BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia

Abstract The survival of chronic lymphocytic leukemia (CLL) cells depends on their interactions with microenvironment components, such as stromal and T cells. Lymph node microenvironment provides protective signals that enable the formation of proliferation centers and favor resistance to conventional chemotherapeutics. One of the key molecules engaged in the communication of CLL cells with their microenvironment is C-X-C chemokine receptor type 4 (CXCR4). The surface expression of CXCR4 is regulated by PIM1 (provirus integration site for Moloney murine leukemia virus) kinase. PIM 1-3 kinases are overexpressed in CLL cells and recent data suggest that targeting PIMs might be a rational therapeutic approach in this type of leukemia. Herein, we assessed associations of PIM kinase expression with clinical characteristics of CLL patients and investigated the consequences of PIM kinase inhibition for cell survival and CXCR4 - dependent signal transduction and migration of primary CLL cells. In primary CLL cells from peripheral blood, PIM2 transcript abundance was higher than PIM1 and PIM3 (p<0.001). PIM2 expression was higher in patients with advanced disease (Rai 3-4, p=0.004). Higher PIM2 expression was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood (p=0.024 and p=0.0059, respectively; Herishanu et al., 2010), suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of PIM kinases in these processes, we assessed whether PIM inhibition interferes with CXCR4 - mediated signaling and migration. We incubated primary CLL cells with a novel pan-PIM inhibitor SEL24-B489 and found decreased phospho-CXCR4 (Ser339) level and decreased CXCR4 surface expression. Primary CLL cells incubated with with SDF1 (500 ng/ml, 15-60 min) exhibited highly increased phosphorylations of mTOR (Ser2448) and Akt (Ser437). Since PIM kinases modulate mTOR signaling, we further investigated whether inhibition of PIM kinases with SEL24-B489 interferes with CXCR4/mTOR pathway. SEL24-B489 blocked baseline phosphorylation level of mTOR negative regulator p-TSC2 (Ser1798). Since TSC2 Ser1798 phosphorylation relieves its suppression on RHEB and promotes mTOR activity, we next assessed PIM inhibitor - induced changes in p-mTOR (Ser2448). Upon SEL24-B489 treatment, mTOR Ser2448 phosphorylation and activity of mTOR downstream substrates (p-Akt Ser473 and p-4EBP1 Thr37/46) markedly decreased. Pre-incubation of CLL cells with 10 uM SEL24-B489 or 10 uM mTOR inhibitor OSI-027 before SDF1 treatment restrained the increase of p-mTOR (Ser2448), p-Akt (Ser473), p-4EBP1 (Thr37/46) and p-TSC2 (Ser1798), and consequently impaired CLL cells migration in the SDF1 gradient. In 4 out of 7 analyzed patients the effect of SEL24-B489 on CLL migration was stronger than the effect of OSI-027 and in the remaining 3 patients, the effects of both inhibitors were similar. Since interactions of CLL cells with their microenvironment block the cytotoxicity of chemotherapeutic agents, we next compared the apoptotic response to SEL24-B489 in CLL cells cultured in the absence or presence of human bone marrow HS5 cells. CLL cells were seeded on HS5 monolayers and treated with SEL24-B489 (5 uM and 10 uM) for 48 hours. In 6 out of 7 cases SEL24-B489 overcame the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells. Taken together, our data demonstrate that CXCR4/SDF1 signal in chronic lymphocytic leukemia cells is transduced through mTOR pathway and that CXCR4 - triggered mTOR activity is modulated by PIM kinases. Pan-PIM inhibitor SEL24-B489 decreased CXCR4 surface expression and SDF-1 - triggered mTOR activity. Finally, SEL24-B489 decreased protective effects of tumor microenvironment and induced CLL cells apoptosis even in the presence of stromal cells. Since overexpression of PIM kinases might be associated with adverse clinical characteristics at diagnosis, PIM inhibition might be a rational therapeutic strategy in CLL. Disclosures Czardybon: Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Other: member of Selvita Scientific Advisory Board.

