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In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 1571-1571

Abstract: Chronic myelogenous leukemia (CML) is a bone marrow-derived hematopoietic neoplasm in which BCR/ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients with CML, leukemic cells can be kept under control by BCR/ABL1 tyrosine kinase inhibitors (TKI), including imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. Nevertheless, resistance or intolerance against one or more of these TKI may occur. Therefore, current research is focusing on novel potential drug targets in CML. A promising class of targets may be epigenetic regulators of cell growth, such as members of the bromodomain and extra-terminal domain (BET) family. The epigenetic reader and BET family member BRD4 has recently been identified as a novel potential drug target in acute myeloid leukemia (AML). However, so far, little is known about the expression and function of BRD4 in CML cells. The aims of the present study were to determine the expression of BRD4 and its downstream target MYC in CML cells and to explore whether BRD4 can serve as a novel drug target in this disease. As determined by qPCR, primary CML cells (chronic phase patients, n=7) as well as the CML cell lines KU812 and K562 expressed BRD4 mRNA. In addition, both CML cell lines stained positive for BRD4 in our immunocytochemistry staining experiments. In one patient with accelerated phase CML, putative leukemic (CD34+/CD38-) stem cells were sorted to near homogeneity and found to express BRD4 mRNA by qPCR. In order to examine the functional role of BRD4 in CML cells, a BRD4-specific shRNA was applied. In these experiments, the shRNA-induced knockdown of BRD4 in KU812 cells and K562 resulted in reduced growth compared to a control shRNA. Furthermore, the BRD4-targeting drug JQ1 was found to inhibit 3H-thymidine uptake and thus proliferation in KU812 cells in a dose-dependent manner (IC50: 0.25-0.75 µM). In addition, we were able to show that JQ1 inhibits growth of primary CML cells with variable IC50 values (0.1-5 µM). However, no substantial growth-inhibitory effects of JQ1 were seen in K562 cells (IC50: 〉 5 µM). As determined by Annexin V/PI staining, JQ1 induced apoptosis in KU812 cells whereas no apoptosis-inducing effect of JQ1 was observed in K562 cells. Nevertheless, we were able to show that both CML cell lines as well as primary CML cells express MYC mRNA, and treatment of KU812 cells or K562 cells with JQ1 resulted in a decreased expression of MYC mRNA and MYC protein. Next, we analyzed whether MYC expression in CML cells can be blocked by BCR/ABL1 TKI. We found that imatinib, nilotinib, dasatinib, and ponatinib decrease MYC mRNA- and MYC protein expression in KU812 and K562 cells. Finally, we found that JQ1 cooperates with imatinib, nilotinib, ponatinib and dasatinib in inhibiting the proliferation of KU812 and K562 cells. Together, our data show that BRD4 serves as a potential new target in CML cells, and that the BRD4 blocker JQ1 cooperates with BCR/ABL1 TKI in inducing growth-inhibition. Whether BRD4 inhibition is a pharmacologically meaningful approach in patients with TKI-resistant CML remains to be determined in clinical trials. Disclosures Sperr: Ariad: Consultancy; Celgene: Consultancy. Zuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V126.23.1571.1571

