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A major problem in SARS-CoV-2-infected patients is the massive tissue inflammation in certain target organs, including the lungs. Mast cells (MC), basophils (BA), and eosinophils (EO) are key effector cells in inflammatory processes. These cells have recently been implicated in the pathogenesis of SARS-CoV-2 infections. We explored coronavirus receptor (CoV-R) expression profiles in primary human MC, BA, and EO, and in related cell lines (HMC-1, ROSA, MCPV-1, KU812, and EOL-1). As determined using flow cytometry, primary MC, BA, and EO, and their corresponding cell lines, displayed the CoV-R CD13 and CD147. Primary skin MC and BA, as well as EOL-1 cells, also displayed CD26, whereas primary EO and the MC and BA cell lines failed to express CD26. As assessed using qPCR, most cell lines expressed transcripts for CD13, CD147, and ABL2, whereas ACE2 mRNA was not detectable, and CD26 mRNA was only identified in EOL-1 cells. We also screened for drug effects on CoV-R expression. However, dexamethasone, vitamin D, and hydroxychloroquine did not exert substantial effects on the expression of CD13, CD26, or CD147 in the cells. Together, MC, BA, and EO express distinct CoV-R profiles. Whether these receptors mediate virus-cell interactions and thereby virus-induced inflammation remains unknown at present.

Systemic mastocytosis (SM) is a hematopoietic neoplasm with a complex pathology and a variable clinical course. Clinical symptoms result from organ infiltration by mast cells (MC) and the effects of pro-inflammatory mediators released during MC activation. In SM, growth and survival of MC are triggered by various oncogenic mutant-forms of the tyrosine kinase KIT. The most prevalent variant, D816V, confers resistance against various KIT-targeting drugs, including imatinib. We examined the effects of two novel promising KIT D816V-targeting drugs, avapritinib and nintedanib, on growth, survival, and activation of neoplastic MC and compared their activity profiles with that of midostaurin. Avapritinib was found to suppress growth of HMC-1.1 cells ( KIT V560G) and HMC-1.2 cells ( KIT V560G + KIT D816V) with comparable IC 50 values (0.1-0.25 µM). In addition, avapritinib was found to inhibit the proliferation of ROSA KIT WT cells, (IC 50 : 0.1-0.25 µM), ROSA KIT D816V cells (IC 50 : 1-5 µM), and ROSA KIT K509I cells (IC 50 : 0.1-0.25 µM). Nintedanib exerted even stronger growth-inhibitory effects in these cells (IC 50 in HMC-1.1: 0.001-0.01 µM; HMC-1.2: 0.25-0.5 µM; ROSA KIT WT : 0.01-0.1 µM; ROSA KIT D816V : 0.5-1 µM; ROSA KIT K509I : 0.01-0.1 µM). Avapritinib and nintedanib also suppressed the growth of primary neoplastic cells in most patients with SM examined (avapritinib IC 50 : 0.5-5 µM; nintedanib IC 50 : 0.1-5 µM). Growth-inhibitory effects of avapritinib and nintedanib were accompanied by signs of apoptosis and decreased surface expression of the transferrin receptor CD71 in neoplastic MC. Finally, we were able to show that avapritinib counteracts IgE-dependent histamine secretion in basophils and MC in patients with SM. These effects of avapritinib may explain the rapid clinical improvement seen during treatment with this KIT inhibitor in patients with SM. In conclusion, avapritinib and nintedanib are new potent inhibitors of growth and survival of neoplastic MC expressing various KIT mutant forms, including D816V, V560G, and K509I, which favors the clinical development and application of these new drugs in advanced SM. Avapritinib is of particular interest as it also blocks mediator secretion in neoplastic MC., Competing Interests: G.H. received honoraria from Novartis. W.R.S. received honoraria from Novartis and Celgene, and a research grant from Lipomed. A.R. and P.V. served as a consultant in a global Novartis trial examining the effects of midostaurin in advanced SM. M.A. received research grants from Blueprint and honoraria from AB Science, Blueprint, and Novartis. P.V. received honoraria from Novartis, Celgene-BMS, Incyte, Pfizer, AOP Orphan, and Blueprint., (AJCR Copyright © 2023.)

