Preclinical Testing Of a Novel Axl-Kinase Inhibitor In Chronic Lymphocytic Leukemia

Abstract The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their ability to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models. In vitro studies: We measured soluble Axl plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in 71 CLL patients and 24 healthy donors. Soluble Axl levels were not significantly higher in CLL patients compared to healthy donors (p=0. 11). However, in CLL patients high sAxl plasma concentrations were differentially expressed with some patients exhibiting normal and others elevated plasma concentrations. The latter showed an association with shorter time to first treatment (p=0.0005) and several poor prognostic markers (e.g. CD38, FISH cytogentics, Binet stage). Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Cellular polarization was analyzed by time-lapse microscopy. We detected cytotoxic effects in a patient-dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (IC50= 0.21 µM vs. 2.88 µM, p<0.05, n=5). The cellular polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 µM, p<0.00001; BMS777607: IC50=6.2µM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5). In vivo studies To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/IL2Rgcnull(NSG) mice were pre-treated with a single intraperitoneal (i.p.) bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the BM of NSG mice by 46% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.0063, BMS777607 p=0.0007; n=4). To evaluate if LDC2636 also exhibits effects in a disease-relevant CLL model, we applied a MEC-1 xenograft model which causes a lethal leukemia in NSG mice. We pretreated the mice with 40mg/kg Axl inhibitor or vehicle-only control i.p. and subsequently transplanted the CLL cell line MEC-1 intravenously. The following four days the mice were injected again with 40mg/kg LDC2636 or vehicle-only i.p. We evaluated the survival time and found that mice treated with LDC2636 lived significantly longer than vehicle-only controls (24 vs. 18 days median survival, p=0.0016, n=15). Mice that received only LDC2636 and no Mec-1 cells did not show any effect. These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches. Disclosures: Schultz-Fademrecht: Lead Discovery Center GmbH: Employment. Unger:Lead Discovery Center GmbH: Employment. Klebl:Lead Discovery Center GmbH: Employment. Choidas:Lead Discovery Center GmbH: Employment.

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Preclinical Testing of a Novel Axl-Kinase Inhibitor in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.1799.1799 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1799-1799

Author(s):

Maria Göbel ◽

Michael Möllmann ◽

Andre Görgens ◽

Ulrich Dührsen ◽

Andreas Hüttmann ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Cell Polarization ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Blue Staining ◽

Nsg Mice

Abstract Abstract 1799 The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their potential to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models. In vitro studies: Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Since a polarized phenotype is required for migration, cell polarization was analyzed by time-lapse video-microscopy. We detected cytotoxic effects in a patient dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (LD50= 1.4 μM vs. 5.2 μM, p<0.004, n=5). Cell polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 μM, p<0.00001; BMS777607: IC50=6.2μM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and cell polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5). In vivo studies: To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/gcnull(NSG) mice were pre-treated with a single intraperitoneal bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the bone marrow of NSG mice by 43% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.046, BMS777607 p=0.0077; n=3). These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches. Disclosures: No relevant conflicts of interest to declare.

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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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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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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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Soluble CD14 is a novel monocyte-derived survival factor for chronic lymphocytic leukemia cells, which is induced by CLL cells in vitro and present at abnormally high levels in vivo

Blood ◽

10.1182/blood-2010-05-284505 ◽

2010 ◽

Vol 116 (20) ◽

pp. 4223-4230 ◽

Cited By ~ 52

Author(s):

Martina Seiffert ◽

Angela Schulz ◽

Sibylle Ohl ◽

Hartmut Döhner ◽

Stephan Stilgenbauer ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

Nuclear Factor Κb ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Nuclear Factor ◽

Soluble Cd14

Abstract Accumulation of leukemic cells in patients with chronic lymphocytic leukemia (CLL) is due to prolonged cell survival rather than increased proliferation. Survival of CLL cells depends on microenvironmental factors. Even though long-lived in vivo, CLL cells rapidly die by spontaneous apoptosis in vitro unless cocultured with stromal cells or their conditioned medium. In the present study, we show that survival of CLL cells is maintained in high cell density cultures, where the main prosurvival activity is delivered by monocytes. Cytokine array and enzyme-linked immunosorbent assay studies revealed increased expression of soluble CD14 by monocytes in the presence of CLL cells. The addition of recombinant soluble CD14 to primary CLL cells resulted in significantly increased cell survival rates, which were associated with higher activity nuclear factor κB. Quantification of serum levels of soluble CD14 revealed abnormally high levels of this protein in CLL patients, indicating a potential role of soluble CD14 in vivo. In summary, the presented data show that monocytes help in the survival of CLL cells by secreting soluble CD14, which induces nuclear factor κB activation in these cells, and that CLL cells actively shape their microenvironment by inducing CD14 secretion in accessory monocytes.