Language: English

Publisher: American Society of Hematology

Publication Date: 2015

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. 1722-1722

Abstract: Chronic myeloid leukemia (CML) is a hematopoietic stem cell neoplasm in which BCR-ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients, leukemic cells can be kept under control by BCR-ABL1 tyrosine kinase inhibitors (TKI). Nevertheless, resistance against one or more TKI may occur. Therefore, research is focusing on novel potential drug targets in CML. We have recently identified the epigenetic reader bromodomain-containing protein 4 (BRD4) as a new therapeutic target in leukemic stem cells (LSC) in acute myeloid leukemia. In the present study, we examine the expression of BRD4 and its downstream effector MYC in CML cells and asked whether BRD4 serves as a drug target in CML cells and whether BRD4-targeting drugs, including JQ1 and newly developed BRD4 degraders (dBET1 and dBET6) are able to overcome LSC resistance in CML. Primary CML cells were obtained from 22 patients with chronic phase (CP) CML and 3 with blast phase (BP) CML. As determined by qPCR and/or immunocytochemistry, the CML cell lines KU812 and K562 as well as primary CML cells expressed BRD4 and MYC. All three BRD4-targeting drugs (JQ1, dBET1 and dBET6) were found to decrease MYC expression in KU812 and K562 cells as assessed by Western blotting. In 3H-thymidine uptake experiments, JQ1 and dBET6 were found to inhibit the proliferation of KU812 in a dose-dependent manner (IC50, JQ1: 100-500 nM; dBET6: 50-100 nM) whereas dBET1 showed only little if any effects on growth of KU812 cells (IC50: 1-5 µM), and in K562 cells, only dBET6 was found to inhibit growth with a reasonable IC50 value (250-500 nM). Corresponding results were obtained when examining drug effects on survival of CML cell lines by Annexin-V/PI staining. All three BRD4-targeting drugs were found to inhibit proliferation of primary CP CML cells with varying IC50 values. As expected, growth-inhibitory effects of dBET6 were more pronounced (IC50: 〈 100 nM) compared to effects seen with JQ1 and dBET1. dBET1 and dBET6 were also found to inhibit growth of primary CML cells obtained from patients with BP CML, whereas JQ1 was not effective. JQ1 also failed to suppress survival on CML CD34+/CD38− LSC. By contrast, dBET1 induced apoptosis in CML LSC at 1 µM and dBET6 induced apoptosis in CML LSC at 0.1 µM. dBET6 induced apoptosis in CML LSC obtained from patients with imatinib-sensitive CML as well as patients with imatinib-resistant CML harboring BCR-ABL1 T315I or BCR-ABL1 F317L. Finally, pre-incubation of CD34+ CP CML cells with dBET6 resulted in reduced leukemic engraftment in NSG mice exhibiting human membrane-bound stem cell factor, SCF [NSG-Tg(hu-mSCF)] 6 months after transplantation (engraftment with CD45+/CD33+/CD19−cells in control mice receiving DMSO-treated cells: 8.1±6.6% vs mice receiving dBET6-treated cells: 1.1±0.6%). To further explore the ability of dBET6 to interfere with LSC resistance in CML, we established a co-culture system mimicking LSC-niche interactions in the osteoblastic niche. In this model, co-culturing K562 cells, KU812 cells or primary CML LSC with the osteoblast-like osteosarcoma cell line CAL-72 resulted in resistance against nilotinib and ponatinib. In this culture system, JQ1 was found to partially restore TKI effects in K562 cells and completely restored TKI effects in KU812 cells. Interestingly, JQ1 was not able to restore TKI effects in primary CML LSC in these co-cultures. However, dBET6 was found to overcome niche cell-induced TKI-resistance of primary CML LSC. Finally, we were able to demonstrate that JQ1, dBET1 and dBET6 inhibit interferon-gamma-induced upregulation of PD-L1 expression in CML LSC. Together we show that BRD4 and MYC are potential new therapeutic drug targets in CML and that the BET-degrader dBET6 overcomes multiple forms of LSC resistance, including i) intrinsic resistance, ii) mutation-induced resistance, iii) niche induced resistance and iv) checkpoint-mediated resistance. Whether BRD4 degradation is also able to overcome TKI-resistance of BCR-ABL1+ LSC in vivo in patients with CML remains to be determined in clinical trials. Disclosures Hoermann: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Wolf:BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding. Mayer:Amgen: Research Funding; Novartis: Research Funding. Zuber:Mirimus Inc.: Consultancy, Other: Shareholder; Boehringer Ingelheim GmbH & Co KG: Research Funding. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2018-99-111126

Language: English

Publisher: American Society of Hematology

Publication Date: 2018

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detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 5503-5503