Patient-related factors are of prognostic importance in acute myeloid leukemia (AML). Likewise, cardiac disorders may limit the tolerance of intensive therapy. Little is known about the prognostic value of N-terminal pro-brain natriuretic peptide (NT-proBNP). We analyzed NT-proBNP levels at diagnosis in 312 AML patients (median age: 61 years; range 17-89 years) treated with 3 + 7-based induction-chemotherapy and consolidation with up to four cycles of intermediate or high-dose ARA-C. NT-proBNP levels were elevated in 199 patients (63.8%), normal (0-125 pg/ml) in 113 (36.2%), and highly elevated (>2000 pg/ml) in 20 patients (6.4%). Median NT-proBNP levels differed significantly among patients with complete remission (153.3 pg/ml), no remission (225.9 pg/ml), or early death (735.5 pg/ml) (p = .002). In multivariate analysis, NT-proBNP, age, and the 2009 European LeukemiaNet (ELN-2009) classification were independent predictors of outcome after induction chemotherapy. Overall survival (OS) differed significantly between patients with normal, moderately elevated, and highly elevated NT-proBNP (p < .001). These differences were observed in all patients and in patients <60 years but not in those ≥60 years. In multivariate analysis, NT-proBNP, age, and ELN-2009 remained independent prognostic variables for OS (p < .01). Together, NT-proBNP is an independent prognostic factor indicating the risk of induction failure, early death, and reduced OS in patients with AML., (© 2023 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Background: Mast cells (MC) and basophils are effector cells of allergic reactions and display a number of activation-linked cell surface antigens. Of these antigens, however, only a few are functionally relevant and specifically expressed in these cells., Objective: We sought to identify MC- and basophil-specific surface molecules and to study their cellular distribution and regulation during cytokine-induced and IgE-dependent activation., Methods: Multicolor flow cytometry was performed to recognize surface antigens and to determine changes in antigen expression upon activation., Results: We identified Siglec-6 (CD327) as a differentially regulated surface antigen on human MC and basophils. In the bone marrow, Siglec-6 was expressed abundantly on MC in patients with mastocytosis and in reactive states, but it was not detected on other myeloid cells, with the exception of basophils and monocytes. In healthy individuals, allergic patients, and patients with chronic myeloid leukemia (CML), Siglec-6 was identified on CD203c + blood basophils, a subset of CD19 + B lymphocytes, and few CD14 + monocytes, but not on other blood leukocytes. CML basophils expressed higher levels of Siglec-6 than normal basophils. IL-3 promoted Siglec-6 expression on normal and CML basophils, and stem cell factor increased the expression of Siglec-6 on tissue MC. Unexpectedly, IgE-dependent activation resulted in downregulation of Siglec-6 in IL-3-primed basophils, whereas in MC, IgE-dependent activation augmented stem cell factor-induced upregulation of Siglec-6., Conclusions: Siglec-6 is a dynamically regulated marker of MC and basophils. Activated MC and basophils exhibit unique Siglec-6 responses, including cytokine-dependent upregulation and unique, cell-specific, responses to IgE-receptor cross-linking., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Basophilia is a crucial prognostic variable in Ph-chromosome-positive chronic myeloid leukemia (CML). The ectoenzyme CD203c is an activation-linked surface antigen that is expressed specifically on basophil-committed progenitor cells and mature basophils. We examined the expression of CD203c on progenitors and/or basophils in 21 healthy donors and 44 patients with CML. As expected, the numbers of CD203c + blood leukocytes were significantly higher in CML patients compared to controls (percentage of CD203c + cells among viable cells in CML at diagnosis: 4.19 ± 3.68% vs. controls: 0.53 ± 0.23%, p < 0.05). Moreover, CML basophils expressed higher levels of CD203c compared to normal basophils (median staining-index in CML at diagnosis: 29.41 ± 19.14 versus controls: 20.44 ± 13.45). We also found that the numbers and percentage of circulating CD203c + cells at diagnosis correlate with the disease-related risk-profile. Incubation of CML basophils with an anti-IgE-antibody resulted in further upregulation of CD203c. After successful treatment with imatinib and/or other BCR::ABL1 inhibitors leading to major or complete molecular responses, the numbers of CD203c + basophils decreased substantially in our CML patients compared to pre-treatment values. Together, CD203c is overexpressed on CML basophils, is further upregulated by IgE receptor cross-linking, and may serve as a biomarker to quantify basophilia in patients with CML at diagnosis and during therapy.