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P2X7 receptor expression in evolutive and indolent forms of chronic B lymphocytic leukemia

Blood ◽

10.1182/blood.v99.2.706 ◽

2002 ◽

Vol 99 (2) ◽

pp. 706-708 ◽

Cited By ~ 131

Author(s):

Elena Adinolfi ◽

Loredana Melchiorri ◽

Simonetta Falzoni ◽

Paola Chiozzi ◽

Anna Morelli ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Receptor Expression ◽

Extracellular Nucleotides ◽

Spontaneous Proliferation ◽

Proliferation Of Lymphocytes ◽

Mediate Cell ◽

And Function ◽

Cell Chronic Lymphocytic Leukemia

Abstract Human leukocytes express a receptor for extracellular nucleotides, named P2X7R, that in lymphocytes can either mediate cell death or proliferation, depending on the level of activation. The authors have investigated P2X7R expression and function in 21 patients affected by B-cell chronic lymphocytic leukemia, 13 with an evolutive and 8 with an indolent variant of the disease. Resting cytoplasmic Ca++ concentration was significantly higher in lymphocytes from patients with the evolutive compared with indolent variant. Furthermore, in the former, P2X7R stimulation triggered a Ca++ influx significantly larger. Higher Ca++ influx correlated with an increased P2X7R expression in the lymphocytes from patients with the evolutive form. Finally, incubation in the presence of extracellular adenosine triphosphate decreased spontaneous proliferation of lymphocytes from patients affected with the evolutive variant but had no effects on lymphocytes from patients with the indolent form. These results suggest that expression and function of P2X7R may correlate with the severity of B-cell chronic lymphocytic leukemia.

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The chemokine receptor CXCR3 is expressed in a subset of B-cell lymphomas and is a marker of B-cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v95.2.627 ◽

2000 ◽

Vol 95 (2) ◽

pp. 627-632 ◽

Cited By ~ 135

Author(s):

Dan Jones ◽

Richard J. Benjamin ◽

Aliakbar Shahsafaei ◽

David M. Dorfman

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Chemokine Receptor ◽

Marginal Zone ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Marginal Zone Lymphoma ◽

Lymphocytic Leukemia ◽

Splenic Marginal Zone Lymphoma ◽

Flow Cytometric

Chemotaxis in leukocytes is mediated through binding of soluble chemokines to transmembrane G-protein coupled receptors. The chemokine receptor CXCR3 has been previously shown to be widely expressed on activated T cells and to mediate T-cell chemotaxis on binding to various ligands, including Mig, IP-10, and ITAC. By using immunohistochemical and flow cytometric analysis, we report that CXCR3 is also expressed on a subset of peripheral blood B cells and in distinct subtypes of B-cell lymphoma. CXCR3 immunohistochemical or flow cytometric expression was seen in 37 of 39 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (diffusely positive in 33 cases), whereas mantle cell lymphoma (30 cases), follicular lymphoma (27 cases), and small noncleaved cell lymphoma (8 cases) were negative in all but 2 cases. Strong CXCR3 expression was also seen in splenic marginal zone lymphoma (14 of 14 cases) and in the monocytoid and plasmacytic cells in extranodal marginal zone lymphoma (15 of 16 cases). This differential expression of CXCR3 in B-cell tumors contrasts with that of another B-cell–associated chemokine receptor, BLR1/CXCR5, which we show here is expressed on all types of B-cell lymphoma tested. We also report that the CXCR3 ligand, Mig, is coexpressed on tumor cells in many cases of CLL/SLL (10 of 13 cases examined) with Mig expression less frequently seen in other B-cell lymphoma subtypes. Coexpression of CXCR3 and its ligand, Mig, may be an important functional interaction in B-CLL, as well as a useful diagnostic marker for the differential diagnosis of small cell lymphomas.

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Ibrutinib restores immune cell numbers and function in first-line and relapsed/refractory chronic lymphocytic leukemia

Leukemia Research ◽

10.1016/j.leukres.2020.106432 ◽

2020 ◽

Vol 97 ◽

pp. 106432 ◽

Cited By ~ 1

Author(s):

Isabelle G. Solman ◽

Lisa K. Blum ◽

Hana Y. Hoh ◽

Thomas J. Kipps ◽

Jan A. Burger ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Immune Cell ◽

Lymphocytic Leukemia ◽

First Line ◽

Cell Numbers ◽

And Function

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Abnormalities in chemokine receptor recycling in chronic lymphocytic leukemia

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03058-9 ◽

2019 ◽

Vol 76 (16) ◽

pp. 3249-3261 ◽

Cited By ~ 3

Author(s):

