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

Blood ◽  
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.

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

2018 ◽  
Vol 215 (2) ◽  
pp. 681-697 ◽  
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.

Comparison of in Vitro Apoptotic Response of Chronic Lymphocytic Leukemia (CLL) Cells to Bcl-2 Antagonist ABT-737 and IAP Antagonist BV6.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4386-4386
Author(s):  
Shinichi Kitada ◽  
Ryuji Yamaguchi ◽  
Laura Z. Rassenti ◽  
Thomas J. Kipps ◽  
John C. Reed
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Patient Treatment ◽  
Altered Expression ◽  
Over Expression ◽  
Iap Antagonist ◽  
Smac Mimetics

Abstract Abstract 4386 Altered expression of Bcl-2-family and IAP-family proteins has been considered to play central roles in apoptosis dysregulation in cancer and leukemia, promoting malignant cell expansion and contributing to chemoresistance. Anti apoptotic Bcl-2-family proteins Bcl-2 and Bcl-XL as well as IAP proteins, including XIAP, have been thoroughly validated as drug discovery targets for cancer and strategies for inhibiting these proteins have been devised based on mimicking their endogenous antagonists, as represented by ABT-737, a fully synthetic Bcl-2/Bcl-XL antagonist developed at Abbott Laboratories, and BV6, bivalent SMAC mimetics developed at Genentech Inc. Chronic lymphocytic leukemia (CLL) is a quintessential example of a human malignancy caused by defective programmed cell death. Over-expression of the Bcl-2 protein is one of the most consistent and prominent etiological factors associated with this disease, whereas over-expression of XIAP protein has been demonstrated in a majority of CLL patients. In this study, we evaluated the pro-apoptotic effects of ABT-737 and BV6 on CLL cells in side-by-side comparisons. We found that the CLL cells of 31 of 42 patients were highly sensitive to ABT-737, inducing potent dose-dependent killing of cells with an IC50 = 52.5 ± 22.5 (mean ± std error). In contrast, the CLL cells of 11 of 42 patients were relatively resistant to ABT-737, requiring higher concentrations to induce apoptosis (IC50 >1 μM). Preliminary statistical analysis was performed to correlate ABT737-sensitive and -resistant CLL with various other characteristics, including patient treatment status, ZAP-70 expression, and VH mutation status, but no significant correlation was observed. In contrast, the CLL cells from all 42 patients examined were relatively resistant to the IAP antagonist BV6, which required high concentrations to induce apoptosis (IC50 > 1 μM). Similar results were obtained using other SMAC mimetic compounds. Analysis of CLL cells for endogenous SMAC protein expression and for the subcellular location of SMAC protein demonstrated that the leukemia cells of 18 of 23 patients expressed high-levels of SMAC, as assessed by immunoblotting. Furthermore, SMAC was located in the cytosolic fraction of these leukemia cells in 7 of 18 cases examined. However, CLL cells with high-levels of cytosolic SMAC had little evidence of concomitant release of cytochrome c from mitochondria and demonstrated little spontaneous apoptosis in vitro. These results suggest that the leukemia cells of some CLL patients may already express cytosolic SMAC, which might explain the relative insensitivity of CLL cells to SMAC mimetics such as BV6. Taken together, these studies suggest that Bcl-2 antagonists such as ABT-737, but not SMAC mimetics such as BV6, may have promising therapeutic activity for most CLL patients. (NIH CA-081534) Disclosures: No relevant conflicts of interest to declare.

Surface IgM of Chronic Lymphocytic Leukemia Cells Displays Unusual Glycans Indicative of Antigen Engagement In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 55-55
Author(s):  
Graham Packham ◽  
Serge Krysov ◽  
Christopher Ian Mockridge ◽  
Kathy N Potter ◽  
Freda K Stevenson
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Variable Region ◽  
Immunoglobulin M ◽  
Lymphocytic Leukemia ◽  
Glycosylation Pattern ◽  
Mature Form