In most patients with chronic myeloid leukemia (CML) clonal cells can be kept under control by BCR::ABL1 tyrosine kinase inhibitors (TKI). However, overt resistance or intolerance against these TKI may occur. We identified the epigenetic reader BRD4 and its downstream-effector MYC as growth regulators and therapeutic targets in CML cells. BRD4 and MYC were found to be expressed in primary CML cells, CD34 + /CD38 - leukemic stem cells (LSC), and in the CML cell lines KU812, K562, KCL22, and KCL22 T315I . The BRD4-targeting drug JQ1 was found to suppress proliferation in KU812 cells and primary leukemic cells in the majority of patients with chronic phase CML. In the blast phase of CML, JQ1 was less effective. However, the BRD4 degrader dBET6 was found to block proliferation and/or survival of primary CML cells in all patients tested, including blast phase CML and CML cells exhibiting the T315I variant of BCR::ABL1. Moreover, dBET6 was found to block MYC expression and to synergize with BCR::ABL1 TKI in inhibiting the proliferation in the JQ1-resistant cell line K562. Furthermore, BRD4 degradation was found to overcome osteoblast-induced TKI resistance of CML LSC in a co-culture system and to block interferon-gamma-induced upregulation of the checkpoint antigen PD-L1 in LSC. Finally, dBET6 was found to suppress the in vitro survival of CML LSC and their engraftment in NSG mice. Together, targeting of BRD4 and MYC through BET degradation sensitizes CML cells against BCR::ABL1 TKI and is a potent approach to overcome multiple forms of drug resistance in CML LSC., (© 2022 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

In most patients with advanced systemic mastocytosis (AdvSM), neoplastic mast cells (MC) express KIT D816V. However, despite their disease-modifying potential, KIT D816V-targeting drugs, including midostaurin and avapritinib, may not produce long-term remissions in all patients. Cyclin-dependent kinase (CDK) 4 and CDK6 are promising targets in oncology. We found that shRNA-mediated knockdown of CDK4 and CDK6 results in growth arrest in the KIT D816V + MC line HMC-1.2. The CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib suppressed the proliferation in primary neoplastic MC as well as in all HMC-1 and ROSA cell subclones that were examined. Abemaciclib was also found to block growth in the drug-resistant MC line MCPV-1, whereas no effects were seen with palbociclib and ribociclib. Anti-proliferative drug effects on MC were accompanied by cell cycle arrest. Furthermore, CDK4/CDK6 inhibitors were found to synergize with the KIT-targeting drugs midostaurin, avapritinib, and nintedanib in inducing growth inhibition and apoptosis in neoplastic MCs. Finally, we found that CDK4/CDK6 inhibitors induce apoptosis in CD34+/CD38- stem cells in AdvSM. Together, CDK4/CDK6 inhibition is a potent approach to suppress the growth of neoplastic cells in AdvSM. Whether CDK4/CDK6 inhibitors can improve clinical outcomes in patients with AdvSM remains to be determined in clinical trials.

