In Vivo and in Vitro Selection of Mitogen Responsive Lymphocytes in Patients with Chronic Lymphocytic Leukemia

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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Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽

10.1182/blood-2003-12-4312 ◽

2004 ◽

Vol 103 (12) ◽

pp. 4389-4395 ◽

Cited By ~ 286

Author(s):

Freda K. Stevenson ◽

Federico Caligaris-Cappio

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Cell Of Origin ◽

Regulatory B Cell ◽

V Genes

Abstract The finding that chronic lymphocytic leukemia (CLL) consists of 2 clinical subsets, distinguished by the incidence of somatic mutations in the immunoglobulin (Ig) variable region (V) genes, has clearly linked prognosis to biology. Antigen encounter by the cell of origin is indicated in both subsets by selective but distinct expression of V genes, with evidence for continuing stimulation after transformation. The key to distinctive tumor behavior likely relates to the differential ability of the B-cell receptor (BCR) to respond. Both subsets may be undergoing low-level signaling in vivo, although analysis of blood cells limits knowledge of critical events in the tissue microenvironment. Analysis of signal competence in vitro reveals that unmutated CLL generally continues to respond, whereas mutated CLL is anergized. Differential responsiveness may reflect the increased ability of post-germinal center B cells to be triggered by antigen, leading to long-term anergy. This could minimize cell division in mutated CLL and account for prognostic differences. Unifying features of CLL include low responsiveness, expression of CD25, and production of immunosuppressive cytokines. These properties are reminiscent of regulatory T cells and suggest that the cell of origin of CLL might be a regulatory B cell. Continuing regulatory activity, mediated via autoantigen, could suppress Ig production and lead to disease-associated hypogammaglobulinemia. (Blood. 2004;103:4389-4395)

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ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood-2015-05-644872 ◽

2016 ◽

Vol 127 (5) ◽

pp. 582-595 ◽

Cited By ~ 104

Author(s):

Marwan Kwok ◽

Nicholas Davies ◽

Angelo Agathanggelou ◽

Edward Smith ◽

Ceri Oldreive ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Synthetic Lethality ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Selective Cytotoxicity ◽

Key Points ◽

Synthetically Lethal

Key PointsATR inhibition is synthetically lethal to TP53- or ATM-defective CLL cells. ATR targeting induces selective cytotoxicity and chemosensitization in TP53- or ATM-defective CLL cells in vitro and in vivo.

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Nurture versus Nature: The Microenvironment in Chronic Lymphocytic Leukemia

Hematology ◽

10.1182/asheducation-2011.1.96 ◽

2011 ◽

Vol 2011 (1) ◽

pp. 96-103 ◽

Cited By ~ 101

Author(s):

Jan A. Burger

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Tyrosine Kinase ◽

B Cell ◽

Disease Progression ◽

Drug Testing ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

In Vivo Models

Abstract Intrinsic factors such as genetic lesions, anti-apoptotic proteins, and aberrant signaling networks within leukemia cells have long been the main focus of chronic lymphocytic leukemia (CLL) research. However, over the past decade, it became increasingly clear that external signals from the leukemia microenvironment make pivotal contributions to disease progression in CLL and other B-cell malignancies. Consequently, increasing emphasis is now placed on exploring and targeting the CLL microenvironment. This review highlights critical cellular and molecular pathways of CLL-microenvironment cross-talk. In vitro and in vivo models for studying the CLL microenvironment are discussed, along with their use in searching for therapeutic targets and in drug testing. Clinically, CXCR4 antagonists and small-molecule antagonists of B cell receptor (BCR)-associated kinases (spleen tyrosine kinase [Syk], Bruton's tyrosine kinase [Btk], and PI3Kδ) are the most advanced drugs for targeting specific interactions between CLL cells and the miocroenvironment. Preclinical and first clinical evidence suggests that high-risk CLL patients can particularly benefit from these alternative agents. These findings indicate that interplay between leukemia-inherent and environmental factors, nature and nurture determines disease progression in CLL.

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Functional and clinical relevance of VLA-4 (CD49d/CD29) in ibrutinib-treated chronic lymphocytic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20171288 ◽

2018 ◽

Vol 215 (2) ◽

pp. 681-697 ◽

Cited By ~ 21

Author(s):

Erika Tissino ◽

Dania Benedetti ◽

Sarah E.M. Herman ◽

Elisa ten Hacken ◽

Inhye E. Ahn ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Progression Free Survival ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

Independent Manner ◽

Btk Inhibitor

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.

