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

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<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>10µM), but unexectedly retained partial sensitivity to venetoclax after IBR treatment (IC50=0.05 µM, p-value <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 <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.

Download Full-text

Development of a Novel Lymph Node-Based 3D Culture System Promoting Chronic Lymphocytic Leukemia Proliferation and Survival

Blood ◽

10.1182/blood-2020-141962 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 47-48

Author(s):

Marco Haselager ◽

Eduard Perelaer ◽

Arnon P. Kater ◽

Eric Eldering

Keyword(s):

Lymph Node ◽

Chronic Lymphocytic Leukemia ◽

In Vitro Culture ◽

Culture System ◽

Research Funding ◽

Lymphocytic Leukemia ◽

2D System

INTRODUCTION. Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in absence of microenvironmental survival signals1. Although co-culture with stromal cells or the addition of soluble factors can increase and extend CLL survival, no system permits the long-term expansion of CLL cells in vitro2. The difficulties of mimicking a physiologic microenvironment supporting CLL cells hinder in vitro studies of proliferation, drug screens and prevent propagation of rare subclones. For other cancers, various types of 3D cultures have been introduced utilizing scaffolds, gels, spheroid cultures and fluidic systems, representing a more accurate representation of the in vivo microenvironment3. Unlike solid tumors, secondary lymphoid tissues where CLL cells proliferate in vivo, do not derive from a single stem cell progenitor. Developing an appropriate 3D in vitro culture system for CLL is of obvious importance and may contribute pathophysiological relevance to study long-term CLL proliferation and more accurate drug screening4,5. Within the field of CLL, attempts have focused on bone marrow stroma, but it may be biologically and clinically more relevant to investigate the lymph node niche as this is the critical site of CLL proliferation6. METHODS. Primary CLL cells were cultured in various 3D systems including hydrogels, hanging drop cultures and ultra-low attachment plates (ULA) plates in parallel to an optimal 2D system, consisting of the culture of primary CLL cells on a monolayer of CD40L-presenting fibroblasts (3T40) or 3T3 negative control fibroblasts. CLL cells were either cultured as PBMCs alone, with or without T cells, or co-cultured with 3T40 or primary lymph node fibroblasts. CLL cells were either stimulated directly with IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28. RESULTS. After testing and comparing multiple systems for the in vitro culture of CLL cells, we optimized a novel CLL culture system utilizing ULA plates creating spheroids of PBMCs isolated from peripheral blood. Without the addition of soluble factors or stroma, primary CLL cells in the ULA 3D model could be maintained in culture for 6 weeks as opposed to 1 week in the 2D system. Aside from significantly promoting CLL survival, cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the ULA 3D model. 3D cultures showed a more consistent and significantly increased CLL proliferation compared to 2D cultures, independent of IGHV mutation status, increasing the average proliferation index of 2.87 to 3.90 (n=10). Additionally, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations (n=6) (Figure 1). Lastly, when PBMCs were stimulated with IL-2, IL-15, IL-21 and CpG, spheroids developed proliferation center-like structures after 4 weeks of culture. CONCLUSIONS. We established a lymph node-based 3D in vitro culture system for CLL leading to increased CLL proliferation and survival compared to 2D systems. The set-up allows long-term expansion of CLL cells in vitro, as well as formation of proliferation center-like structures. We are currently optimizing drug resistance studies, expansion of specific CLL subclones and performing competition experiments. References: 1. Hamilton et al., Mimicking the tumour microenvironment: three different co-culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells, 2012. 2. Asslaber et al., Mimicking the microenvironment in chronic lymphocytic leukaemia - where does the journey go?, 2013. 3. Gurski et al., 3D Matrices for Anti-Cancer Drug Testing and Development, 2010. 4. Nunes et al., 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs, 2019. 5. Aljitwai et al., A novel three-dimensional stromal-based model for in vitro chemotherapy sensitivity testing of leukemia cells, 2014. 6. Van Gent et al., In vivo dynamics of stable chronic lymphocytic leukemia inversely correlates with somatic hypermutation levels and suggest no major leukemic turnover in bone marrow, 2008. Disclosures Kater: Genentech: Research Funding; Abbvie: Research Funding; Roche: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Eldering:Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding.

Download Full-text

Mapping the Targets of Dasatinib in Chronic Lymphocytic Leukemia Reveals Distinct Roles for Abl and Btk in Drug Resistance and Adhesion, and Explains Clinical Effects On Lymph Node Reduction

Blood ◽

10.1182/blood.v120.21.3900.3900 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3900-3900

Author(s):

Eric Eldering ◽

Christian R Geest ◽

Martin FM de Rooij ◽

Nora Liu ◽

Bogdan I Florea ◽

...

Keyword(s):

Drug Resistance ◽

Lymph Node ◽

Chronic Lymphocytic Leukemia ◽

Broad Spectrum ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Clinical Effects ◽

Open Label

Abstract Abstract 3900 In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche. This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Blood ◽

10.1182/blood-2021-149442 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 248-248

Author(s):

Alice Bonato ◽

Riccardo Bomben ◽

Supriya Chakraborty ◽

Giulia Felician ◽

Claudio Martines ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Pi3k Inhibitor ◽

Leukemia Cells ◽

Wild Type ◽

Clonal Composition

Abstract Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021). To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P<0.05 for each condition). To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P<0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P<0.001, with 1.0 μM IBR: 28% vs 53%, P<0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001). To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with >10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting. Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. Figure 1 Figure 1. Disclosures Marasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

Download Full-text

Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia

Hematology ◽

10.1182/asheducation.v2012.1.88.3801172 ◽

2012 ◽

Vol 2012 (1) ◽

pp. 88-96 ◽

Cited By ~ 26

Author(s):

Adrian Wiestner

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Absolute Lymphocyte Count ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Cell Trafficking

Abstract Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that depend on host factors in the tissue microenvironment for survival and proliferation. In vitro, CLL cells rapidly undergo apoptosis unless microenvironmental factors are provided that support their survival. Signaling pathways activated in the microenvironment in vivo include the B-cell receptor (BCR) and NF-κB pathways. Thus, CLL is a disease “addicted to the host” and is dependent on pathways that promote normal B-cell development, expansion, and survival; this is particularly true in the case of the BCR signaling cascade. Small-molecule inhibitors of kinases that are essential for BCR signal transduction abrogate the stimulating effects of the microenvironment on CLL cells. The orally administered tyrosine kinase inhibitors fostamatinib and ibrutinib and the phosphatidylinositol 3-kinase inhibitor GS-1101 have induced impressive responses in relapsed and refractory CLL patients, mostly with moderate side effects. Reductions in lymphadenopathy and splenomegaly are seen within weeks and are frequently accompanied by a transient rise in absolute lymphocyte count that is asymptomatic and probably the result of changes in CLL cell trafficking. This review discusses the biologic basis for kinase inhibitors as targeted therapy of CLL and summarizes the exciting early clinical experience with these agents.

Download Full-text

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.

Download Full-text

CLL Cells from Ibrutinib-Induced Lymphocytosis of Relapsed/Refractory Chronic Lymphocytic Leukemia Patients Are Responsive to Obinutuzumab, but Not Rituximab, Ex Vivo

Blood ◽

10.1182/blood.v126.23.4157.4157 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4157-4157 ◽

Cited By ~ 2

Author(s):

Loïc Ysebaert ◽

Christian Klein ◽

Anne Quillet-Mary

Keyword(s):

Lymph Node ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Peripheral Blood ◽

Absolute Number ◽

Lymphocytic Leukemia ◽

Viable Cell Number ◽

Cluster 2

Abstract Introduction: Ibrutinib is an irreversible first-in-class inhibitor of BTK (Bruton tyrosine kinase) approved for the therapy of relapsed/refractory chronic lymphocytic leukemia (R/R CLL). The drug mediates a transient increase in circulating CLL cells together with reduction in spleen and lymph node size, by both cellular mobilization and apoptosis of resident CLL cells (Herman SE, et al. Blood 2014;123:3286-95). These events occur with important patients' inter-variability (Herman SE, et al. Leukemia 2014;28:2188-96), one cluster of patients presents with greater peak lymphocytosis (resolving between 1 to more than 6 months), while another cluster presents with rapid resolution of lymphocytosis and lymph node/spleen size within 2 months. Upon such dramatic shifts in disease distribution the first 2 months of therapy (and sometimes lasting >6-12 months), the question of phenotypic changes, sensitivity to monoclonal antibodies (MoAbs), and subclonal diversity of circulating cells remains central for further combination studies. In this study, we evaluated changes in CD5, CD19, and CD20 expression in vitro/in vivo, and peripheral blood side population (SP) cells (a fraction highly enriched in chemorefractory cells, Gross E, et al. Leukemia 2010;24:1885-92) upon ibrutinib therapy. We also investigated whether patterns of lymphocytosis may predict for response to rituximab (RTX) or obinutuzumab (GA101). Methods: R/R CLL patients (n=25) median prior lines=4, range=2-8), PBMCs were collected before ibrutinib initiation and after 1 and 2 months of therapy. PBMC were seeded at 10 x 106 cells/mL in culture medium and treated for 7 days with 10µg/mL control IgG1 (trastuzumab), RTX or obinutuzumab. The specific percentage of remaining B cells in MoAbs-treated samples was calculated as (absolute number in treated samples/absolute number in control samples) x 100. For each condition, absolute number of remaining B cells =total viable cell number (trypan blue exclusion determination) x % of viable CD19+/CD5+ lymphocytes (flow cytometry determination). For statistical analyses, Student's test (paired, two-sided) was used (*p<0.05;**p<0.01;***p<0.001). Results: We firstanalyzed patterns ofabsolute lymphocytes count ( ALC) across 23 patients receiving ibrutinib (Fig 1a) to classify them into two clusters as previously published (Fig 1b): Cluster 1 and cluster 2 did not differ significantly in terms of initial lymphocytosis, line of therapy, gender, karyotype, IgHV. Interestingly, the SP fraction in peripheral blood was significantly increased (median: 5/microL before ibrutinib, 10/microL at peak lymphocytosis), suggesting mobilization of resident SP cells, although no apoptosis was detected (in vitro or in vivo) with ibrutinib. We next assessed CD5, CD19 and CD20 levels in vitro (n=22) and in vivo (n=15) upon ibrutinib therapy. In vitro, ibrutinib significantly reduced CD20 (Fig 2a) and CD19 surface expression, but not CD5; nevertheless anti-CD20 MoAbs still had activity in vitro (Fig 2b). Expression levels were not linked to clusters 1 or 2. Finally we compared RTX- and obinutuzumab-induced B-cell depletion before administration of ibrutinib, and at various sampling time points (1 to 6 months). Obinutuzumab induced significantly superior depletion at various timepoints than RTX. More interestingly, when analysis was performed from paired samples before/during ibrutinib therapy from the same ibrutinib-exposed patients, only obinutuzumab-induced depletion was increased in cluster 2 (Fig 3). Conclusions: Ongoing and planned clinical studies evaluate the combination of ibrutinib and obinutuzumab in CLL (first-line and relapsed). Some concerns have emerged due to published preclinical data showing that ibrutinib can interfere with efficacy of therapeutic antibodies. Here, we suggest that ibrutinib-exposed CLL cells, despite wide inter-patient heterogeneity, are targetable with obinutuzumab. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Klein: Roche: Employment.

Download Full-text

Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2012-05-423194 ◽

2012 ◽

Vol 120 (24) ◽

pp. 4684-4691 ◽

Cited By ~ 105

Author(s):

Adrian Wiestner

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Absolute Lymphocyte Count ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Cell Trafficking

AbstractChronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that depend on host factors in the tissue microenvironment for survival and proliferation. In vitro, CLL cells rapidly undergo apoptosis unless microenvironmental factors are provided that support their survival. Signaling pathways activated in the microenvironment in vivo include the B-cell receptor (BCR) and NF-κB pathways. Thus, CLL is a disease “addicted to the host” and is dependent on pathways that promote normal B-cell development, expansion, and survival; this is particularly true in the case of the BCR signaling cascade. Small-molecule inhibitors of kinases that are essential for BCR signal transduction abrogate the stimulating effects of the microenvironment on CLL cells. The orally administered tyrosine kinase inhibitors fostamatinib and ibrutinib and the phosphatidylinositol 3-kinase inhibitor GS-1101 have induced impressive responses in relapsed and refractory CLL patients, mostly with moderate side effects. Reductions in lymphadenopathy and splenomegaly are seen within weeks and are frequently accompanied by a transient rise in absolute lymphocyte count that is asymptomatic and probably the result of changes in CLL cell trafficking. This review discusses the biologic basis for kinase inhibitors as targeted therapy of CLL and summarizes the exciting early clinical experience with these agents.

