Kooperativer Bibliotheksverbund

In: Blood, American Society of Hematology, Vol. 138, No. 7 ( 2021-08-19), p. 544-556

Abstract: Bruton tyrosine kinase (BTK) inhibitors are highly active drugs for the treatment of chronic lymphocytic leukemia (CLL). To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines (pS), threonines (pT), and tyrosines (pY) (pS:pT:pY). Expression of 83 proteins differed between unmutated immunoglobulin heavy-chain variable region (IGHV) CLL (UM-CLL) and mutated IGHV CLL (M-CLL). Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition, and survival. One protein, myristoylated alanine-rich C-kinase substrate (MARCKS), showed striking differences in expression and phosphorylation level in UM-CLL vs M-CLL. MARCKS sequesters phosphatidylinositol-4,5-bisphosphate, thereby affecting central signaling pathways and clustering of the B-cell receptor (BCR). Genetically induced loss of MARCKS significantly increased AKT signaling and migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in patients with CLL treated with acalabrutinib.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.2020009165

Language: English

Publisher: American Society of Hematology

Publication Date: 2021

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 1947-1947

Abstract: Background: CLL patients frequently suffer relapse after an initially successful chemotherapy. This distinct resistance towards chemotherapy is thought to be caused by microenvironmental stimulation. Within the tumor microenvironment (TME) cells are not only stimulated by well-known external stimuli like CD40 ligand (CD40L) or activation of the B cell receptor (BCR), but are also exposed to hypoxia, which was found in the bone marrow and lymphatic tissue. Despite the known importance of hypoxia in solid tumors, its impact on survival and treatment response in CLL is still poorly understood. Methods: We have established a novel in vitro model for the CLL microenvironment, which considers both the external stimulation by CD40L and the hypoxic oxygen levels (1% O2). Treatment efficacy of different drugs in normoxia (21% O2) and hypoxia were determined by AnnexinV/7-AAD staining and subsequent FACS analysis. The underlying molecular mechanisms were analyzed via qRT-PCR and immunoblot. Furthermore B-cell lines Raji, Ramos and Mec-1 were continuously exposed to increasing concentrations of fludarabine or the BH3 mimetic ABT-737. After establishment of resistance the molecular adaptation was assessed and correlated to the changes induced by hypoxia. Results: Hypoxia is known to protect solid cancers from chemotherapy. In our model we made similar observations for CLL, since sensitivity to the classical DNA-targeting drugs fludarabine and bendamustine was reduced under hypoxic conditions. Interestingly, the tyrosine kinase inhibitor ibrutinib did not benefit from hypoxia either. However, this resistance was overcome by the mitochondria-targeting BH3 mimetics ABT-199 and ABT-737, whose effect was pronounced under hypoxia. We reveal that this effect was caused by an uncoupling of major signaling pathways. Under hypoxic conditions the activity of Akt, ERK1/2 and NFκB was reduced, while p38 MAPK became hyperphosphorylated. Phospho-p38 (pp38) downregulated Mcl-1 levels, which are the main regulator of sensitivity towards BH3 mimetics. Despite the known heterogeneity in between CLL patients this effect was found in most samples analyzed. The functional importance was underlined by the observation that pharmacological inhibition of p38 MAPK could reconstitute Mcl-1 levels and thereby resistance in hypoxia. The relevance of the pp38-Mcl-1 axis for ABT efficacy was emphasized by findings in B-cell lines with acquired resistance. Each ABT-resistant clone of the three tested cell lines induced p38 activity and decreased Mcl-1 levels. In contrast, in the fludarabine-resistant clones the pp38-Mcl-1 axis was not altered. Conclusion: These are the first experiments providing evidence that hypoxia has a crucial impact on survival and response to chemotherapy in CLL. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agents, while the small molecules ABT-199 and ABT-737, which specifically target mitochondria, efficiently eradicate CLL cells within the microenvironment. Furthermore, we identified the pp38-Mcl-1 axis to be a major determinant of sensitivity to these BH3 mimetics, which warrants further evaluation of p38 as a novel biomarker for prediction of sensitivity to BH3 mimetics. Disclosures No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V124.21.1947.1947

Language: English

Publisher: American Society of Hematology

Publication Date: 2014

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 354-354

Abstract: Background: The immunoglobulin-like protein TOSO, which has been found to serve as Fc receptor for IgM (FcµR), was shown by us and others to be overexpressed on CLL cells and only weakly expressed on more aggressive B-NHL. However the functional role of TOSO on lymphomagenesis has not been explored so far. Methods: To determine the role of TOSO on lymphoma development, we took advantage of the Eµ-TCL1 transgenic mice, which usually end up with an aggressive (IgVH unmutated) CLL-like phenotype. We generated a novel B cell-specific conditional knockout (KO) mouse model in which EµTCL1 mice (TC or control in the following) were crossbred with TOSO-floxed mice, expressing Cre recombinase under the control of the CD19 promoter (EµTCL1;Tosofl/fl;Cd19cre/wtor TCT in the following). TCT mice were further compared with p53 conditional knockout (EµTCL1;Tp53fl/fl;Cd19cre/wt or TCP). Results: In this study, we compared kinetics, overall survival and phenotype of lymphoma/CLL in TC, TCT and TCP mice. Interestingly, TCTmice developed a very aggressive phenotype and resulted in significantly shorter overall survival compared to TC mice (TCT 274 days vs. TC 346 days; p 〈 0.0001). As expected, mice lacking p53 (TCP) died even more rapidly than TCT mice (median survival: TCP 233 days). Initially, all three genotypes (TC, TCT, TCP) developed a CLL phenotype, exhibiting a CD19 and CD5 positive malignant clone. In the TCT mice, shorter overall survival is accompanied by a stronger increase of blood leukocytes. Flow cytometry analysis confirmed a strong increase of leukemic CD19/CD5-positive B cells in the blood of TCT mice. With only 20 weeks of age, leukemic cells already made up 37.5 % (SD ± 15.47; n=14) of lymphocytes (TC: 14.3 % SD ± 9.81; n=31). At the age of 36 weeks, TCT mice showed even a 3.6-fold elevated malignant cell count compared to control mice (n=35 TC, n=14 TCT; p=0.006). All TCT mice developed a splenomegaly, with spleen weight (p=0.01) and size (p=0.018) significantly increased in 36 week old TCT mice (n=7) compared to TC mice (n=7) and comparable to those from TCP mice. Interestingly, between week 28 and 36, we could observe that most of the TCT mice start losing CD19+ cells in the blood in contrast to TC and TCP mice. Immunohistochemistry revealed the expansion of malignant cells with pleomorphic nuclei and abundant cytoplasm in the spleen and bone marrow, as we know it from Richter`s transformation. To understand the rapid development of leukemia in TCT mice, we first determined the role of the BCR in this model. Interestingly, flow cytometry revealed a higher surface IgM expression (MFI: TCT 9,27; TC 2,05). In addition, in vitro assays revealed a significantly higher resistance of TCT cells towards PI3K inhibition (Idelalisib and Duvelisib) compared to TC cells. To further rule out the role of TOSO under "germinal-center conditions", we stimulated primary human CLL cells with CD40L expressing feeder cells and IL-4. Interestingly, both stimuli (either alone or in combination) resulted in almost complete loss of TOSO on CLL cells. Moreover, we uncovered, that the TOSO promoter is counteractively regulated by NF-κB and BCL6. Furthermore, our data illustrate that DNA hypomethylation of the TOSO promoter is a discriminating characteristic in CLL patients compared to healthy donors, thus explaining the significantly enhanced expression levels. Thus, both, epigenetic regulation and altered NF-κB/ BCL6 expression are critical pathogenetic steps in the development of CLL and aggressive B-NHL by regulating TOSO expression. Conclusion: The transformation of CLL into more aggressive malignancies is still not fully understood. Our data reveal that the loss of TOSO might play a major role in Richter's transformation by upregulation of the BCR and by mimicking the germinal-center phenotype. Disclosures Fingerle-Rowson: Roche: Employment. Wendtner:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann-La Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hallek:GSK: Research Funding; Mundipharma: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Gilead: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V128.22.354.354

Language: English

Publisher: American Society of Hematology

Publication Date: 2016

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 583-583

Abstract: Rationale: The BTK inhibitor ibrutinib has proved to be highly effective in both treatment naїve and refractory or relapsed CLL patients. In contrast to most other therapeutic options, where unmutated IGVH correlates with adverse prognosis, response to ibrutinib is independent of IGVH mutation status. This compulsorily raises the question why CLL cells with an unmutated IGVH are equally susceptible to BTK inhibition. One key aspect might be understanding the global (phospho)proteome of UM-CLL and M-CLL cells which has not been investigated so far. Methods: We performed a comprehensive proteomics and phosphopreoteomics analysis of 14 CLL samples with unmutated or mutated IgVH using quantitative mass spectrometry (MS). ITRAQ labelling and TiO2 enrichment/HILIC fractionation were utilized for this approach. Results: Altogether we identified 9184 phosphopeptides corresponding to 2854 proteins. We found the typical pS:pT:pY ratio, with 89% of all detected phosphopeptides containing a phosphorylated serine (pS), 10% a phosphorylated threonine (pT) and 1% a phosphorylated tyrosine (pY) in CLL samples. Strikingly, UM-CLL showed a higher basal phosphorylation level than M-CLL samples with significantly higher phosphorylation of 92 out of 102 identified proteins (P 〈 0.05) Interestingly, ibrutinib reverted this phosphorylation pattern predominantly in UM-CLL but not M-CLL samples and led to a shift of the ratio towards phosphotyrosine in UM-CLL (pS:pT:pY in %: 30:9:61) but not in M-CLL (pS:pT:pY: 78:7:15). Most of the indentified phosphopeptides clustered in pathways that regulate migration and motility and cell survival and death. Regarding the BCR pathway, we identified known and novel p-sites: Lyn (8 sites), PIK3AP1 (5), PLCG2 (5), Syk (4), BLK (4), PIK3R4 (2), LCK (2), BTK (1), PIK3C2B (1), PIK3C3 (1), PIK3CD (1). In this context a novel molecule, MARCKS attracted our attention. We identified three different p-sites (S101, T150 and S170) which were differentially phosphorylated in M-CLL and UM-CLL. Moreover, our proteome approach revealed distinct expression levels of 38 proteins out of 3466 isolated proteins between the two groups (1,5-fold changes; P 〈 0.01), among them MARCKS. Expression of MARCKS was significantly higher in M-CLL samples. MARCKS, a PKC substrate, was shown to play a critical role in invasiveness and metastasis of various cancer types, but its role in CLL is unclear. Since MARCKS has not been associated with CLL, we proved our findings from MS in a larger cohort of CLL patients both on protein level (n=36) and on transcription level (n=337). Strikingly, shorter PFS of CLL patients (n=337) undergoing chemoimmunotherapy correlates with low expression of MARCKS independently of the mutational status. We further investigated the cellular function of MARCKS in CLL cells utilizing CRISPR/Cas9 to generate KO cells. We were able to show that MARCKS regulates migration towards CXCL12 and that a loss of MARCKS leads to significantly increased migration. As MARCKS is upstream of AKT at the plasma membrane, we wondered if its expression might be relevant for AKT signalling. Importantly, we found that AKT phosphorylation (S473) was significantly upregulated in MARCKS KO cells indicating that MARCKS is involved in AKT regulation. Since MARCKS seems to be involved in tumor microenvironment (TME) interaction, we determined the influence of TME stimuli on MARCKS regulation. Interestingly, MARCKS was upregulated by CD40:CD40L interaction but phosphorylated upon BCR stimulation in both M-CLL and UM-CLL as assessed by immunoblot. Furthermore, we identified MARCKS to be targeted by ibrutinib, as phosphorylation at S170 was reduced upon ibrutinib treatment. Conclusion: For the first time we could show a comprehensive picture of the phosphoproteome and proteome of UM-CLL and M-CLL samples. Strikingly, the basal phosphorylation level was significantly higher in UM-CLL and was more susceptible to ibrutinib treatment. Our findings reveal a relevant association of MARCKS expression with CLL prognosis, supported by the functional evidence that MARCKS acts upstream as novel modulator of AKT signalling and controls migration towards CXCL12. These data indicate that MARCKS is a novel and relevant target of ibrutinib especially in the context of the TME. Disclosures Bahlo: Roche: Honoraria, Other: Travel Grants. Fischer:Roche: Other: Travel support. Wendtner:Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding. Stilgenbauer:Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hallek:Pharmacyclics: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2018-99-118235

Language: English

Publisher: American Society of Hematology

Publication Date: 2018

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Genetics in Medicine, Elsevier BV, Vol. 25, No. 11 ( 2023-11), p. 100928-

Type of Medium: Online Resource

ISSN: 1098-3600

URL: Article

DOI: 10.1016/j.gim.2023.100928

Language: English

Publisher: Elsevier BV

Publication Date: 2023

detail.hit.zdb_id: 2063504-7

SSG: 12

In: The American Journal of Human Genetics, Elsevier BV, Vol. 106, No. 6 ( 2020-06), p. 872-884

Type of Medium: Online Resource

ISSN: 0002-9297

URL: Article

DOI: 10.1016/j.ajhg.2020.04.016

Language: English

Publisher: Elsevier BV

Publication Date: 2020

detail.hit.zdb_id: 1473813-2

SSG: 12

In: Orphanet Journal of Rare Diseases, Springer Science and Business Media LLC, Vol. 9, No. 1 ( 2014-12)

Type of Medium: Online Resource

ISSN: 1750-1172

URL: Article

DOI: 10.1186/s13023-014-0108-6

Language: English

Publisher: Springer Science and Business Media LLC

Publication Date: 2014

detail.hit.zdb_id: 2225857-7

In: Genetics in Medicine, Elsevier BV, Vol. 20, No. 6 ( 2018-06), p. 599-607

Type of Medium: Online Resource

ISSN: 1098-3600

URL: Article

DOI: 10.1038/gim.2017.154

Language: English

Publisher: Elsevier BV

Publication Date: 2018

detail.hit.zdb_id: 2063504-7

SSG: 12

In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 3918-3918

Abstract: Abstract 3918 Background: CLL cells circulating in the peripheral blood are sensitive to therapy while malignant cells residing in the microenvironment survive and are the source of relapse. One of the strongest microenvironmental stimuli is CD40 ligand (CD40L)-CD40 interaction, which induces proliferative/anti-apoptotic genes in CLL cells, protecting them from apoptosis and many cytotoxic drugs. Despite the evident importance of CD40 activation further stimuli have to be considered, especially hypoxia. Lymph nodes, particularly those being infiltrated by malignant cells, show a low oxygen tension ( 〈 1%). Prior CLL investigations never took this important factor into account, hence the impact of hypoxia on cell survival and drug-resistance is still unrevealed. Methods: We have established an in vitro model, which mimics hypoxic conditions and CD40L-CD40 interaction, in order to understand the molecular basis of drug resistance of CLL cells resident in the microenvironment. CLL cells were cultured on CD40L feeder cells and kept up to 96 hours in hypoxia (1% O2) or normoxia (21% O2). We determined how proliferation rates in CLL are affected by these conditions and subsequently applied several drugs to investigate differences in drug efficacy between normoxia and hypoxia. Apoptosis was determined by AnnexinV/7AAD-staining and subsequent flow cytometry. Expression of potential target molecules was determined by qRT-PCR and Western Blotting. Results: Hypoxia is known to protect malignant cells in solid cancers from chemotherapy. We made similar observations, since classical DNA-targeting drugs were inefficient to kill CLL cells under hypoxic conditions. However, we identified ABT-737, which affects mitochondrial integrity, to be even more efficient under hypoxic conditions compared to normoxia. In order to explain this discrepancy we investigated the expression of several mitochondrial localized anti-/proapoptotic genes on RNA and protein level. We show that the de-regulation of BclXL and Mcl-1 under hypoxic conditions is essential for ABT-737 sensitivity. BclXL deregulation depends on a general reduction in protein translation in hypoxic cells. Mcl-1 protein expression differs from its mRNA expression, hence we expected regulation subsequent to protein synthesis. Indeed we could identify an increased activity of the proteasome in hypoxia, as Mcl-1 is a short-lived protein with a rapid proteasomal turnover this is a feasible explanation for the observed downregulation. Interestingly, hypoxia has a great impact on proliferation of primary CLL cells under different stimuli in vitro. Conclusion: These are the first experiments investigating the impact of oxygen tension on survival and response to chemotherapy of CLL cells. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agent Fludarabine and Bendamustine. Furthermore we point out that small molecules like ABT-737, which specifically target mitochondria, might be efficient in targeting CLL cells protected by hypoxia and CD40L-CD40 interaction within the microenvironment. Development of novel in vitro models like ours will help us understand the specific molecular changes induced by microenvironmental stimuli and their impact on drug efficacy. These findings will allow us to identify novel therapeutic targets. M.Hu. and L.P.F. contributed equally to this work Disclosures: No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V120.21.3918.3918

Language: English

Publisher: American Society of Hematology

Publication Date: 2012

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detail.hit.zdb_id: 80069-7

In: Scientific Reports, Springer Science and Business Media LLC, Vol. 9, No. 1 ( 2019-11-15)

Abstract: An acute increase in blood flow triggers flow-mediated dilation (FMD), which is mainly mediated by endothelial nitric oxide synthase (eNOS). A long-term increase in blood flow chronically enlarges the arterial lumen, a process called arteriogenesis. In several common human diseases, these processes are disrupted for as yet unknown reasons. Here, we asked whether β1 integrin, a mechanosensory protein in endothelial cells, is required for FMD and arteriogenesis in the ischemic hindlimb. Permanent ligation of the femoral artery in C57BL/6 J mice enlarged pre-existing collateral arteries and increased numbers of arterioles in the thigh. In the lower leg, the numbers of capillaries increased. Notably, injection of β1 integrin-blocking antibody or tamoxifen-induced endothelial cell-specific deletion of the gene for β1 integrin ( Itgb1 ) inhibited both arteriogenesis and angiogenesis. Using high frequency ultrasound, we demonstrated that β1 integrin-blocking antibody or endothelial cell-specific depletion of β1 integrin attenuated FMD of the femoral artery, and blocking of β1 integrin function did not further decrease FMD in eNOS-deficient mice. Our data suggest that endothelial β1 integrin is required for both acute and chronic widening of the arterial lumen in response to hindlimb ischemia, potentially via functional interaction with eNOS.

Type of Medium: Online Resource

ISSN: 2045-2322

URL: Article

DOI: 10.1038/s41598-019-53137-x

Language: English

Publisher: Springer Science and Business Media LLC

Publication Date: 2019

detail.hit.zdb_id: 2615211-3