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Relationship between in vitro and in vivo Radiosensitivity of Lymphocytes in Chronic Lymphocytic Leukemia

Acta Haematologica ◽

10.1159/000205618 ◽

1988 ◽

Vol 80 (3) ◽

pp. 129-133 ◽

Cited By ~ 5

Author(s):

Robert Schrek ◽

William R. Best ◽

Stefano Stefani

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia

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Effect of a thymic factor on T lymphocytes in B cell chronic lymphocytic leukemia: in vitro and in vivo studies

Blood ◽

10.1182/blood.v64.3.667.667 ◽

1984 ◽

Vol 64 (3) ◽

pp. 667-671 ◽

Cited By ~ 7

Author(s):

F Lauria ◽

D Raspadori ◽

S Tura

Keyword(s):

T Cell ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

B Cell ◽

Proliferative Response ◽

Lymphocytic Leukemia ◽

Before And After ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abnormalities of T lymphocytes in B cell chronic lymphocytic leukemia (B-CLL) have been extensively documented by several immunologic investigations. Following recent studies pointing to the favorable effect of TP-1, a partially purified extract of calf thymus, on the T cell-mediated immunity of several diseases, including Hodgkin's disease, we have used monoclonal antibodies and the enriched T lymphocytes of 16 untreated B-CLL patients to evaluate the proportion of T cell subsets before and after the administration of TP-1. In addition, the proliferative response to phytohemagglutinin (PHA) and the helper function in a pokeweed mitogen (PWM) system were assessed. In ten cases, the effect of TP-1 was also studied in vitro by evaluating the same parameters before and after incubation of B-CLL T cells with the drug. The study demonstrated that in vivo administration of TP-1 increases significantly (P less than .001) the proportion of the defective helper/inducer T cell population (OKT4-positive cells) in B-CLL, leading to a near normal OKT4/OKT8 ratio. Furthermore, the improved phenotypic profile was accompanied by an increased proliferative response to PHA and, in particular, by a significant increase (P less than .01) of T helper capacity; this increase was, however, insufficient to enable the normalization of the serum immunoglobulin levels. The in vitro incubation of B-CLL T lymphocytes did not succeed in producing significant modifications in distribution and function.

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Dissecting the Prognostic Significance and Functional Role of Progranulin in Chronic Lymphocytic Leukemia

Cancers ◽

10.3390/cancers11060822 ◽

2019 ◽

Vol 11 (6) ◽

pp. 822 ◽

Cited By ~ 1

Author(s):

Lena Schulze-Edinghausen ◽

Claudia Dürr ◽

Selcen Öztürk ◽

Manuela Zucknick ◽

Axel Benner ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Prognostic Significance ◽

Lymphocytic Leukemia ◽

Cancer Associated Fibroblast ◽

Survival Gene ◽

Potential Use

Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eμ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eμ-TCL1 leukemia cells to bone marrow chimeric Grn−/− mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn−/− chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease.

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Complement alternative pathway activation by chronic lymphocytic leukemia cells: its role in their hepatosplenic localization

Blood ◽

10.1182/blood.v63.2.463.463 ◽

1984 ◽

Vol 63 (2) ◽

pp. 463-467 ◽

Cited By ~ 12

Author(s):

F Praz ◽

G Karsenty ◽

JL Binet ◽

P Lesavre

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Alternative Pathway ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Complement Alternative Pathway ◽

Acetylneuraminic Acid ◽

Pathway Activation ◽

Do So

Abstract Using affinity-purified 125I-F(ab')2 anti-human C3, we have investigated the ability of various leukemic cells to activate complement. Lymphocytes from patients with chronic lymphocytic leukemia (CLL) activated the alternative pathway, but cells from patients with other forms of leukemia or normal lymphocytes did not do so. The amount of C3 deposited on the CLL cells was significantly higher in patients with organomegaly (i.e., splenomegaly and/or hepatomegaly). Activation of complement by CLL cells as assessed by C3 deposition on the membrane occurred both in vivo and in vitro and was not related to the N- acetylneuraminic acid content of the membrane.

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Chronic Lymphocytic Leukemia B Cells Can Undergo Somatic Hypermutation and Intraclonal Immunoglobulin VHDJH Gene Diversification

Journal of Experimental Medicine ◽

10.1084/jem.20011693 ◽

2002 ◽

Vol 196 (5) ◽

pp. 629-639 ◽

Cited By ~ 74

Author(s):

Carmela Gurrieri ◽

Peter McGuire ◽

Hong Zan ◽

Xiao-Jie Yan ◽

Andrea Cerutti ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Lymphocyte ◽

Somatic Hypermutation ◽

Point Mutations ◽

Single Strand Conformation Polymorphism ◽

Lymphocytic Leukemia ◽

Gene Diversification

Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.

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