Download Full-text

Nonconventional Gene Expression within the NF-κb Signaling Pathway Induced By Poly(propylene)Imine Glycodendrimers in Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v128.22.5595.5595 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5595-5595

Author(s):

Ida Franiak-Pietryga ◽

Kinga Ostrowska ◽

Dietmar Appelhans ◽

Henryk Maciejewski ◽

Maria Bryszewska ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Signaling Pathway ◽

Research Funding ◽

Target Genes ◽

Early Stage ◽

Lymphocytic Leukemia ◽

P Value

Abstract Introduction The nuclear factor kappa-light-chain enhancer of activated B-cells (NF-κB) signaling pathway is constitutively active in a variety of cancers, including chronic lymphocytic leukemia (CLL). The importance of this signaling pathway identifies it as a prime therapeutic target, however the complexity and potential side effects of inhibiting NF-κB have thus far made the clinical use of NF-κB inhibitors a relatively unexplored resource in this disease. There are a few combined therapies available for the treatment of CLL includes chemotherapy with agents such as chlorambucil, cyclophosphamide, fludarabine and bendamustine, along with immunotherapy including rituximab and alemtuzumab. None available therapy for CLL is curative. Nanotechnology, a new and promising field of scientific research, may be of use in medicine and the pharmaceutical industry. Dendrimers, nanoparticles of dendritic architecture, can interact effectively and specifically with cell components. We have already proved the influence of PPI-G4-OS-M3 dendrimers in cultures in vitro on CLL cells apoptosis.Herein, the objective was to evaluate how MEC-1 cells survival in vitro is affected by influence on NF-κB pathway by PPI-G4-OS-M3 dendrimer comparing to FA. Material and methods Dendrimer, in which approximately 35% of peripheral amino groups, was coated with maltotriose have been defined as PPI-G4-OS-M3 and was used in concentration of 8 mg/ml (the IC50 value for this dendrimer). 'OS' abbreviation stands for the open shell structure of carbohydrate-modified dendrimers. The molar mass of this PPI dendrimer was 31000 g/mol. Fludarabine (FA, Genzyme) in concentration of 1.6 µM, based on previous studies, was used. MEC-1 (DSMZ no. ACC 497) was used as a homogenous cell line with del(17p)(11q). In cultures the percentage of apoptotic cells was verified using AnnV and PI by means of flow cytometer. Cells predominated in the early stage of apoptosis.A microarray gene expression (Agilent SurePrint Technologies) was performed. Samples were hybridized to a whole human genome microarray 8x60K. Arrays were scanned on Agilent DNA Microarray Scanner. Data were deposited at Gene Expression Omnibus (GEO) (accession number GSE68094).Analysis of differential expression of genes was done with the limma method (Smyth, G. K., 2004) as implemented in R/Bioconductor software. We used the FDR multiple testing adjustment. We declared as differentially expressed the genes with FDR-adjusted p-value <0.1, which means that 10% of genes declared as DE are expected to be false positives. Results Dendrimer induced expression of REL, RELB and NFKBIB genes. In contrast, FA monotherapy resulted in significant differences in gene expression of cellular pathway-dependent transcription factor NF-kB. The most significant differences in the function of the FA and dendrimer are reflected in different levels of expression of three genes: NFKBIA, BCL3 and CHUK. Conclusion Constitutive NF-κB signaling contributes to cell growth, proliferation and survival. CLL cells have high basal levels of NF-κB compared with normal B cells. The activity is variable in CLL patients, correlates with in vitro cell survival, and importantly, increased levels of NF-κB activity enhanced resistance to the purine analogues FA (del17p). Therefore, disruption of NF-κB signaling and downstream target genes either promoted or repressed is an important strategy to pursue to disrupt drug resistance in CLL. The study indicates that the use of PPI dendrimers modified maltotriose may be the key to developing therapies deliberates CLL. The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371from the National Science Centre, Poland. Disclosures Robak: Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding.

Download Full-text

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.

Download Full-text

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)

Download Full-text