µl and spleen tumor burden of 500-1000 million leukemic cells. Resistance could not be attributed to the mutational landscape of BTK and PLCγ2. Mutations in p53, Myc, Notch1, Atm, and Sf3b1 were detected but found to be intrinsic in individual ancestors and further present in the follow-up transplantations. RNA-seq analysis revealed 863 differentially expressed genes in mice with acquired resistance to acalabrutinib (compared to untreated, Figure 1) and 114 shared differentially expressed genes in mice with acquired or transplantation-based resistance. Gene set enrichment analysis demonstrated the presence of MAPK signaling and NF-kB signaling gene signatures within the resistant tumors. In particular, acquired resistance was accompanied by changes in the B cell receptor downstream components. We also compared the murine 863 gene signature to human samples from acalabrutinib resistant patients (progression on drug versus baseline) and found 13 genes are differently expressed in both, human and mouse. Validation for this gene expression data is ongoing and updated results will be presented. Conclusion: We have developed novel TCL1-tg based murine models of acalabrutinib resistance. Our models provide well-characterized tools to study primary as well as acquired BTK inhibitor resistance and allow the current testing of potential new therapeutic approaches to overcome resistance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal