Expression of ZAP-70 Does Not Accelerate Leukemia Development and Progression in the Eμ-TCL1 Transgenic Mouse Model of Chronic Lymphocytic Leukemia

Abstract Abstract 925 One of the most relevant prognostic factors in chronic lymphocytic leukemia (CLL) is expression of the protein tyrosine kinase ZAP-70. Typically, patients whose leukemic cells express ZAP-70 at 30–100% of the levels in normal T cells have aggressive disease, whereas patients whose leukemic cells do not express ZAP-70 or express only low levels of this protein have indolent disease. Previously, we and others demonstrated that ZAP-70 modulates B-cell receptor signaling and thus affects the capacity of the leukemic cells to respond to antigen stimulation. However, a direct link between an altered antigen response and CLL pathogenesis has still not been established and, more importantly, the question whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of aggressive CLL still remains to be answered. We have now addressed these issues by analyzing in vivo the impact of forced expression of ZAP-70 on the development and behavior of leukemias that arise in the Eμ-TCL1 transgenic (tg) mouse model of CLL. This model is characterized by the development of antigen-driven leukemias that resemble human CLL in many aspects but are always ZAP-70-negative. To force the expression of ZAP-70 in TCL1 leukemias, we generated two tg lines with targeted expression of ZAP-70 in the B cell compartment (ZAP70high and ZAP70low) and crossed them with Eμ-TCL1 tg mice. B cells in ZAP70high tg mice express similar levels of ZAP-70 as normal mouse T cells, whereas the levels of ZAP-70 in B cells of ZAP70lowtg mice are approximately 10 times lower. Both cohorts of Eμ-TCL1/ZAP70 double tg mice developed characteristic TCL1 leukemias. Eμ-TCL1/ZAP70low tg mice developed leukemias with onset and rate of progression similar to their ZAP-70-negative littermates, indicating that low levels of ZAP-70 do not alter the development and behavior of the disease. Surprisingly, Eμ-TCL1/ZAP70high tg mice developed leukemias with an approximately 2 month delay compared to their ZAP-70-negative Eμ-TCL1 tg littermates, which was contrary to the expectation that high ZAP-70 expression will accelerate leukemia development. The delay in leukemia development was especially evident at 6 months of age, when leukemic cells could be detected in the PB of 77% (10/13) of Eμ-TCL1 tg mice and only 24% (4/17) of Eμ-TCL1/ZAP70hightg mice (P=0.011). Since ZAP-70 expression can affect the migratory and adhesion capacity of human CLL cells in vitro, we first investigated if the delayed appearance of leukemic cells in the PB of Eμ-TCL1/ZAP70high tg mice could be due to increased retention of the leukemic cells in the lymphoid tissues. Assessment of tumor burden in the spleen, peritoneal cavity (PC), bone marrow and PB of 7 months old mice showed that the number of tumor cells in each compartment was significantly lower in Eμ-TCL1/ZAP70hightg mice than their Eμ-TCL1 littermates, suggesting that the delay in leukemia appearance is not caused by increased tissue retention but rather by reduced tumor growth. To investigate if ZAP-70 impairs tumor growth by affecting proliferation, we performed in vivo BrdU incorporation analysis of leukemic cells from spleen and PC of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice. Spleen and PC samples were analyzed because they are the major sites of leukemia proliferation in Eμ-TCL1 tg mice. Interestingly, while the percentage of proliferating leukemic cells in the spleens of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice was similar (mean % of BrdU+ cells ±SD: 6.81 ±1.67 and 6.15 ±2.92, respectively; P=n.s.), the percentage of proliferating leukemic cells in the PC of Eμ-TCL1/ZAP70high tg mice was significantly lower (mean % of BrdU+cells ±SD: 1.74 ±1.05 and 0.56 ±0.39, respectively; P=0.024). In summary, this study shows that ZAP-70 expression, per se, is unable to accelerate leukemia development and progression in an established in vivo model of CLL and suggests that ZAP-70 is not directly responsible for the greater disease severity in the poor prognosis subset of CLL. In addition, this study reveals that ZAP-70 in certain tissue environments can function as a negative regulator of leukemic cell proliferation, contrary to the widespread perception of ZAP-70 as a positive regulator of leukemic cell responses. Disclosures: No relevant conflicts of interest to declare.

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Development of a Transgenic Mouse Model to Study the Role of ZAP-70 in the Development and Progression of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.2830.2830 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2830-2830

Author(s):

Stefania Gobessi ◽

Sara Bennardo ◽

Pablo G Longo ◽

Brendan Doe ◽

Dimitar G Efremov

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Mouse Model ◽

B Cell ◽

Lymphocytic Leukemia ◽

Bcr Signaling ◽

Leukemia Development ◽

Low Levels ◽

The Impact

Abstract Abstract 2830 The protein tyrosine kinase ZAP-70 is an important prognostic factor in chronic lymphocytic leukemia (CLL). Patients that are considered ZAP-70-positive typically express 30–100% of the levels of ZAP-70 in T-cells, whereas in the remaining patients ZAP-70 is either not expressed or is expressed at lower levels. ZAP-70-positive patients have more aggressive disease and shorter survival than patients with low or absent ZAP-70. In vitro experiments with human lymphoma cell lines and primary CLL B-cells have shown that ZAP-70 is involved in B cell receptor (BCR) signaling, indicating that overexpression of ZAP-70 could affect the capacity of the leukemic cells to respond to antigen stimulation. Despite the strong association between ZAP-70 expression and prognosis, it is still not clear whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of more aggressive CLL. To further address this issue, we generated transgenic (tg) mice that express different levels of ZAP-70 in B cells. In these mice expression of the murine ZAP-70 transgene is targeted to the B cell compartment by a VH or a CD19 promoter (VH-ZAP70 and CD19-ZAP70 tg mice, respectively). B cells in CD19-ZAP70 tg mice express the same levels of ZAP-70 as normal murine T cells, whereas the levels of ZAP-70 in B cells of VH-ZAP70 tg mice are approximately 10 times lower. Immunophenotyping analysis of spleen and peritoneal cavity samples from wild type, VH-ZAP70 and CD19-ZAP70 tg mice did not reveal significant differences in the percentage of follicular (FO), marginal zone (MZ) and B1 B cells, indicating that ectopic expression of ZAP-70 does not affect normal B cell development and maturation. In terms of BCR signal transduction, no abnormalities were detected in VH-ZAP70 tg mice, suggesting that low levels of ZAP-70 do not affect BCR signaling. In contrast, B cells from CD19-ZAP70 tg mice showed altered phosphorylation of several molecules downstream of the BCR, such as Syk and BLNK, whereas phosphorylation of Cbl was not affected. To investigate the impact of ZAP-70 expression on leukemia development and progression, we crossed VH-ZAP70 and CD19-ZAP70 tg mice with Eμ-TCL1 tg mice. The latter mice develop leukemias that are considered a mouse model of human CLL. These leukemias are CD5+, express unmutated IGHV genes and stereotyped polyreactive BCRs, but are always ZAP-70-negative. VH-ZAP70/Eμ-TCL1 tg mice (n=11) have been followed for over a year and did not show any differences with respect to their Eμ-TCL1 littermates (n=10). Both groups, starting from the age of 7–8 months, developed leukemias with a similar rate of progression and impact on survival, suggesting that low levels of ZAP-70 do not affect the behavior of the disease. The cohort of CD19-ZAP70/Eμ-TCL1 tg mice was more recently established. These animals are currently 4 months old and still do not show signs of leukemia development. Data from the extended follow-up of these mice will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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Chronic Lymphocytic Leukemia-Exosomes Switch Endothelial and Mesenchymal Stromal Cells into Cancer-Associated Fibroblasts to Sustain Leukemic Cell Survival

Blood ◽

10.1182/blood.v124.21.2927.2927 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2927-2927 ◽

Cited By ~ 1

Author(s):

Jerome Paggetti ◽

Franziska Haderk ◽

Martina Seiffert ◽

Bassam Janji ◽

Yeoun Jin Kim ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

B Lymphocytes ◽

Stromal Cells ◽

Leukemic Cell ◽

Lymphoid Organs ◽

Lymphocytic Leukemia ◽

Leukemic Cells

Abstract Chronic lymphocytic leukemia (CLL), the most common hematologic malignancy in Western countries, is mostly affecting the elderly over 65 year-old. CLL is characterized by the accumulation of mature but non-functional B lymphocytes of clonal origin in the blood and the primary lymphoid organs. CLL was previously considered as a relatively static disease resulting from the accumulation of apoptosis-resistant but quiescent B lymphocytes. However, recent studies using heavy water labeling indicated that CLL is in fact a very dynamic disease with alternation of proliferation phases and peripheral circulation. A focus on the trafficking of CLL cells in vivo has shown that leukemic cells circulate between the blood and the lymphoid organs but have a preference for the bone marrow. Recent next-generation sequencing of CLL cells indicated the presence of different genetic subclones. This intraclonal heterogeneity observed in CLL subpopulations may be in part determined by the interactions that leukemic cells entertain with their microenvironment when B cells migrate into the lymph nodes and the bone marrow. Indeed, tumor-stroma interactions are not only providing signals necessary for leukemic cells survival but may also influence the clonal architecture and evolution. One of these interactions involves CLL-derived exosomes. Here, we show that CLL-exosomes efficiently transfer nucleic acids, including functional microRNAs, and proteins, including MHC-Class II molecules and B-cell specific proteins, to bone marrow mesenchymal stem cells and endothelial cells. CLL-exosomes also activate signaling pathways, including PI3K and NF-κB pathways, in these stromal cells. As a consequence, gene expression is strongly modified indicating a switch towards a cancer-associated fibroblast phenotype. Functionally, exosome-stimulated stromal cells show a striking actin cytoskeleton remodeling characterized by the formation of stress fibers, and enhanced proliferation, motility and angiogenic properties. We also identified several proteins synthesized and secreted by stromal cells that promote leukemic cell adhesion and survival ex vivo. To confirm the involvement of CLL-exosomes in CLL pathology in vivo, MEC-1-eGFP cells were subcutaneously injected into immunocompromised NSG mice together with CLL-exosomes. We observed a significant increase in tumor size and a reduction in survival of exosome-treated animals. Flow cytometry analysis of selected organs indicated an enrichment in leukemic cells in the kidney, providing a potential explanation to the renal failures observed in CLL patients. In conclusion, the communication between CLL cells and stromal cells may be a critical factor influencing CLL progression by promoting leukemic cell survival. This study demonstrates the crucial role of exosomes as mediators of the communication between leukemic cells and their microenvironment. Exosomes could thus represent a suitable target for therapeutic intervention in CLL. Disclosures No relevant conflicts of interest to declare.

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Immunoliposomal Delivery of Mir-29b By Targeting Tumor Antigen ROR1 Induces Epigenetic Reprograming in Human-ROR1-Expressed Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.1743.1743 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1743-1743

Author(s):

Chi-Ling Chiang ◽

Frank W Frissora ◽

Zhiliang Xie ◽

Xiaomeng Huang ◽

Rajeswaran Mani ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Mouse Model ◽

Cell Line ◽

Tumor Antigen ◽

Targeted Delivery ◽

Leukemia Cell ◽

Leukemic Cell ◽

Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL), characterized by accumulation of CD5+CD19+sIgM+ B lymphocytes in peripheral blood and lymphoid organs, is classified into indolent and aggressive forms. Patients with indolent CLL generally survive 5 to 10 years and do not require treatment until severe symptoms, while those with aggressive CLL show resistant to standard treatment and survive less than 24 months. While emerging B cell antigen receptor directed therapies are promising, resistance to such therapies pose problems warranting novel therapeutic approaches. MicroRNA (miR) profiling revealed lower expression of miR-29b in aggressive CLL associated with survival, drug resistance and poor prognosis via its up-regulation of anti-apoptotic proteins myeloid leukemia cell differentiation protein 1 (Mcl1) and oncogenic T-cell leukemia 1 (Tcl1). Thus, specific overexpression of miR-29b in B-CLL cells could be a potential therapy for aggressive CLL patients. Despite the promise, short circulation half-life, limited cellular uptake and off-target effects on non-desirable tissues pose a challenge for miR-based therapies. To promote efficiency and specificity of miR-29b delivery, we developed neutral immunonanoparticles with selectivity to CLL via targeting tumor antigen ROR1, which is expressed in over 95% of CLL but not normal B cells. We optimized a novel 2A2-immunoliposome (2A2-ILP) recognizing surface ROR1 on primary CLL cell to promote internalization and miR-29b uptake (n=6, p=0.042*). About 20-fold increased uptake of miR-29b was achieved with 2A2-ILP-miR-29b formulation compared to control. Further ROR1 targeted delivery of miR29b resulted in significant downregulation of DNMT1 and DNMT3a mRNA and protein (n=3, DNMT1: p= 0.0115*; DNMT3a: p=0.0231*, SP1; p=0.0031**) in primary CLL cells and a human CLL cell line OSU-CLL. Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week post- treatment with 2A2-ILP-miR-29b (n=3, p=0.0003***). To further study the in vivo ROR1-targeting efficiency of 2A2-ILP-miR-29b, we used our recently described Eµ-hROR1x Tcl1 CLL mouse model that develops CLL like disease with human ROR1 antigen in leukemic CD19+CD5+ B cells. Using hROR1+CD19+CD5+ leukemic cell engraftment model, we showed significant in-vivo efficacy of ROR1-ILP-miR-29b formulation associated with a) decreased number of circulating leukemic B220+CD5+ cells b) reduced splenomegaly (p=0.0461*, 2A2-29b: n=9; PBS: n=8) c) with extended survival (p=0.0075**, 2A2-29b: n=9; IgG-29b: n=7; 2A2-SC: n=7; PBS: n=8). In summary, 2A2-ILP effectively delivered functional miR-29b, resulting in downregulation of DNMT1 and DNMT3a, reduction of hypermethylation and anti-leukemic activity. Ongoing studies are aimed at understanding miR-29b mediated in-vivo methylome reprograming using our novel hROR1xTcl1 transgenic mouse model and ROR1-targeted miR-29b delivery formulation. Figure 1. Figure 1. Disclosures Byrd: Acerta Pharma BV: Research Funding.

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CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eμ-TCL1 mice through a CD40L-independent mechanism

Blood Advances ◽

10.1182/bloodadvances.2020003795 ◽

2021 ◽

Vol 5 (14) ◽

pp. 2817-2828

Author(s):

Matteo Grioni ◽

Arianna Brevi ◽

Elena Cattaneo ◽

Alessandra Rovida ◽

Jessica Bordini ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Antigen Specificity

Abstract Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+ T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/− mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/− mice, and TCL1+/+CD40−/− mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

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In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor

Blood ◽

10.1182/blood-2010-10-311845 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4736-4745 ◽

Cited By ~ 51

Author(s):

Greta Maria Paola Giordano Attianese ◽

Virna Marin ◽

Valentina Hoyos ◽

Barbara Savoldo ◽

Irene Pizzitola ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

B Lymphocytes ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Car T Cells ◽

Car T

Abstract Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19+CD5+CD20dim B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR+ T cells showed specific cytotoxic activity against CD23+ tumor cell lines (average lysis 42%) and primary CD23+ CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR+ T cells released inflammatory cytokines (1445-fold more TNF-β, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR+ T cells. Redirected T cells were also effective in vivo in a CLL Rag2−/−γc−/− xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR+ T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR+ T cells represent a selective immunotherapy for the elimination of CD23+ leukemic cells in patients with CLL.

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The Tumor Microenvironment-Dependent Transcription Factors AHR and HIF-1α Are Dispensable for Leukemogenesis in the Eµ-TCL1 Mouse Model of Chronic Lymphocytic Leukemia

Cancers ◽

10.3390/cancers13184518 ◽

2021 ◽

Vol 13 (18) ◽

pp. 4518

Author(s):

Susanne Gonder ◽

Anne Largeot ◽

Ernesto Gargiulo ◽

Sandrine Pierson ◽

Iria Fernandez Botana ◽

...

Keyword(s):

Transcription Factors ◽

Tumor Microenvironment ◽

Chronic Lymphocytic Leukemia ◽

Mouse Model ◽

Hypoxia Inducible Factor ◽

The Elderly ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Knock Out

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in the elderly and is characterized by the accumulation of mature B lymphocytes in peripheral blood and primary lymphoid organs. In order to proliferate, leukemic cells are highly dependent on complex interactions with their microenvironment in proliferative niches. Not only soluble factors and BCR stimulation are important for their survival and proliferation, but also the activation of transcription factors through different signaling pathways. The aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF)-1α are two transcription factors crucial for cancer development, whose activities are dependent on tumor microenvironment conditions, such as the presence of metabolites from the tryptophan pathway and hypoxia, respectively. In this study, we addressed the potential role of AHR and HIF-1α in chronic lymphocytic leukemia (CLL) development in vivo. To this end, we crossed the CLL mouse model Eµ-TCL1 with the corresponding transcription factor-conditional knock-out mice to delete one or both transcription factors in CD19+ B cells only. Despite AHR and HIF-1α being activated in CLL cells, deletion of either or both of them had no impact on CLL progression or survival in vivo, suggesting that these transcription factors are not crucial for leukemogenesis in CLL.

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The Valproic Acid-Fludarabine Combination Induces a Synergistic Response in Chronic Lymphocytic Leukemia Via a Mechanism Involving the Lysosomal Protease Cathepsin B.

Blood ◽

10.1182/blood.v120.21.2892.2892 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2892-2892

Author(s):

Ju-Yoon Yoon ◽

David Szwajcer ◽

Ganchimeg Ishdorj ◽

Pat Benjaminson ◽

James B Johnston ◽

...

Keyword(s):

Valproic Acid ◽

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Cathepsin B ◽

Leukemic Cell ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Lysosomal Protease

Abstract Abstract 2892 Chronic Lymphocytic Leukemia (CLL) is the most common haematological malignancy in the western world. Fludarabine, a nucleoside analogue, is commonly used to treat Chronic Lymphocytic Leukemia (CLL) in untreated and relapsed CLL. However, patients commonly develop resistance to fludarabine. We hypothesize that the addition of Valproic Acid (VPA), an inhibitor of histone deacetylases (HDACs), can improve fludarabine-based therapy. The VPA-Fludarabine combination induced a synergistic response in human leukemic cells and primary CLL cells. Fludarabine also interacted synergistically with three other HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), Trichostatin A, and sodium butyrate, while the synergy was not observed with valpromide, the VPA analogue that does not inhibit HDACs. We confirmed that fludarabine treatment activates caspases-8, -9 and caspase-3, and we also show that fludarabine treatment activates caspase-2, an upstream caspase that has been implicated in cell death associated with lysosome membrane permeabilization (LMP). Activation of all four caspases was enhanced by the addition of VPA. Enhanced activation of caspases was associated with down-regulation of two prominent anti-apoptotic proteins, Mcl-1 and XIAP. The down-regulation of Mcl-1 and XIAP was dependent on the lysosomes, as their alkalinization using either chloroquine or NH4Cl partially stabilized both proteins, leading to reduced apoptosis. Chemical inhibition of a specific lysosomal protease, cathepsin B, using CA074-Me, was sufficient to stabilize Mcl-1 and XIAP, reduce caspase activation and apoptosis. Treatment with fludarabine or the VPA-fludarabine combination led to the loss of lysosome integrity, as visualized by fluorescent staining, thus suggesting a leakage of the lysosomal content into the cytosol in response to the drugs. Addition of purified cathepsin B to leukemic cell lysates led to the reduction in protein levels of Mcl-1, XIAP and pro-caspase-2, thus suggesting that the re-localization of cathepsin B into the cytosol is sufficient to drive cell death. VPA treatment enhanced cathepsin B levels in both leukemic cell lines and primary CLL cells. When cathepsin B activity was examined using zRR-AMC, a fluorogenic substrate of cathepsin B, VPA also increased cathepsin B activity, and this activity was abolished by the addition of CA074-Me. In parallel with the in vitro/ex vivo experiments, we had launched a phase II clinical trial at CancerCare Manitoba. Six relapsed CLL patients who had received at least one prior therapy with fludarabine were examined. No responses were seen after 28 days using VPA alone, in line with the in vitro observation of minimal cytotoxicity of VPA at low doses. However, in five patients who continued on VPA with fludarabine, three patients showed a >50% fall in lymphocyte/lymph node size after receiving five cycles of the combination. When the leukemic cells from VPA-treated CLL patients were examined, VPA administration induced increased levels of histone-3 acetylation and cathepsin B in vivo. In summary, a novel mechanism for fludarabine cytotoxicity has been elucidated, where fludarabine induces a loss of lysosomal integrity, leading to cathepsin B-dependent cell death. VPA interacted with fludarabine synergistically, and this synergy was associated with the VPA-induced increase in VPA level and activity. VPA induced increase in histone-3 acetylation and cathepsin B in vivo, and this induction of cathepsin B is likely to be contributing to the clinical response observed in fludarabine-relapsed/refractory CLL patients. Disclosures: Off Label Use: Valproic acid as adjunct therapy in Chronic Lymphocytic Leukemia. Johnston:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Evaluation of vecabrutinib as a model for non-covalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia

Blood ◽

10.1182/blood.2021011516 ◽

2021 ◽

Author(s):

Billy Michael Chelliah Jebaraj ◽

Annika Müller ◽

Rashmi Priyadharshini Dheenadayalan ◽

Sascha Endres ◽

Philipp M. Roessner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Inhibitory Effect ◽

Lymphocytic Leukemia ◽

Transfer Model ◽

Treatment Benefit ◽

Btk Inhibitors

Covalent Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop by a mutation in cysteine 481 of BTK (C481S), which prevents the irreversible binding of the drugs. In the present study we performed pre-clinical characterization of vecabrutinib, a next generation non-covalent BTK inhibitor, with ITK inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wildtype BTK. In the murine Eµ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, while the naïve populations were increased. Of importance, vecabrutinib treatment significantly reduced frequency of regulatory CD4+ T-cells (Tregs) in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on activation and proliferation of isolated T-cells. Lastly, combination treatment of vecabrutinib with venetoclax was found to augment treatment efficacy, significantly improve survival and lead to favourable reprogramming of the microenvironment in the murine Eµ-TCL1 model. Thus, non-covalent BTK/ITK inhibitors such as vecabrutinib may be efficacious in C481S BTK mutant CLL, while preserving the T-cell immunomodulatory function of ibrutinib.

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P66Shc: A Pleiotropic Regulator of B Cell Trafficking and a Gatekeeper in Chronic Lymphocytic Leukemia

Cancers ◽

10.3390/cancers12041006 ◽

2020 ◽

Vol 12 (4) ◽

pp. 1006 ◽

Cited By ~ 2

Author(s):

Laura Patrussi ◽

Nagaja Capitani ◽

Cosima T. Baldari

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Chemokine Receptors ◽

Adaptor Protein ◽

Leukemic Cell ◽

Lymphocytic Leukemia ◽

Cell Trafficking ◽

Gene Encoding ◽

Cell Invasiveness

Neoplastic B cells from chronic lymphocytic leukemia patients (CLL) have a profound deficiency in the expression of p66Shc, an adaptor protein with pro-apoptotic and pro-oxidant activities. This defect results in leukemic B cell resistance to apoptosis and additionally impinges on the balance between chemokine receptors that control B cell homing to secondary lymphoid organs and the sphingosine phosphate receptor S1PR1 that controls their egress therefrom, thereby favoring leukemic B cell accumulation in the pro-survival lymphoid niche. Ablation of the gene encoding p66Shc in the Eµ-TCL1 mouse model of human CLL enhances leukemogenesis and promotes leukemic cell invasiveness in both nodal and extranodal organs, providing in vivo evidence of the pathogenic role of the p66Shc defect in CLL pathogenesis. Here we present an overview of the functions of p66Shc in B lymphocytes, with a specific focus on the multiple mechanisms exploited by p66Shc to control B cell trafficking and the abnormalities in this process caused by p66Shc deficiency in CLL.

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Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival

Blood ◽

10.1182/blood-2002-02-0558 ◽

2002 ◽

Vol 100 (8) ◽

pp. 2973-2979 ◽

Cited By ~ 164

Author(s):

Anne J. Novak ◽

Richard J. Bram ◽

Neil E. Kay ◽

Diane F. Jelinek

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

B Lymphocyte ◽

Leukemic Cell ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Aberrant Expression ◽

B Lymphocyte Stimulator

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

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