Abstract: Background: Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy with features of both a myelodysplastic syndrome and a myeloproliferative neoplasm.The pathogenesis of CMML is incompletely understood due to the large heterogeneity of molecular aberrations in genes involved in epigenetic regulation, RNA-splicing and signal transduction including components of RAS and JAK2 signaling. Functional tests may be important to better estimate the contribution of a particular molecular aberration in the pathogenesis of the malignancy. We have originally demonstrated extensive in vitro formation of myeloid colonies (CFU-GM) without addition of exogenous growth factors in a subset of patients with CMML (Geissler et al, Leuk Res 1988). We reported that this spontaneous CFU-GM colony formation in CMML is a GM-CSF dependent in vitro phenomenon (Geissler et al, J Exp Med 1996) and could also show in a small retrospective study that CMML patients with high spontaneous CFU-GM growth ( 〉 100/105 PBMNC) have a worse prognosis compared to patients with low myeloid colony formation (Sagaster et al, Ann Hematol 2004) suggesting a clinical significance of our observation. In juvenile myelomonocytic leukemia, in which molecular aberrations are mainly restricted to the RASopathy genes including NRAS, KRAS, NF1, CBL and PTPN11, spontaneous formation of CFU-GM due to GM-CSF-specific hypersensitivity is a hallmark feature of disease, which has been included in the diagnostic criteria. We therefore speculated that high spontaneous myeloid colony formation in CMML might also reflect hyperactivation of the RAS signaling pathway. Aim: Our aim was to study the correlation between spontaneous myeloid colony formation and the presence of mutations in RASopathy genes in patients with CMML. Moreover the relationship of high autonomous CFU-GM formation with phenotypic features of CMML and its clinical outcome was investigated. Patients and Methods: In this study we included 137 CMML patients of the "Austrian Biodatabase for CMML (ABCMML)" in whom CFU-GM data and/or molecular data were available. CFU-GM growth in the absence of exogenous cytokines was assessed in a central laboratory using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Molecular characterization was also performed in a central laboratory using NGS with amplicon-based target enrichment of 39 CMML associated genes. Assuming that clones that are too small are unlikely to significantly impact hematopoiesis only mutations with an allele burden of ≥20% were considered positive in this analysis. Clinical and hematological data were obtained from patients records. Results:High spontaneous CFU-GM growth (≥100/105 PBMNC) was found in 38/135 (28%) CMML patients, of whom 3 were already transformed into secondary AML at the time of in vitro culture testing. There was a significant correlation between high CFU-GM formation in vitro and the presence of mutations in genes involved in the RAS signaling pathway. The incidence of RAS pathway mutations was 72% in CMML patients with high colony growth and 31% in patients with low spontaneous CFU-GM formation (p 〈 0.0001). As shown in Table 1 high spontaneous myeloid colony formation was associated with increased WBC counts, increased blast cells, increased LDH, more pronounced splenomegaly and inferior survival (Fig. 1). There was no significant difference regarding autonomous CFU-GM growth in CMML patients with molecular aberrations in genes of epigenetic regulation and RNA-splicing, respectively. High spontaneous CFU-GM was never observed in CMML patients in whom the JAK2 V617F mutation was the only molecular aberration in signaling pathways (0/8 patients). Furthermore the in vitro conversion from a growth factor dependent to a growth factor independent phenotype by RAS but not by JAK2 could be demonstrated in BaF3 cells (Fig. 2). Conclusion: Our findings indicate that high spontaneous in vitro myeloid colony formation is associated with the presence of RAS pathway mutations, leukocytosis, splenomegaly and reduced survival. These results suggest that CMML with high spontaneous colony growth is a mainly RAS pathway driven malignancy resulting in myeloproliferation and inferior outcome. This may have clinical implications concerning therapeutic strategies aimed at targeting the hyperactive RAS signaling pathway in these patients. Disclosures Geissler: Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celegene: Honoraria, Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V128.22.5503.5503

Language: English

Publisher: American Society of Hematology

Publication Date: 2016

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detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 101, No. 1 ( 2003-01-01), p. 375-375