Laura Patrussi ◽

Nagaja Capitani ◽

Cosima T. Baldari

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Chemokine Receptor ◽

Lymphocytic Leukemia ◽

Receptor Recycling

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p66Shc deficiency in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia enhances leukemogenesis by altering the chemokine receptor landscape

Haematologica ◽

10.3324/haematol.2018.209981 ◽

2019 ◽

Vol 104 (10) ◽

pp. 2040-2052 ◽

Cited By ~ 5

Author(s):

Laura Patrussi ◽

Nagaja Capitani ◽

Cristina Ulivieri ◽

Noemi Manganaro ◽

Massimo Granai ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Mouse Model ◽

Chemokine Receptor ◽

Lymphocytic Leukemia

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Lenalidomide Reduces Survival of Chronic Lymphocytic Leukemia Cells in Primary Co-Cultures by Altering the Myeloid Microenvironment

Blood ◽

10.1182/blood.v120.21.3894.3894 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3894-3894

Author(s):

Angela Schulz ◽

Claudia Dürr ◽

Thorsten Zenz ◽

Stephan Stilgenbauer ◽

Peter Lichter ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Drug Effects ◽

Surface Expression ◽

Lymphocytic Leukemia ◽

Cell Surface Expression ◽

Clinical Activity ◽

Altered Expression

Abstract Abstract 3894 Chronic lymphocytic leukemia (CLL) cells are highly dependent on their microenvironment. External stimuli provided by bone marrow stromal cells or non-malignant leukocytes are required for their survival and proliferation. Interestingly, peripheral blood-derived monocytes differentiate in the presence of CLL cells to so-called Nurse-like cells (NLCs), which are round or fibroblast-shaped adherent cells that were shown to promote survival of CLL cells in vitro and to exist in lymph nodes of CLL patients. In search of new therapeutic options for patients with CLL, the immunomodulatory drug lenalidomide turned out to have significant clinical activity in CLL. Lenalidomide does not induce apoptosis in CLL cells directly, but is rather believed to act via the microenvironment. Several studies described that it alters cytokine levels and the activation status of the cells. Further, a CLL-specific T-cell defect was shown to be repaired by lenalidomide, which might represent a major activity of this drug in CLL. However, its mechanism of action seems to be complex and is not well understood. As monocytes as well as NLCs are very effective in maintaining survival of CLL cells, we aimed to investigate whether lenalidomide interferes with these supportive cell-cell interactions. To do this, we established primary co-cultures of monocytes and CLL cells in the presence or absence of lenalidomide and observed a significantly decreased viability of CLL cells after 14 days of treatment, suggesting an impact of this drug on the survival support of NLCs. Therefore, we analyzed the immunophenotype of NLCs by flow cytometry, as well as the secretion of cytokines in the co-cultures by ELISA and antibody-coupled bead arrays. Among the effects induced by lenalidomide, we observed reduced cell surface expression of the MHC II protein HLA-DR on NLCs as well as lower levels of the chemokine CCL2, but higher levels of IL-10 in the culture supernatant, indicating an altered inflammatory milieu in the co-cultures. The enhanced IL-10 levels resulted in an increase in STAT1 phosphorylation in CLL cells as measured by Western blot analysis. As a consequence, enhanced expression of the adhesion molecule ICAM-1 (CD54) and an altered expression of cytoskeletal genes (e.g. RHOC and CORO1B) were observed in CLL cells after lenalidomide treatment. Chemotaxis assays using transwell culture dishes and SDF1-α as chemoattractant revealed an impaired migratory potential of lenalidomide-treated CLL cells, which was not due to reduced expression of the SDF1-α receptor CXCR4. In summary, our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug effects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide impairs the migratory potential of CLL cells which may affect circulation and homing of CLL cells in vivo. Disclosures: No relevant conflicts of interest to declare.