Abstract Abstract 55 Several lines of evidence support the idea that surface immunoglobulin M (sIgM) plays a key role in determining the clinical behavior of chronic lymphocytic leukemia (CLL). For example, the presence of somatic mutations in immunoglobulin variable region genes is a strong prognostic marker with unmutated CLL (U-CLL) associated with a poor outcome relative to mutated CLL (M-CLL). U-CLL also generally express higher levels of sIgM and retain the ability to signal via this receptor. In this study, we used surface biotinylation to analyse sIgM in CLL and discovered that it exists in two forms with differing mobility on SDS-PAGE. Treatment with glycosidases revealed that these forms were due to different N-glycosylation patterns in the μ constant region. One form is similar to that of normal B cells in bearing mature complex glycans common to most cell surface glycoproteins. The other is an immature mannosylated form more characteristic of endoplasmic reticulum (ER)-located μ chains. CLL cells expressed variable proportions of the immature mannosylated form and quantitative analysis demonstrated that, on average, the proportion of mannosylated sIgM was approximately 2-fold higher (p=0.006) in U-CLL compared to M-CLL. Although normal B cells isolated from blood expressed only the mature form of sIgM, in vitro treatment with anti-μ resulted in upregulation of the immature form, suggesting that glycan modification is a consequence of antigen exposure. Consistent with this, in vitro incubation of CLL cells was associated with increased expression of the mature form of sIgM. Phosphotyrosine analysis demonstrated that both forms of sIgM were able to signal following sIgM engagement in vitro. Taken together, these findings support the concept that CLL cells are continuously exposed to antigen in vivo. This process leads to a change in the N-glycosylation pattern of the re-expressed sIgM so that a mannosylated form predominates, especially in U-CLL. Strikingly, expression of mannosylated sIgM is also characteristic of follicular lymphoma, where it is constitutively displayed via N-glycosylation sites in the Ig variable region (Radcliffe et al. J Biol Chem. 2007; 282, 7405-15). Persistent mannosylation of sIgM appears to be a feature common to several B-cell malignancies, suggesting a role in pathogenesis. Disclosures: No relevant conflicts of interest to declare.

IL4 and CD40L Prevent Apoptosis of Chronic Lymphocytic Leukemia Cells Via Intracellular pSTAT6 and NFkB Signaling and Not Via Receptor Kinetics

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3893-3893
Author(s):  
Daniel Mertens ◽  
Nupur Bhattacharya ◽  
Sarah Häbe ◽  
Hartmut Döhner ◽  
Stephan Stilgenbauer
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conditioned Medium ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Malignant Cells ◽  
Downstream Signaling ◽  
Receptor Kinetics

Abstract Abstract 3893 Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental input for their extended survival in vivo, but the underlying molecular mechanism is still unclear. Compared to non-malignant B-cells, CLL cells are more responsive to contact dependent complex stimuli like coculture on bone marrow derived stromal cell lines of both human (p<0.0001) and murine origin (p<0.01), but also to soluble factors (human conditioned medium p<0.0001, murine conditioned medium p<0.001, all student′s t-test). In order to understand the intrinsic difference of the anti-apoptotic phenotype of CLL cells, the signalling circuitry of the malignant cells was modelled. Compared to candidate ligands like SDF-1 (at concentrations between 10–1000ng/ml), BAFF (250–1000ng/ml), APRIL (250–1000ng/ml) and soluble anti-IgM (1–25μg/ml), the factors CD40L (10–2000ng/ml) and IL4 (0.1–10ng/ml) were the most efficient ligands in rescuing CLL cells from spontaneous death in vitro. The dose response of IL4 and CD40L displayed different saturation and cooperativity between CLL cells and non-malignant B-cells. Using IL4, saturation was reached both for CLL cells and B-cells at 0.2pM, but at 52% survival (+/− 8%) for CLL cells and 28% (+/−7%) for B-cells, and the estimated dissociation constant Kd was 0.01pM for both ligands. For CD40L, CLL cell survival reached saturation at 40nM, while no saturation was reached for B-cells. Intriguingly, B-cells showed cooperativity in their response to CD40L, with a cooperativity coefficient of 2.0 and a Kd of 70pM, while cooperativity for CD40L was lost in CLL cells (Kd of only 2.6pM). This pointed towards distinct differences in ligand-receptor interactions or in downstream signaling between CLL cells and non-malignant B-cells. However, high-throughput spatial analysis with a microscope-coupled cytometer did not show differences of receptor quantity or receptor distribution between malignant and non-malignant cells. In contrast, quantity and phosphorylation levels of downstream signalling nodes like STAT6 (measured by flow cytometry and validated by Western-blot) and the activity of NF-kB (p65 binding to DNA measured by oligonucleotide-coupled ELISA) were higher in CLL cells compared to B-cells from healthy donors. Therefore, the defect in IL4 and CD40L signalling that leads to an enhanced survival in CLL cells is likely caused by changes in the intracellular circuitry. Disclosures: No relevant conflicts of interest to declare.

Microrna-34a As a Marker for Fludarabine Resistance and Impairment of p53-Pathway in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3883-3883
Author(s):  
Marek Mraz ◽  
Katerina Cerna ◽  
Veronika Mayerova ◽  
Katerina Musilova ◽  
Karla Plevova ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Absolute Quantification ◽  
P53 Mutation ◽  
Fold Change ◽  
Lymphocytic Leukemia ◽  
Apoptotic Pathway ◽  
P53 Pathway

Abstract Abstract 3883 Background. MicroRNAs (miRNAs) are known to be involved in the pathogenesis of chronic lymphocytic leukemia (CLL) and affect its clinical course (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012). Moreover, we and others have shown that several miRNAs are down-regulated in the aggressive CLL subtype harboring p53 aberration (Mraz et al. Leukemia, 2009). The role of miRNAs in primary or acquired resistance to therapy in CLL, however, is poorly understood. Aim. In this study, we screened for miRNAs that are induced by fludarabine-mediated apoptosis in vitro, and we suggested that differences in the expression of one of the identified miRNAs (miR-34a) can be used to distinguish patients with impairment of the p53-apoptotic pathway. Results. Ten primary CLL B-cell samples (purity>95% of CD5+19+ cells) were treated in vitro with fludarabine dose (IC50 dose of 3.5 ug/mL, 48hrs), and the expression of 750 miRNAs was subsequently profiled (TaqMan miRNA Cards, ABI). In comparison with untreated control samples, 15 miRNAs were induced by fludarabine (fold change>1.5, SAM FDR<0.05). The most prominently up-regulated miRNA was miR-34a (fold change 3.7, P=0.003), which is a known p53 down-stream target (He et al. Nature, 2007). We then compared miR-34a up-regulation post fludarabine treatment to the decrease in cell viability (wt-p53 samples, N=20). This revealed that miR-34a induction was significantly higher in CLL samples more sensitive to fludarabine and suggested its role in the apoptotic effects of fludarabine in B-cells. Moreover, the up-regulation of miR-34a was also observed in vivo in samples obtained from fifty FCR-treated CLL patients (fold change 2.2, P<0.0001, analyzed at day 0 and 3 of FCR). These data encouraged us to develop an assay for absolute quantification of miR-34a which would allow determining the copy numbers of miR-34a, defining precise cut-offs, and comparing miR-34a levels during the course of the disease in one patient. We designed synthetic RNA oligos that were used to construct standard curves for both miR-34a and a normalization gene (RNU48). Using this assay, we profiled the expression of miR-34a in a cohort of CLL patients (N=200) to define a cut-off value that would discriminate therapy resistant cases. The distribution of miR-34a expression in the cohort ranged from 1 to 81820 molecules (per 10e6 copies of RNU48). Significantly, miR-34a levels below 2500 copies (N=47) were correlated with shorter overall survival (9.6 years vs. not-reached, HR 2.2 [CI 1.1–4.5], P=0.03). Subsequently, the expression of miR-34a was compared in samples stratified by known prognostic markers: chromosomal aberrations (del17p13, del11q23, tris.12, and del13q14), IgHV status, expression of CD38, CD49d, age, gender, and Rai stage. The lower miR-34a levels were only associated with the deletion of 17p13 locus that includes the p53 gene (N=18, fold change −3.4, P=0.003). Remarkably, CLL samples with sole p53 mutation not accompanied by p53 deletion (N=13) also expressed low levels of miR-34a compared to wt-p53 (P=0.005, fold change −2.7). Most notably, 77% (10/13) of these samples had miR-34a levels below the cut-off of 2500 copies. We further validated our observations and assay by analyzing miR-34a expression in paired samples from 12 CLL cases that acquired p53 aberration during the course of the disease. This emphasized that miR-34a expression decreased in all cases after occurrence of p53 mutation (P=0.0008, fold change −6.1). Additionally, the effect of miR-34a up-regulation on therapy response is currently being investigated in a cohort of FCR-treated patients (N=50). Conclusions. Our data provide complex evidence for the use of miR-34a as a marker of fludarabine-resistant disease. MicroRNA-34a quantification can identify p53 mutated cases that would not be recognized by FISH (mutation not accompanied by del17p13), and miR-34a down-regulation can be used as a sensor for acquisition of p53 abnormality during the course of the disease. This can be accomplished without treatment of cells with gamma-irradiation, which was previously used to identify functional impairment of the p53-pathway (Pettitt et al. Blood, 2001; Mous et al. Leukemia, 2009; Lin et al. CCR, 2012). The described assay for absolute quantification of miR-34a encourages further inter-laboratory validation. IGA MZCR NT11218–6/2010, CZ.1.07/2.3.00/20.0045, CZ.1.05/1.1.00/02.0068 Disclosures: No relevant conflicts of interest to declare.

Divergence in CD19-Mediated Signaling Unfolds Intra-Clonal Diversity in Chronic Lymphocytic Leukemia Which Correlates with Disease Progression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4570-4570
Author(s):  
Yair Herishanu ◽  
Sigi Kay ◽  
Nili Dezorella ◽  
Chava Perry ◽  
Varda Deutsch ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Disease Progression ◽  
Conflicts Of Interest ◽  
Clonal Diversity ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Fraction ◽  
Akt Phosphorylation

Abstract Abstract 4570 Emerging data on intra-clonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. Here we report intra-clonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into responding (CD19-R) and non-responding (CD19-N) sub-populations. Engagement of CD19 by anti-CD19 antibody rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA and exhibited distinct morphological features. Both sub-populations reacted to IgM stimulation in a similar manner and exhibited similar levels of Akt phosphorylation, pointing to functional signaling divergence within the B-cell receptor. CD19 unresponsiveness was partially reversible, where CD19-N cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19-signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow-derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with more than 18.5% of CD19-R cell fraction had a shorter median time to treatment compared to patients with less than 18.5% of CD19-R cell fraction. Conclusions: Divergence in CD19-mediated signaling unfolds both inter-patient and intra-clonal diversity in CLL. This signaling diversity is associated with physiological implications including the location of the cells and disease progression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of the TCL-1 transgenic mouse as a preclinical drug development tool for human chronic lymphocytic leukemia

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1334-1338 ◽  
Author(s):  
Amy J. Johnson ◽  
David M. Lucas ◽  
Natarajan Muthusamy ◽  
Lisa L. Smith ◽  
Ryan B. Edwards ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Drug Development ◽  
Transgenic Mouse ◽  
Subsequent Development ◽  
Lymphocytic Leukemia ◽  
New Drugs ◽  
Clinical Activity ◽  
Suitable Animal Model

Abstract Drug development in human chronic lymphocytic leukemia (CLL) has been limited by lack of a suitable animal model to adequately assess pharmacologic properties relevant to clinical application. A recently described TCL-1 transgenic mouse develops a chronic B-cell CD5+ leukemia that might be useful for such studies. Following confirmation of the natural history of this leukemia in the transgenic mice, we demonstrated that the transformed murine lymphocytes express relevant therapeutic targets (Bcl-2, Mcl-1, AKT, PDK1, and DNMT1), wild-type p53 status, and in vitro sensitivity to therapeutic agents relevant to the treatment of human CLL. We then demonstrated the in vivo clinical activity of low-dose fludarabine in transgenic TCL-1 mice with active leukemia. These studies demonstrated both early reduction in blood-lymphocyte count and spleen size and prolongation of survival (P = .046) compared with control mice. Similar to human CLL, an emergence of resistance was noted with fludarabine treatment in vivo. Overall, these studies suggest that the TCL-1 transgenic leukemia mouse model has similar clinical and therapeutic response properties to human CLL and may therefore serve as a useful in vivo tool to screen new drugs for subsequent development in CLL.

scholarly journals Chronic lymphocytic leukemia cells receive RAF-dependent survival signals in response to CXCL12 that are sensitive to inhibition by sorafenib

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 882-889 ◽  
Author(s):  
Davorka Messmer ◽  
Jessie-F. Fecteau ◽  
Morgan O'Hayre ◽  
Ila S. Bharati ◽  
Tracy M. Handel ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Molecular Mechanisms ◽  
Lymphocytic Leukemia ◽  
Mitogen Activated Protein Kinase ◽  
Clinical Activity ◽  
Mitogen Activated Protein ◽  
Chemokine Cxcl12 ◽  
New Treatments

Abstract The chemokine CXCL12, via its receptor CXCR4, promotes increased survival of chronic lymphocytic leukemia (CLL) B cells that express high levels of ζ-chain–associated protein (ZAP-70), a receptor tyrosine kinase associated with aggressive disease. In this study, we investigated the underlying molecular mechanisms governing this effect. Although significant differences in the expression or turnover of CXCR4 were not observed between ZAP-70+ and ZAP-70− cell samples, CXCL12 induced greater intracellular Ca2+ flux and stronger and more prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase/ERK kinase (MEK) in the ZAP-70+ CLL cells. The CXCL12-induced phosphorylation of ERK and MEK in ZAP-70+ CLL cells was blocked by sorafenib, a small molecule inhibitor of RAF. Furthermore, ZAP-70+ CLL cells were more sensitive than ZAP-70− CLL cells to the cytotoxic effects of sorafenib in vitro at concentrations that can readily be achieved in vivo. The data suggest that ZAP-70+ CLL cells may be more responsive to survival factors, like CXCL12, that are elaborated by the leukemia microenvironment, and this sensitivity could be exploited for the development of new treatments for patients with this disease. Moreover, sorafenib may have clinical activity for patients with CLL, particularly those with ZAP-70+ CLL.

scholarly journals Ibrutinib Treatment in CLL Interrupts CD40 Signaling Capacity and Sensitizes CLL Cells to Venetoclax

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1545-1545
Author(s):  
Karoline Kielbassa ◽  
Marco Haselager ◽  
Danique Bax ◽  
Julie Dubois ◽  
Mark-David Levin ◽  
...  
Keyword(s):  
Lymph Node ◽  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Lymphocytic Leukemia ◽  
Cell Surface Expression ◽  
P Value ◽  
Cd40 Expression

Abstract Background: For proliferation and survival, chronic lymphocytic leukemia (CLL) cells depend on interactions with cells and soluble factors present in the tumor microenvironment (TME). These interactions also increase expression of B-cell leukemia/lymphoma-2 (Bcl-2) proteins, including Bcl-XL, Mcl-1 and Bfl-1, thereby reducing drug sensitivity. In the VISION HOVON141 clinical trial, patients with relapsed or refractory CLL are treated with 2 cycles of Bruton tyrosine kinase inhibitor ibrutinib (IBR) lead-in followed by 13 cycles of IBR + Bcl-2 inhibitor venetoclax (VEN) combination before randomization. The combination of IBR + VEN may have synergistic anti-tumor effects, since IBR forces CLL cells from lymph node (LN) to the blood (PB) where they become fully dependent on Bcl-2, and succumb to VEN. To support a mechanistic basis for this premise, we investigated changes in expression of Bcl-2 proteins, effects of TME-mimicking signals, and venetoclax sensitivity before/after 2 months IBR treatment. Results: At baseline, lymph node emigrant cells (CXCR4dim/CD5high) have higher Bfl-1 expression, in addition to earlier reported Bcl-XL and Mcl-1 expression than cells immigrating back to the lymph node (CXCR4high/CD5dim)(Haselager et al., 2020). After 2 months of IBR treatment a clear reduction in all four pro-survival proteins was observed (N=17 p&lt;0.001). Despite these changes, VEN sensitivity was not different in unstimulated PB CLL cells obtained at baseline versus at 2 months of IBR treatment (N=8; IC50=0.001µM). In contrast, CD40 stimulated CLL cells obtained at baseline are fully resistant to VEN (IC50&gt;10µM), but unexectedly retained partial sensitivity to venetoclax after IBR treatment (IC50=0.05 µM, p-value &lt;0.0001; Figure 1A). This suggested reduced CD40 signalling capacity and was accompanied by reduced ability to upregulate Bcl-XL, Mcl-1, and Bfl-1 expression. Indeed, cell surface expression, as well as level of CD40 activation as measured by CD95 induction, was clearly reduced after 2 months IBR. Importantly, these effects occurred in vivo, as IBR did not directly affect CD40 signaling in vitro. These data imply that under IBR treatment, when cells cannot (re-)enter LN sites, a factor is lacking that maintains or induces CD40 expression. In search of stimuli that can augment CD40 signaling capacity, it was found that BCR stimulation had no effect on CD40 expression. In contrast, TLR9 stimulation via CpG led to increased CD40 expression in CLL cells (N=7, p-value &lt;0.0001)(Figure 1B). These findings align well with recent data which indicate a role for TLR9 signalling in vivo by unmethylated mitochondrial DNA in CLL (Kennedy et al., 2021). Taken together, these data provide a mechanistic explanation, by which a triad of signaling pathways not only involving BCR but also CD40 and TLR9 is interrupted by IBR (Figure 1C). Discussion/conclusion: IBR treatment broadly affects Bcl-2 family protein expression and CD40 signaling in vivo. The combined data indicate a novel aspect of IBR efficacy, namely its capacity to interrupt TLR9-induced CD40 upregulation, which normally primes CLL cells in the LN environment for drug resistance. This also suggests that drugs that inhibit TLR9 signaling may synergize with IBR. References Haselager, M. V., et al (2020). Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL. Blood, 136(25), 2918 Kennedy, E., et al (2021). TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target. Blood, 137(22), 3064 Figure 1 Figure 1. Disclosures Levin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Westerweel: Novartis: Research Funding; Incyte: Consultancy; BMS / Celgene: Consultancy; Pfizer: Consultancy. Niemann: CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Novo Nordisk Foundation: Research Funding; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding.

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

Blood ◽  
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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