Type of Medium: Online Resource

ISSN: 1528-0020 , 0006-4971

URL: Article

DOI: 10.1182/blood-2002-08-2557

Language: English

Publisher: American Society of Hematology

Publication Date: 2003

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 4223-4223

Abstract: Chronic myelomonocytic leukemia (CMML) is a stem cell-derived hematopoietic neoplasm characterized by dysplasia, uncontrolled expansion of monocytic (progenitor) cells in the bone marrow (BM) and in the peripheral blood (PB), and an increased risk of progression to secondary acute myeloid leukemia (sAML). Patients with advanced CMML and sAML are often highly resistant to therapy and their prognosis is dismal. It is thought that drug resistance in myeloid malignancies is a quality of leukemia stem cells (LSC), but little is known about CMML-initiating and propagating LSC. We investigated the phenotype and functional behavior of putative CMML-initiating cells in 15 patients with CMML (7 females, 8 males; median age 73 years; range 45-82 years) and 6 with sAML following CMML (1 female, 5 males; median age 67.5 years; range 66-76 years). BM and/or PB samples were examined by multicolor flow cytometry using antibodies against CD34, CD38 and various additional surface markers and target antigens. In a subset of patients, putative stem and progenitor cells (CD34+ cells, CD34+/CD38─ cells and CD34+/CD38+ cells) were FACS-sorted to high purity ( 〉 95%) and were employed in xenotransplantation experiments or in drug testing experiments. We found that CMML-initiating and propagating LSC reside within the CD34+/CD38─ fraction of the malignant clone. Whereas highly purified CD34+ cells engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG) 1Eav/MloySzJ (NSGS) mice with full-blown CMML (engraftment rate 44.8±26.0%), no CMML was produced by the bulk of CD34- monocytic cells (engraftment rate 0.8±0.5%; p=0.002). CMML engraftment was also detectable when transplanting unselected mononuclear cells (engraftment rate 19.7±10.9%). By contrast, no leukemic engraftment was produced by CD38+ CMML fractions (engraftment rate 0.1±0.1%; p=0.003), indicating that the NSGS-repopulating CMML LSC reside specifically in a CD34+/CD38- fraction of the clone. In sAML, both the CD34+/CD38- cell fraction (engraftment rate 92.2±6.2%) and the CD34+/CD38+ fraction (engraftment rate 80.5±7.2%) produced engraftment with AML blasts in NSGS mice. In a next step, we established the cell surface phenotype of CD34+/CD38- LSC in CMML and sAML. As assessed by multicolor flow cytometry, CD34+/CD38- CMML cells invariably expressed CD33/Siglec-3, CD117/KIT, CD123/IL3RA, CD133/AC133, CD135/FLT3, and IL-1RAP. In a subset of patients, CMML LSC also expressed CD52 (9/11 patients; 81%), CD114 (3/7 patients; 43%), CD184 (9/12 patients; 75%), CD221 (8/11 patients; 73%) and/or CLL-1 (7/13 patients; 54%). CMML LSC did not express CD25 or CD26. However, in patients with sAML, LSC also displayed CD25 (median fluorescence intensity, MFI: CMML: 0.9 vs. sAML: 23.0; p 〈 0.001). Compared to hematopoietic stem cells in normal BM (NBM), CMML LSC displayed slightly increased levels of CD117/KIT (MFI CMML: 32.5 vs. MFI NBM: 15.0; p=0.019), CD135/FLT3 (MFI CMML: 1.9 vs. MFI NBM: 0.8; p=0.001), CD184/CXCR4 (MFI CMML: 1.6 vs. MFI NBM 0.9; p=0.027), and IL-1RAP (MFI CMML: 1.6 vs. MFI NBM: 0.8; p=0.004). No correlations between surface-marker expression on LSC and the type of CMML (CMML-0/1/2 or dysplastic vs. proliferative CMML) or the clinical course were found. To confirm the clinical relevance of expression of surface target antigens on CMML LSC, we applied the CD33-targeted drug gemtuzumab-ozogamicin (GO). As assessed by combined staining for LSC (CD34+/CD38-) and AnnexinV/DAPI, incubation of CMML LSC with GO (0.001-1 µg/ml) resulted in dose-dependent apoptosis in all donors tested, and the same result was obtained in the monoblastic cell lines THP-1 (GO at 1 µg/ml: 94.2±1.5% vs. control: 12.7±2.2%, p 〈 0.05) and Mono-Mac-1 (GO at 1 µg/ml: 56.4±12.1% vs. control: 10.1±0.6% p 〈 0.05). In conclusion, LSC in CMML and sAML reside within CD34+/CD38─ cell populations that express distinct profiles of surface markers and target antigens. During progression of CMML into sAML, LSC apparently acquire CD25. Characterization of CMML LSC and LSC in sAML should facilitate their enrichment and the development of LSC-eradicating therapies. Disclosures Hoermann: Novartis: Honoraria; Roche: Honoraria. Sperr:Celgene: Consultancy, Honoraria; Novartis: Honoraria. Sill:Astex: Other: Advisory board; Novartis: Other: Advisory board; AbbVie: Other: Advisory board; Astellas: Other: Advisory board. Geissler:Novartis: Honoraria; AOP: Honoraria; Roche: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Ratiopharm: Honoraria; Celgene: Honoraria; Abbvie: Honoraria; Pfizer: Honoraria. Deininger:Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Ascentage Pharma: Consultancy, Honoraria; Fusion Pharma: Consultancy; TRM: Consultancy; Sangoma: Consultancy; Adelphi: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy. Valent:Pfizer: Honoraria; Blueprint: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Deciphera: Honoraria, Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2019-122530