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Microenvironment-Induced Expression of PIM Kinases Supports Chronic Lymphocytic Leukemia Cells Survival and Promotes CXCR4-mTOR Pathway Dependent Migration

Blood ◽

10.1182/blood.v128.22.3239.3239 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3239-3239 ◽

Cited By ~ 3

Author(s):

Emilia Bialopiotrowicz ◽

Patryk Gorniak ◽

Bartosz Pula ◽

Monika Noyszewska-Kania ◽

Hanna Makuch-Lasica ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Peripheral Blood ◽

Surface Expression ◽

Mtor Pathway ◽

Lymphocytic Leukemia ◽

Apoptotic Response ◽

Pim Kinases

Abstract Lymph node microenvironment provides chronic lymphocytic leukemia (CLL) cells with pro-survival and protective signals, fostering resistance to conventional chemotherapeutics. CLL cells overexpress oncogenic PIM kinases, which modulate proteins engaged in transcription, translation, apoptosis, cell cycle and adhesion/motility (Mol Cancer Ther 2014, 13: 1231-45). Herein, we searched for the link between tumor microenvironment and PIMs expression, compared the clinical characteristics of CLL patients with high versus low expression of PIM kinases, and investigated the consequences of their inhibition with newly developed pan-PIM inhibitor, SEL24-B489 in primary CLL cells. We first evaluated the expression of PIM kinases in CD19+ cells derived from 88 newly diagnosed CLL cases. Patients with unmutated IGHV status exhibited significantly higher PIM1 transcript levels than patients with mutated IGHV genes. Subjects with advanced CLL (Binet C) exhibited higher PIM2 expression than patients in Binet A/B stage. Significantly higher PIM2 transcript abundance at the time of diagnosis was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood, suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of microenvironment in the control of PIM expression, peripheral blood CLL cells were incubated with anti-IgM or CD40 ligand. Both stimuli induced PIM1 and PIM3 expression. Co-culture of CLL cells with stromal cell (HS5) monolayers promoted the expression of PIM3 isoform. We next assessed the consequences of PIM inhibition in CLL cells using novel pan-PIM inhibitor, SEL24-B489. Incubation with SEL24-B489 decreased phosphorylation of PIM substrates, p-FOXO1/3a(T24/T32) and p-4EBP1(S65), and induced dose-dependent apoptosis in 27 out of 28 analyzed cases, regardless of the IGHV mutation status and including relapsed patients. Of note, SEL24-B489 induced higher apoptotic response in primary CLL cells than referential pan-PIM inhibitor AZD1208. CLL cells with 17p13 deletion and obtained from chemo-refractory patients were also vulnerable to SEL24-B489, suggesting that functional p53 is not required for execution of SEL24-B489-mediated apoptosis. Importantly, SEL24-B489 was not toxic for cells derived from healthy donors. Since microenvironmental cues increase expression of PIM kinases, we hypothesized that interactions with stromal cells might hinder the in vitro activity of the PIM inhibitor. To explore this possibility, we compared apoptotic response to SEL24-B489 in CLL cells co-cultured on HS5 monolayers and CLL cells grown without the stromal support. In 6 out of 7 tested cases, SEL24-B489 overrode the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells. PIM1 was shown to regulate CLL cells migration through CXCR4(S339) phosphorylation (Mol Cancer Ther 2014, 13: 1231-45). Accordingly, SEL24-B489 decreased phospho-CXCR4(S339), CXCR4 surface expression, and impaired CLL cells migration in the CXCL12 gradient. Surprisingly, decrease in the CXCR4 surface expression after SEL24-B489 was relatively modest when compared to the effect of this inhibitor on CXCL12-directed migration. We found that incubation of CLL cells with CXCL12 led to increase in the phosphorylation of mTOR(S2448) and Akt(S473). SEL24-B489 reduced the levels of p-mTOR(S2448), p-Akt(S473), p-4EBP1(T37/T46) and p-TSC2(S1798), revealing inhibitory effect on mTOR pathway. Pre-incubation of CLL cells with an mTOR inhibitor similarly restrained CXCL12-mediated mTOR activity and led to impaired CLL cells migration, uncovering the key role of mTOR axis in CXCR4-dependent migration. Thus, SEL24-B489 impairs the CLL cell migration by inhibiting CXCR4 surface expression and the CXCR4-triggered mTOR pathway. Taken together, we show that microenvironment signals increase expression of PIM kinases, supporting CLL cell survival and migration. Inhibition of PIM kinases impairs CXCR4-dependent migration and leads to CLL cells death, regardless of the p53 status. Targeting PIM kinases in CLL patients will likely release the cells from microenvironmental niches and might be a rational therapeutic strategy. Disclosures Warzocha: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Czardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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