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MiR-181b in Chronic Lymphocytic Leukemia B Cells Is Regulated By Cellular Interaction with CD4+ T Cells and Increases the CTL Toxicity Versus the Leukemic Clone

Blood ◽

10.1182/blood.v126.23.4134.4134 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4134-4134

Author(s):

Mirco di Marco ◽

Serena Veschi ◽

Rosa Visone ◽

Giuseppe Leone ◽

Paola Lanuti ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd4 T Cells ◽

Lymphocytic Leukemia ◽

Healthy Donors ◽

Leukemic Clone

Abstract Clinical progression of chronic lymphocytic leukemia (CLL) is characterized by gradual reduction of the ratio T/B cells, along with immune cell dysfunction due, at least in part, to T cell defects, such as decreased expression of CD40L and reduced signaling via the TCR CD3. This compromise the ability of T cells to respond and to eliminate leukemic cell from CLL patients. Enhanced activation of either allogenic or autologous T cells can drive the death of CLL cells in vitro and in human subjects. Changes in microRNAs expression also characterize clinical progression of CLL with a strong decrease of miR-181b/a and miR-130a associated with the more aggressive phase of the disease. The miR-181b targets anti-apoptotic proteins, such as BCL-2 and MCL1 and its expression correlates with those protein levels in CLL. In this study we demonstrate that the expression of those microRNAs in CLL-B cells, are regulated by T cells. We co-cultured allogenic pure CLL-B cells with either activated (CD2, CD3 and CD28 antibodies, used to mimic antigen-presenting cells) or not activated CD4+ T cells from healthy donors. We observed a significant increase of mir-181b/a and miR-130a expression in CLL B-cells after co-culture with activated CD4+ T cells in 8 out of 11 cases. A significant increase of these miRs was also determined in purified CLL B-cells after 4 days activation of peripheral blood mononuclear cells (PBMCs) from CLL patients, even if in minor rate. By the use of specific antibodies, co-culture with Hela CD40 expressing cells and transwell experiments, we established that this effect is a T/B contact-dependent signaling mediated through CD40L-CD40 interaction. We determine that increased expression of the 3 miRs occurs at the transcriptional level. Since the expression of miR-181b showed the most significant variation in previous experiments it was selected for further analyses. We next investigated the in vivo role of the miR-181b in highly immunodeficient mice. The CLL cell line, MEC-01, infected with either the LV-miR-181b_coGFP or the LV-CTRL_coGFP was intravenously inoculated in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were sacrificed after 4 weeks and assayed for percentage of GFP+ cells in bone marrow and spleen compartments. The miR-181b did not show any specific effect into the leukemic clone. However when the same cells were inoculated in an environment hosting mature T cells, miR-181b consistently influences the death of leukemic cells (Fig 1B), suggesting that T cells are required to potentiate the apoptotic role of this miRNA. To explain what we observed in vivo, we mixed in vitro MEC-01 infected with either the LV-miR-181b or the LV-CTRL and CD8+ T cells from healthy donors. After few hours of contact T cells showed stronger cytotoxic effect on MEC-01 carrying miR-181b as compared to the control. Mixed lymphocyte reaction CD40L-activated CLL and T cells is used to generate effector CTLs. Therefore we grew T cell with CD40L-activated MEC-01 in which the expression of miR-181b was either shut down by lentiviral vector or unchanged as control. After one week, we monitored by cytofluorimetry the CD38 surface marker on T cells since its expression has been associated with more active CTLs and, by ELISA, the release of IL-10, the inhibitor of the potent inducer of CTLs INF-g. We demonstrate that activated MEC-01 with higher expression of miR-181b leads to an increase of the cell number expressing CD38 and this was accompanied by a reduced release of IL-10 from B cells through down-regulation of c-FOS, which we show to be target of the miR-181b and to promote the transcription of the IL-10. In conclusion, our data suggest a role of the miR-181b in the immune response against CLL-B cells. We show that an efficient activation of CD4+ T cells through CD3-complex pathway and a right CD40L-CD40 interaction lead to a significant increase of the some miRNAs deregulated over the progression of chronic lymphocytic leukemia, namely miR-181b. This miRNA potentiates the cytotoxicity of T cells favoring the killing of the leukemic clone. Disclosures No relevant conflicts of interest to declare.

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Targeting of HSP70/HSF1 Axis Abrogates In Vitro Ibrutinib-Resistance in Chronic Lymphocytic Leukemia

Cancers ◽

10.3390/cancers13215453 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5453

Author(s):

Federica Frezzato ◽

Andrea Visentin ◽

Filippo Severin ◽

Serena Pizzo ◽

Edoardo Ruggeri ◽

...

Keyword(s):

Heat Shock ◽

Chronic Lymphocytic Leukemia ◽

Resistance Mechanisms ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Heat Shock Factor 1 ◽

Ibrutinib Resistance ◽

In Cells

The Btk inhibitor ibrutinib has significantly changed the management of chronic lymphocytic leukemia (CLL) patients. Despite its clinical efficacy, relapses occur, and outcomes after ibrutinib failure are poor. Although BTK and PLCγ2 mutations have been found to be associated with ibrutinib resistance in a fair percentage of CLL patients, no information on resistance mechanisms is available in patients lacking these mutations. The heat shock protein of 70kDa (HSP70) and its transcription factor heat shock factor 1 (HSF1) play a role in mediating the survival and progression of CLL, as well as taking part in drug resistance in various cancers. We demonstrated that resveratrol and related phenols were able to induce apoptosis in vitro in leukemic cells from CLL untreated patients by acting on the HSP70/HSF1 axis. The same was achieved in cells recovered from 13 CLL patients failing in vivo ibrutinib treatment. HSP70 and HSF1 levels decreased following in vitro treatment, correlating to apoptosis induction. We suggest an involvement of HSP70/HSF1 axis in controlling resistance to ibrutinib in CLL cells, since their inhibition is effective in inducing in vitro apoptosis in cells from ibrutinib refractory patients. The targeting of HSP70/HSF1 axis could represent a novel rational therapeutic strategy for CLL, also for relapsing patients.

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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.bloodjournal632463 ◽

1984 ◽

Vol 63 (2) ◽

pp. 463-467 ◽

Cited By ~ 1

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

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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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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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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