Language: English

Publisher: American Society of Hematology

Publication Date: 2019

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. 3189-3189

Abstract: Background:Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy of the elderly with a heterogenous molecular pathophysiology. Whereas mutations in components of the RAS pathways are among the most common somatic mutations in CMML the JAK2 V617F mutation which is a typical finding in polycythemia vera and around 50% of patients with essential thrombocythemia and primary myelofibrosis, respectively, is by far less frequently detected in CMML but can be consistently found in a subgroup of patients in larger series. Due to the fact that JAK2 V617F-positive CMML is a rare disease the clinical, hematological and in vitro growth characteristics of this entity are poorly investigated. In the "Austrian Biodatabase for Chronic Myelomonocytic Leukemia (ABCMML)" we retrospectively and prospectively collect clinical, biologic, and molecular information of patients with CMML from different centers in a real life setting. Aims:Our aim was to characterize the clinical, hematological, molecular and biologic features of CMML patients harboring a JAK2 V617F mutation. Methods:The diagnosis of CMML was established according to diagnostic criteria of the World Health Organization (WHO) classification of 2008 (Vardiman et al, Blood 2009). Clinical and hematological data were obtained from patients records. For molecular characterization we used next-generation sequencing with amplicon-based target enrichment of 39 CMML associated genes. Only mutations with an allele burden of 〉 10% were considered positive in this analysis. Autonomous colony-forming units granulocyte/macrophage (CFU-GM) growth in the absence of exogenous cytokines was assessed using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Results:Up to now targeted NGS data are available in 116 patients and in vitro culture data in 75 patients respectively. We identified 13 CMML patients who had a JAK2 V617F mutation with an allele frequency 〉 10%. Clinical, hematological, and biologic characteristics in these patients were compared with 103 patients who had NGS sequencing and were negative for the JAK2 V617F mutation. As shown in Table 1 JAK2 V617F-positive CMML patients had significantly higher WBC counts, higher hemoglobin values, higher platelet counts and more pronounced splenomegaly as compared to JAK2 V617F-negative patients. On the other hand the percentage on monocytes in peripheral blood and the numbers of CFU-GM growing in vitro without addition of exogenous growth factors were lower in CMML patients with the JAK2 V617F mutation as compared to patients without this mutation. The majority of JAK2 V617F-positive patients had additional mutations that can be also found in JAK2 V617F-negative patients, in particular mutations in genes of epigenetic regulation and RNA-splicing, respectively. As shown in Figure 1 there was a trend towards a better survival of patients with the JAK2 V617F mutation as compared to JAK2 V617F-negative patients (p=0.05). In a JAK2 V617F-positive CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we were able to demonstrate the disappearance of constitutional symptoms and a durable spleen response lasting for over 56 months (Fig. 2). Conclusion:Out data show that CMML patients with the JAK2 V617F mutation have hematological, biologic and clinical characteristics different from JAK2 V617F-negative CMML patients. These findings suggest that JAK2 V617F-positive CMML patients should be regarded as a distinct subgroup which may benefit from specific targeted treatments. Disclosures Geissler: Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celegene: Honoraria, Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V128.22.3189.3189

Language: English

Publisher: American Society of Hematology

Publication Date: 2016

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detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 2917-2917

Abstract: In allogeneic transplantation it is a major goal to maintain a stable long term engraftment. However, several factors like viral infections, drug toxicity or low stem cell numbers may hamper hematopoietic recovery or may even cause a late graft failure after successful initial engraftment. Hematopoietic growth factors or additional stem cell donations (boosts) may help to overcome this problem. In this retrospective analysis, the efficacy of boosts with highly purified peripheral stem cells either from closely matched unrelated or from haploidentical related donors was evaluated in pediatric patients with stagnant hematopoiesis ( & lt;1500 leucocytes or & lt;50 000 platelets between 30–90 days posttransplant, n=11) or with initially increasing and then decreasing hematopoiesis (n=3). Patients with Graft rejection or hemophagocytosis were excluded. Diagnoses were: acute leukemias (n=10), nonmalignatn diseases (n=4). Most boosts were taken from the original graft: if the grafts contained & gt;10x106 (unrelated donors) or & gt;20x106/kg BW CD34+ cells, surplus stem cells were purified with an immunomagnetic selection method using antiCD34 or antiCD133 coated microbeads and cryopreserved. Three haploidentical donors were asked for a second donation. Our aim was to maintain stable and complete hematopoiesis after transplantation without inducing acute or chronic GvHD. A total of 18 boosts were given between 30 and 290 days after transplantation with a median number of 5 (0.6–34) x 106 CD34+/CD133+ progenitor cells/kg and only 2200 (100–25000) residual T-cells/kg. All patients had a complete donor chimerism and no reconditioning or posttransplant pharmacological immunosuppression was administered. Patients who received G-CSF were excluded. Due to the low number of T cells, acute GvHD & gt;grade I and also chronic GvHD could be completely avoided even in haploidentical donors. Leukocyte counts, neutrophile counts, lymphocyte counts (and platelet counts in patients with thrombocytopenia) within the week before infusion of the boosts were compared with cell counts 4 and 8 weeks after infusion, using the paired T-test. A significant increase of all cell counts was observed (medina numbers pre boosting: 900/μl, 312/μl, 142/μl; 4 weeks post boosting: 2065/μl, 1264/μl, 317/μl; 8 weeks post boosting:2570/μl, 1780/μl, 385/μl; p & lt;0.05 for all comparisons). Furthermore, independence from platelet transfusion could be achieved. Conclusions: Boosts with purified stem cells and low T-cell contamination ( & lt;10000 T-cells/kg) can be administered after transplantation without the risk of inducing GvHD, even in mismatched donors. No pharmacological immunosuppression was necessary. Thus, the method represents a safe tool and can improve donor derived hematopoiesis.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V106.11.2917.2917

Language: English

Publisher: American Society of Hematology

Publication Date: 2005

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detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 1965-1965

Abstract: Systemic mastocytosis (SM) is a myeloid neoplasm defined by abnormal growth and pathologic accumulation of neoplastic mast cells (MC) in various internal organs. The indolent variant of SM (ISM) is associated with an almost normal life expectancy. By contrast, the prognosis in advanced SM, including SM with an associated hematologic neoplasm (AHN), aggressive SM (ASM), and MC leukemia (MCL) is poor with short survival times. Most patients with SM express the D816V-mutated variant of KIT, which confers resistance against several tyrosine kinase inhibitors (TKI), including imatinib. Midostaurin is a TKI that is effective against KIT D816V. However, despite encouraging clinical efficacy, this drug cannot produce continuous complete remission in all patients. One problem in advanced SM is that the AHN component of the disease, especially when progressing into acute myeloid leukemia (AML) is often drug-resistant. The aims of this study were to evaluate the effects of the multi-kinase inhibitor DCC-2618 on proliferation and survival of primary neoplastic mast cells, various mast cell lines and other malignant and non-malignant cell types that may play a role in advanced SM. As assessed by 3H-thymidine-uptake, DCC-2618 was found to inhibit the proliferation of all human MC lines tested, with lower IC50 values measured in HMC-1.1 cells lacking KIT D816V (11.2±4.3 nM) and ROSAKIT WT cells (61±11 nM) than in KIT D816V+ HMC-1.2 cells (147±68 nM) and ROSAKIT D816V cells (133±43 nM). DCC-2618 also produced growth inhibition in the multi-resistant MCL lines MCPV-1.1 (164±72 nM), MCPV-1.2 (256±167 nM), MCPV-1.3 (124±46 nM), and MCPV-1.4 (235±114 nM). In addition, DCC-2618 was found to inhibit the proliferation of primary neoplastic bone marrow MC obtained from patients with SM including MCL (Figure). We also found that DCC-2618 induces apoptosis in HMC-1 cells and ROSA cells, and to a lesser degree in MCPV-1 cells as determined by light microscopy and AnnexinV/PI staining. Moreover, DCC-2618 was found to block phosphorylation of KIT in all MC lines tested. In a next step, we explored the effects of DCC-2618 on growth of other leukemia cell lines as well on vascular endothelial cells. In these experiments, we were able to show that DCC-2618 inhibits the proliferation of the FIP1L1-PDGFRA+ eosinophilic leukemia cell line EOL-1 (IC50 2±0.6 nM) and the FLT3 ITD-mutated AML cell lines MV4-11 (IC50 130±18 nM) and MOLM-13 (IC50 110±26 nM). DCC-2618 also induced apoptosis in EOL-1, MV-411, and MOLM-13 cells. Moreover, DCC-2618 was found to inhibit the growth of cultured human vascular endothelial cells, suggesting that the drug may also counteract SM-related neo-angiogenesis in SM. DCC-2618 did not inhibit the proliferation of the immature AML cell line KG1 and the monoblastic cell line U937, but was found to block proliferation in primary leukemic monocytes in patients with monoblastic AML or chronic myelomonocytic leukemia (CMML) which may have clinical implications as CMML and AML are the most prevalent types of AHN in advanced SM. Finally, we were able to show that the major DCC-2618-metabolite, DP-5439, is equally effective in producing growth inhibition in all cell lines tested as well as in primary neoplastic MC compared to DCC-2618 (Figure). In summary, our data show that DCC-2618 is a new potent multi-targeted TKI that counteracts growth of neoplastic MC as well as growth and survival of leukemic monocytes, AML blasts, eosinophils, and endothelial cells in vitro. Whether DCC-2618 is also able to inhibit the growth of neoplastic MC and other leukemic (AHN) cells in vivo in patients with advanced SM remains to be determined in clinical trials. Indeed, a first Phase I clinical trial examining the effects of DCC-2618 in SM has recently been initiated. Figure Figure. Disclosures Valent: Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V128.22.1965.1965

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. 112, No. 4 ( 2008-08-15), p. 1374-1381

Abstract: The spectrum of entities, the therapeutic strategy, and the outcome of mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) differs between children and adolescents on the one hand and adult patients on the other. Whereas adult maB-NHLs have been studied in detail, data on molecular profiling of pediatric maB-NHLs are hitherto lacking. We analyzed 65 cases of maB-NHL from patients up to 18 years of age by gene expression profiling, matrix comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and immunohistochemistry. The majority of the analyzed pediatric patients were treated within prospective trials (n = 49). We compared this group to a series of 182 previously published cases of adult maB-NHL. Gene expression profiling reclassified 31% of morphologically defined diffuse large B-cell lymphomas as molecular Burkitt lymphoma (mBL). The subgroups obtained by molecular reclassification did not show any difference in outcome in children treated with the NHL-Berlin-Frankfurt-Muenster (BFM) protocols. No differences were detectable between pediatric and adult mBL with regard to gene expression or chromosomal imbalances. This is the first report on molecular profiling of pediatric B-NHL showing mBL to be much more prominent in children than suggested by morphologic assessment. Based on molecular profiling mBL is a molecularly homogeneous disease across children and adults.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2008-01-136465

