Novel Tyrosine Kinase Inhibitors Trigger Drug Sensitivity of Primary CLL Cells by Controlling Glucosylation of Ceramides

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3905-3905
Author(s):  
Janine Schwamb ◽  
Valeska Feldhaus ◽  
Michael Baumann ◽  
Michaela Patz ◽  
Susanne Brodesser ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Treatment Options ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Published Data ◽  
The Novel ◽  
Apoptosis Resistance

Abstract Abstract 3905 Background: Apoptosis resistance of chronic lymphocytic leukemia (CLL) cells is mediated by several pro-survival stimuli. In particular, engagement of the B-cell receptor (BCR), CD40-CD40 ligand (CD40L) interaction or stimulation by interleukin-(IL)-4 were identified as major factors to regulate chemoresistance. Sphingolipids are known to be involved in several metabolic pathways involved in chemoresitance. Therefore, we focused on ceramide as pro-apoptotic molecule and its counterpart glucosylceramide, which rather contributes to proliferation and survival. Methods and Results: Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of pro-apoptotic ceramide in BCR/IL-4/CD40L-stimulated primary CLL cells compared to untreated controls (p=0.0258, p=0.0478, p=0.0114). Anti-apoptotic glucosylceramide levels were significantly increased after BCR cross-linking (p=0.0435) while other stimuli caused no relevant change in glucosylceramide expression. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via the enzyme UDP-glucose ceramide glucosyltransferase (UGCG) (p=0.0001). Besides specific UGCG inhibitors, we could show for the first time that IgM-mediated UGCG expression was significantly inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which were able to revert IgM-induced apoptosis resistance of CLL cells. Recently published data revealed sphingolipids to be essential for mediation of apoptosis via mitochondria. Therefore, we chose ABT-737 – a well-known and also mitochondria-targeting drug – as candidate partner for PI3Kδ and BTK inhibition. When combining each tyrosine kinase inhibitor with ABT-737, a synergistic apoptotic effect could be documented, even under protection by BCR stimulation. Conclusion: In summary, we could demonstrate that sphingolipids are critically involved in CLL pathogenesis. UGCG could be identified as drugable target by the novel kinase inhibitors CAL-101 and PCI-32765 resulting in even synergistic apoptosis following additional application of ABT-737. Sphingolipids seem to offer further targets providing novel treatment options in CLL. C.M.W. and L.P.F. contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

scholarly journals B-cell receptor triggers drug sensitivity of primary CLL cells by controlling glucosylation of ceramides

Blood ◽  
2012 ◽  
Vol 120 (19) ◽  
pp. 3978-3985 ◽  
Author(s):  
Janine Schwamb ◽  
Valeska Feldhaus ◽  
Michael Baumann ◽  
Michaela Patz ◽  
Susanne Brodesser ◽  
...  
Keyword(s):  
B Cell ◽  
Kinase Inhibitors ◽  
Treatment Options ◽  
Cell Receptor ◽  
Cd40 Ligand ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Interleukin 4 ◽  
Sphingolipid Metabolism ◽  
Apoptosis Resistance

Abstract Survival of chronic lymphocytic leukemia (CLL) cells is triggered by several stimuli, such as the B-cell receptor (BCR), CD40 ligand (CD40L), or interleukin-4 (IL-4). We identified that these stimuli regulate apoptosis resistance by modulating sphingolipid metabolism. Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of proapoptotic ceramide in BCR/IL-4/CD40L–stimulated primary CLL cells compared with untreated controls. Antiapoptotic glucosylceramide levels were significantly increased after BCR cross-linking. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via UDP-glucose ceramide glucosyltransferase (UGCG). Besides specific UGCG inhibitors, our data demonstrate that IgM-mediated UGCG expression was inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which reverted IgM-induced resistance toward apoptosis of CLL cells. Sphingolipids were recently shown to be crucial for mediation of apoptosis via mitochondria. Our data reveal ABT-737, a mitochondria-targeting drug, as interesting candidate partner for PI3Kδ and BTK inhibition, resulting in synergistic apoptosis, even under protection by the BCR. In summary, we identified the mode of action of novel kinase inhibitors CAL-101 and PCI-32765 by controlling the UGCG-mediated ceramide/glucosylceramide equilibrium as a downstream molecular switch of BCR signaling, also providing novel targeted treatment options beyond current chemotherapy-based regimens.

scholarly journals Acalabrutinib in the treatment of chronic lymphocytic leukemia: a review of recent evidence

2021 ◽  
Vol 23 (2) ◽  
pp. 332-338
Author(s):  
Andrei A. Petrenko ◽  
Maria I. Kislova ◽  
Elena A. Dmitrieva ◽  
Eugene A. Nikitin
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
Real Life ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Phase Iii ◽  
Multicenter Studies ◽  
Bruton Tyrosine Kinase

Chronic lymphocytic leukemia (CLL) treatment landscape has changed dramatically with the recently developed drugs targeting the B-cell receptor (BCR) signalling pathway. Acalabrutinib, a second generation Bruton tyrosine kinase inhibitor, was approved in 2020 in Russia for the treatment of patients with CLL. Acalabrutinib was developed as a more selective Bruton tyrosine kinase inhibitor then ibrutinib. This drug is aimed at reducing the adverse events that limit the use of ibrutinib, such as atrial fibrillation and bleeding. Phase I/II multicenter studies have demonstrated the efficacy and safety of acalabrutinib monotherapy in untreated CLL patients and in patients with relapsed/refractory CLL and ibrutinib intolerance. Phase III trials, ASCEND and ELEVATE-TN, compared acalabrutinib monotherapy and a combination of acalabrutinib and obinutuzumab versus standard therapies and demonstrated improved efficacy and tolerability of acalabrutinib. A phase III trial comparing acalabrutinib and ibrutinib monotherapy (ELEVATE-RR) is ongoing. The results of this study along with real-life clinical data could determine the place of acalabrutinib in CLL treatment.

scholarly journals Correlations between Mutations in Cancer-Related Genes, Therapy Responses and Outcomes of the 3 rd Generation Tyrosine Kinase-Inhibitor (TKI) in Persons with Chronic Myeloid Leukemia Failing Prior TKI-Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 308-308
Author(s):  
Xiaoshuai Zhang ◽  
Zongru Li ◽  
Yazhen Qin ◽  
Robert Peter Gale ◽  
Xiaojun Huang ◽  
...  
Keyword(s):  
High Risk ◽  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Functional Enrichment ◽  
Molecular Response

Abstract Background Most, but not everyone with chronic myeloid leukaemia (CML) responds to imatinib or 2 nd-generation tyrosine kinase-inhibitors (TKIs). Mutations in cancer-related genes and in other than ABL1 may explain variable responses and outcomes to the 3 rd-generation TKIs including ponatinib and olverembatinib. Aim Interrogate correlations between mutations in cancer-related genes and therapy responses and outcomes to 3 rd-generation TKIs. Methods We used deep targeted sequencing for cancer-related mutations and Sanger sequencing for BCR::ABL1 on DNA samples from 167 subjects with CML failing to the prior imatinib and/or 2 nd-generation TKI-therapy and just before receiving a 3 rd-generation TKI. Gene ontology (GO) analysis was used to evaluate functional enrichment in GO terms among mutated genes. Optimal cut-offs for variant allele frequencies (VAFs) of the common mutations were determined by analyzing receiver-operator characteristic (ROC) curves. A Cox multi-variable regression model was used to identify correlations between mutations in cancer-related genes and therapy responses and outcomes of 3 rd-generation TKI-therapy. Results 167 subjects in chronic phase (n = 125) and accelerated phase (n = 42) received ponatinib (n = 28) or olverembatinib (n = 139) therapy. 27 subjects were exposed to imatinib; 79, a 2 nd-generation TKI; 61, imatinib and a 2 nd generation TKI. 142 (85%) subjects had ABL1 mutations including ABL1T315I (n = 116) or others (n = 26). 163 subjects had other cancer-related mutations which were evaluated in epigenetic regulators (n = 150), transcription factors (n = 84), cell signaling (n = 42), tumor suppressors (n = 39), protein kinases (n = 27), chromatin modification (n = 9) and DNA damage repair (n = 3) related-genes according to functional enrichment. The top 10 mutations were ASXL1 (n = 115), RUNX1 (n = 12), KMT2D (n = 12), PHF6 (n = 8), KMT2C (n = 8), IKZF1 (n = 8), STAT5A (n = 8), DNMT3A (n = 7), TET2 (n = 6) and BCOR (n = 6). 20 subjects had high-risk additional chromosomal abnormalities (ACAs). Frequency of BCR::ABL1 mutations was inversely- (p < 0.001) and of cancer-related mutations directly-related (p = 0.009) to increasing exposure to prior TKI therapies. These relationships were especially so for mutations in KMT2C (p = 0.06), DNMT3A (p = 0.09), KDM6A (p = 0.06) and TNFAIP3 (p = 0.08). BCR::ABL1 (82% vs. 95%, p = 0.03), RUNX1 (5% vs. 14%, p = 0.04), KMT2C (3% vs. 10%, p = 0.08) and IKZF1 (3% vs. 10%, p = 0.10) were more common in accelerated phase. With a median follow-up of 34 months (interquartile range [IQR], 12-40 months), 95 and 71 subjects achieved a complete cytogenetic response (CCyR) and major molecular response (MMR). 18 subjects transformed to accelerated (n = 8) or blast (n = 10) phases, 16 died of disease progression (n = 12) or other causes (n = 4). 3-year cumulative incidences of CCyR and MMRwere 65% (95% Confidence Interval [CI], 58, 71%) and 52% (43, 61%). 3-year probabilities of progression-free survival (PFS) and survival were 88% (81, 92%) and 91% (85, 95%). Mutations in tumor suppressor genes were more common in subjects not achieving a CCyR (27% vs. 19%, p = 0.01). In multi-variable analyses ASXL1 mutation with a VAF ≥ 17% and a PHF6 mutation were significantly associated with lower cumulative incidences of CCyR (p < 0.001 and p = 0.032) and MMR (p < 0.001 and p = 0.04). Moreover, subjects with BCR-ABL1T315I mutation had significantly higher cumulative incidences of CCyR (p = 0.07) and MMR (p = 0.04) than those with no BCR-ABL1 mutation and other BCR-ABL1 non-T315I mutation. Increasing age, more Ph 1-chromosome-positive cells, the best prior therapy-response < partial cytogenetic response (PCyR) and more TKI-therapies were associated with poor responses. STAT5A mutation was significantly associated with worse PFS (p = 0.002) and survival (p < 0.001), RUNX1 mutation (p = 0.006), high-risk ACAs (p = 0.07) and accelerated phase (p = 0.002) with worse PFS and increasing age (p = 0.05) and comorbidity(ies) (p = 0.05) with wosre survival. Conclusions ASXL1 mutations with a VAF ≥ 17% and PHF6 mutations were associated with poor responses of the 3 rd-generation TKI-therapy. STAT5A and RUNX1 mutations and high-risk ACAs were also associated with worse outcomes in persons receiving a 3 rd-generation TKI. These data should help physicians select people to receive 3 rd-generation TKIs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia

Hematology ◽  
2012 ◽  
Vol 2012 (1) ◽  
pp. 88-96 ◽  
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.

EGFR mutation analysis and treatment outcomes with tyrosine kinase inhibitors in EGFR mutants: The Manchester Lung Cancer Group Experience.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e18095-e18095
Author(s):  
Rahul Peck ◽  
Elizabeth Connolly ◽  
Paul Taylor ◽  
Corinne Faivre-Finn ◽  
Fiona Hellen Blackhall ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Egfr Mutation ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Response To Treatment ◽  
Smoking History ◽  
Egfr Mutations ◽  
Published Data ◽  
Duration Of Treatment

e18095 Background: The presence of Epidermal Growth Factor Receptor (EGFR) activating mutations in patients with NSCLC was first described in 2005. Tumours exhibiting these mutations show sensitivity to treatment with an oral Tyrosine Kinase Inhibitor. Testing for EGFR mutations in patients with non-squamous NSCLC began in the Greater Manchester and Cheshire network in the last quarter of 2009. Methods: We audited the notes of consecutive patients who were identified with an activating EGFR mutation by the Central Manchester Genetics Laboratory between November 2009 and October 2011. Results: A total of 110 mutations were identified in tumour tissue from 98 patients. 13.6% were in exon 18, 38.2% in exon 19, 15.4% in exon 20 and 32.8% in exon 21. 65% of patients were female. The median age was 69 years (36-89). Notes were available for 85 patients, 59 of whom received treatment with an EGFR TKi. 7 had previously received radical treatment and 19 never received treatment. 7% were current smokers, 40% were ex-smokers, 30.6% had never smoked and smoking history was not documented in 22.4%. An initial response to treatment was seen in 55%, with stable disease in 15%.The mean duration of treatment was 7.6 months (2 weeks – 23 months), with 24 patients still receiving a TKi at the time of data analysis. The most commonly seen toxicities were diarrhoea and rash. Only 1 patient had no documented toxicity from their TKi. 17 patients (29%) had treatment discontinued or interrupted because of toxicity. In 8 of these, treatment was re-introduced at a reduced dose, and 3 patients went back to full dose. Conclusions: Our results confirm that treatment with a TKi is effective for those patients whose tumours harbour EGFR mutations, with a side effect profile consistent with published data.

scholarly journals BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor–induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3

Blood ◽  
2009 ◽  
Vol 113 (10) ◽  
pp. 2302-2311 ◽  
Author(s):  
Amanda Nordigården ◽  
Maria Kraft ◽  
Pernilla Eliasson ◽  
Verena Labi ◽  
Eric W.-F. Lam ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Bone Marrow Cells ◽  
Treatment Options ◽  
Leukemic Cell ◽  
Therapeutic Interventions ◽  
Leukemic Cells ◽  
Leukemic Cell Lines ◽  
Induced Apoptosis

Abstract Constitutively activating internal tandem duplications (ITD) of FLT3 (FMS-like tyrosine kinase 3) are the most common mutations in acute myeloid leukemia (AML) and correlate with poor prognosis. Receptor tyrosine kinase inhibitors targeting FLT3 have developed as attractive treatment options. Because relapses occur after initial responses, identification of FLT3-ITD–mediated signaling events are important to facilitate novel therapeutic interventions. Here, we have determined the growth-inhibitory and proapototic mechanisms of 2 small molecule inhibitors of FLT3, AG1295 or PKC412, in hematopoietic progenitor cells, human leukemic cell lines, and primary AML cells expressing FLT3-ITD. Inactivation of the PI3-kinase pathway, but not of Ras–mitogen-activated protein (MAP) kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced up-regulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from gene-targeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor, where loss of Puma only provided transient protection from apoptosis, but loss of Bim preserved clonal survival upon FLT3-ITD inhibition.

scholarly journals Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia

Blood ◽  
2012 ◽  
Vol 120 (24) ◽  
pp. 4684-4691 ◽  
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.

scholarly journals Cardiovascular Toxicities of Tyrosine Kinase Inhibitor in CML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-6
Author(s):  
Samip R Master ◽  
Richard Preston Mansour
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Line Treatment ◽  
Front Line ◽  
First Line ◽  
First Line Treatment

Background: Cardiovascular (CV) toxicity is a known toxicity of tyrosine kinase inhibitors (TKI) used for chronic myeloid leukemia (CML). Imatinib, dasatinib, nilotinib and bosutinib are all approved for first line treatment for CML. We did a retrospective analysis on adverse effects (AE) of TKIs that has been made available to public by the FDA. Methods: The FDA has made the data on AEs of various treatments available to general public through the FDA Adverse Events Reports System (FAERS) public dashboard. We investigated the CV AEs of various TKIs for the years 2017-2019. Results: The percentage of CV AE compared to total AEs reported for Imatinib, Dasatinib, Bosutinib , Nilotinib and Ponatinib were 7.2%, 10.5%, 15.8%, 23.4% and 23.5% respectively. The percentage of CV AE leading to death for Imatinib, Dasatinib, Bosutinib , Nilotinib and Ponatinib were 8.3%, 9.1%, 9.1%, 13.7 % and 18.6% respectively. Conclusions: Out of the reported cases of AEs to TKIs approved for front line CML, nilotinib appears to have more CV AE compared to imatinib, dasatinib and bolutinib. Imatinib appears to have least CV AE out of the total AEs reported Disclosures No relevant conflicts of interest to declare.

scholarly journals Bruton tyrosine kinase inhibitors for the frontline treatment of chronic lymphocytic leukemia

2020 ◽  
Vol 27 (6) ◽  
Author(s):  
V. Banerji ◽  
A. Aw ◽  
S. Robinson ◽  
S. Doucette ◽  
A. Christofides ◽  
...  
Keyword(s):  
High Risk ◽  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Variable Region ◽  
Tp53 Gene ◽  
Cell Receptor ◽  
Response To Therapy ◽  
Lymphocytic Leukemia ◽  
Btk Inhibitors

Chronic lymphocytic leukemia (CLL) is the most commonly diagnosed adult leukemia in Canada. Biologic heterogeneity of CLL can be observed between patients which results in variable disease trajectory and response to therapy. Notably, patients with high-risk features such as the presence of deletions in chromosome 17p, aberrations in the TP53 gene, or unmutated immunoglobulin heavy chain variable region genes have inferior outcomes and response to standard chemoimmunotherapy compared to patients without these features. Novel agents which target the B cell receptor signalling pathway, such as Bruton’s tyrosine kinase (BTK) inhibitors have demonstrated clinical efficacy and safety in patients with treatment-naïve CLL, particularly in those with high-risk features. However, due to the current lack of head-to-head trials comparing BTK inhibitors, selection of the optimal BTK inhibitor for patients with CLL is unclear and requires the consideration of multiple factors. This review focuses on the efficacy, safety, and pharmacological features of the BTK inhibitors that are approved or are under clinical development and discusses the practical considerations for the use of these agents in the Canadian treatment landscape.

Recruitment of PKC-Beta to Lipid Rafts Mediates Apoptosis-Resistance in Chronic Lymphocytic Leukemia Expressing Zap-70.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2354-2354
Author(s):  
Ingo Ringshausen ◽  
Michaela Wagner ◽  
Gloria Lutzny ◽  
Madlen Oelsner ◽  
Yvonne Feuerstacke ◽  
...  
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Apoptosis Resistance ◽  
Constitutive Activation ◽  
Clinical Phase ◽  
Highly Active

Abstract Abstract 2354 Poster Board II-331 A defect in the programmed cell death, apoptosis, is implemented in the pathogenesis of CLL. About ten years ago, it became evident that patients with CLL can be divided into those with an indolent course of the leukaemia and those which suffer from a more aggressive disease, typically requiring frequent chemotherapy and ultimately develop a chemotherapy-refractory state. The latter group of patients aberrantly express the T-cell associated protein ZAP-70. The object of this study was to identify the molecular differences underlying the pathogenesis of these two CLL subgroups. To study differences in the apoptotic program we used primary CLL cells derived from untreated ZAP-70 negative and positive patients. Here we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor (BCR) and recruits protein kinase C-beta (PKC-beta) into lipid raft domains only in patients with an aggressive variant of the disease. Subsequently, PKC-beta is activated and shuttles from the plasma membrane into the mitochondria. By using co-immunoprecipitation experiments and PKC-beta specific small molecule inhibitors we unravel that the anti-apoptotic protein Bcl-2 and its antagonistic BH3-protein Bim are putative substrates for PKC-beta. PKC-beta mediated phosphorylation of Bcl-2 augments its anti-apoptotic function by increasing its ability to sequester more pro-apoptotic Bim. In addition, the phosphorylation of Bim by PKC-beta leads to its proteasomal degradation. Therefore, high levels of phospho-Bcl-2 and low levels of Bim are a hallmark of ZAP-70 positive, aggressive CLL. Importantly, posttranscriptional modifications of Bcl-2 seem to outweigh the absolute expression of Bcl-2 with respect to the suppression of apoptosis. We demonstrate that these cells are strongly protected from chemotherapy-induced cytotoxic stress. Our data indicate that the constitutive activation of PKC-beta is directly involved in the apoptotic defect in ZAP-70 positive CLL. We finally show that targeting PKC-beta is an attractive approach to the treatment of CLL patients. Enzastaurin is a PKC-beta specific inhibitor and currently tested in clinical phase I/II trials for cancer patients. Our data demonstrate that this compound is highly active in CLL cells and augments the cytotoxic effects of standard chemotherapeutic drugs. Our results provide evidence that the constitutive activation of PKC-beta is directly implicated in the pathogenesis of aggressive CLL by altering the function of the apoptosis-regulating proteins Bcl-2 and Bim. These changes confer cells to a more anti-apoptotic state with aggressiveness of the disease. Targeting PKC-beta with small-molecule inhibitors like Enzastaurin might offer a new therapeutic strategy to control or even cure CLL. Disclosures: No relevant conflicts of interest to declare.

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