Language: English

Publisher: American Society of Hematology

Publication Date: 2008

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. 4241-4241

Abstract: Chronic myeloid leukemia (CML) is a stem cell disease characterized by BCR-ABL1. Most patients in chronic phase (CP) CML achieve long-lasting molecular responses when treated with BCR-ABL1 tyrosine kinase inhibitors (TKI). However, resistance against TKI occurs in a subset of patients. Several molecular mechanisms, including BCR-ABL1 mutations, contribute to TKI resistance. For imatinib-resistant patients, second- and third generation TKI, including nilotinib, dasatinib, bosutinib, and ponatinib, are available. Using these drugs, it is now possible to cover most of the known BCR-ABL1 mutations, including the multi-resistant mutation T315I. Ponatinib, a third generation TKI, induces growth-inhibitory effects in drug-resistant patients even if T315I is expressed. However, not all mutant forms of BCR-ABL1 are responsive to ponatinib. Moreover, it has been described that multiple secondary mutations in BCR-ABL1, especially T315I-involving compound mutations, confer resistance against ponatinib. Furthermore, resistance against TKI may develop independent of BCR-ABL1 mutations. Therefore, drug combinations covering a broad range of targets, are currently under investigation with the aim to overcome drug resistance in advanced CML. Bardoxolone methyl (CDDO-Me) is an oleanane triterpenoid that has been described to induce ROS generation and to suppress a number of survival-related molecules, including AKT, mTOR, and STAT3. The aim of the current project was to evaluate the anti-leukemic effects of CDDO-Me in TKI-resistant CML cells. As assessed by 3H-thymidine uptake experiments, CDDO-Me was found to inhibit growth of various CML cell lines, including K562, an imatinib resistant sub-clone of K562, KU812, and imatinib-resistant KCL22 cells (IC50: 0.1-0.5 µM). These effects were accompanied by induction of apoptosis as assessed by staining for AnnexinV and propidium iodide. Furthermore, CDDO-Me was found to block the growth of Ba/F3 cells harboring the BCR-ABL1 mutations T315I, E255K, G250E, H396P, or F359V as well as Ba/F3 cells expressing TKI-resistant compound mutations, such as T315I/E255V, T315I/F311L, T315I/F359V, or T315I/G250E (IC50: 0.1-0.25 µM). The anti-proliferative effects of CDDO-Me were also confirmed in primary CML cells isolated from 13 patients with chronic phase (CP) CML (4 TKI-resistant patients, 3 with BCR-ABL1 mutations), one in blast phase (BP), and one suffering from ponatinib-resistant Ph+ ALL harboring BCR-ABL1T315I/E255K. IC50 values were comparable between samples isolated from freshly diagnosed patients (IC50: 0.1-0.5 µM) and samples isolated from heavily pre-treated patients, (IC50: 0.1-0.5 µM) suggesting that BCR-ABL1 mutations do not influence responses to this drug. In consecutive experiments, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with second- or third-generation BCR-ABL1 TKI. The combination ´CDDO-Me+ponatinib´ was found to be effective in Ba/F3 cells expressing various BCR-ABL1 mutations, including T315I-involving compound mutations. We also found that the combination ´CDDO-Me+TKI´ leads to simultaneous dephosphorylation of STAT3 and STAT5. To clarify whether this drug action contributes to the synergistic drug-interactions observed, we performed experiments with shRNA directed against STAT3 or STAT5 and the specific STAT5-inhibitor AC-3-019. Knockdown of STAT3 was found to produce synergistic effects with TKI and with AC-3-019 in K562 and KCL22 cells, whereas STAT5-knockdown sensitized CML cells against CDDO-Me, pointing to a new effective concept of dual STAT3+STAT5 inhibition. However, CDDO-Me was also found to increase expression of heme-oxygenase-1 (HO-1), a heat-shock-protein known to trigger drug resistance and cell survival in CML cells. We therefore combined CDDO-Me with the HO-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects in human CML cells and BCR-ABL1+Ba/F3 cells. Moreover, SMA-ZnPP was found to sensitize KU812 cells and Ba/F3 cells expressing BCR-ABL1T315I/F311L against the combination ´CDDO-Me+TKI´. Together, combined targeting of STAT3, STAT5, and HO-1 overcomes multiple forms of TKI resistance in highly resistant CML clones expressing BCR-ABL1T315I or T315I-containing compound mutations. Whether such drug combinations are effective in vivo in TKI-resistant patients remains to be elucidated. Disclosures Hoermann: Ariad: Honoraria; Gilead: Research Funding; Amgen: Honoraria; Novartis: Honoraria. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Deininger:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lion:Amgen: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria. Valent:Ariad: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Amgen: Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V128.22.4241.4241

Language: English

Publisher: American Society of Hematology

Publication Date: 2016

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7