During progression of myeloid neoplasms, the basophil compartment may expand substantially and in some of these patients, a basophilic leukemia is diagnosed. In patients with Ph-chromosome+ chronic myeloid leukemia, acceleration of disease is typically accompanied by marked basophilia. In other myeloid neoplasms, secondary leukemic expansion of basophils is rarely seen. We report on 5 patients who suffered from a myelodysplastic syndrome, myeloproliferative neoplasm, or acute leukemia and developed a massive expansion of basophils during disease progression. In 4 of 5 patients, peripheral blood basophil counts reached 40%, and the diagnosis "secondary basophilic leukemia" was established. As assessed by flow cytometry, neoplastic basophils expressed CD9, CD18, CD25, CD33, CD63, PD-L1, CD123, and CLL-1. In addition, basophils were found to display BB1 (basogranulin), 2D7, tryptase and KIT. In 4 of 5 patients the disease progressed quickly and treatment with azacitidine was started. However, azacitidine did not induce major clinical responses, and all patients died from progressive disease within 3 Y. In in vitro experiments, the patients´ cells and the basophilic leukemia cell line KU812 showed variable responses to targeted drugs, including azacitidine, venetoclax, hydroxyurea, and cytarabine. A combination of venetoclax and azacitidine induced cooperative antineoplastic effects in these cells. Together, secondary basophilic leukemia has a poor prognosis and monotherapy with azacitidine is not sufficient to keep the disease under control for longer time-periods. Whether drug combination, such as venetoclax+azacitidine, can induce better outcomes in these patients remains to be determined in future clinical studies., (Copyright © 2021. Published by Elsevier Inc.)

Chronic myelomonocytic leukemia (CMML) is a stem cell-derived neoplasm characterized by dysplasia, uncontrolled expansion of monocytes, and substantial risk to transform to secondary acute myeloid leukemia (sAML). So far, little is known about CMML-initiating cells. We found that leukemic stem cells (LSC) in CMML reside in a CD34 + /CD38 - fraction of the malignant clone. Whereas CD34 + /CD38 - cells engrafted NSGS mice with overt CMML, no CMML was produced by CD34 + /CD38 + progenitors or the bulk of CD34 - monocytes. CMML LSC invariably expressed CD33, CD117, CD123 and CD133. In a subset of patients, CMML LSC also displayed CD52, IL-1RAP and/or CLL-1. CMML LSC did not express CD25 or CD26. However, in sAML following CMML, the LSC also expressed CD25 and high levels of CD114, CD123 and IL-1RAP. No correlations between LSC phenotypes, CMML-variant, mutation-profiles, or clinical course were identified. Pre-incubation of CMML LSC with gemtuzumab-ozogamicin or venetoclax resulted in decreased growth and impaired engraftment in NSGS mice. Together, CMML LSC are CD34 + /CD38 - cells that express a distinct profile of surface markers and target-antigens. During progression to sAML, LSC acquire or upregulate certain cytokine receptors, including CD25, CD114 and CD123. Characterization of CMML LSC should facilitate their enrichment and the development of LSC-eradicating therapies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Ponatinib is a tyrosine kinase inhibitor (TKI) directed against BCR-ABL1 which is successfully used in patients with BCR-ABL1 T315I + chronic myeloid leukemia (CML). However, BCR-ABL1 compound mutations may develop during therapy in these patients and may lead to drug resistance. Asciminib is a novel drug capable of targeting most BCR-ABL1 mutant-forms, including BCR-ABL1 T315I , but remains ineffective against most BCR-ABL1 T315I + compound mutation-bearing sub-clones. We demonstrate that asciminib synergizes with ponatinib in inducing growth-arrest and apoptosis in patient-derived CML cell lines and murine Ba/F3 cells harboring BCR-ABL1 T315I or T315I-including compound mutations. Asciminib and ponatinib also produced cooperative effects on CRKL phosphorylation in BCR-ABL1-transformed cells. The growth-inhibitory effects of the drug combination 'asciminib+ponatinib' was further enhanced by hydroxyurea (HU), a drug which has lately been described to suppresses the proliferation of BCR-ABL1 T315I + CML cells. Cooperative drug effects were also observed in patient-derived CML cells. Most importantly, we were able to show that the combinations 'asciminib+ponatinib' and 'asciminib+ponatinib+HU' produce synergistic apoptosis-inducing effects in CD34 + /CD38 - CML stem cells obtained from patients with chronic phase CML or BCR-ABL1 T315I + CML blast phase. Together, asciminib, ponatinib and HU synergize in producing anti-leukemic effects in multi-resistant CML cells, including cells harboring T315I+ BCR-ABL1 compound mutations and CML stem cells. The clinical efficacy of this TKI combination needs to be evaluated within the frame of upcoming clinical trials., Competing Interests: K.V.G.: honoraria from Novartis, Incyte, BMS, Pfizer and AbbVie. K.B.: employment at Oncopeptides AB. T.L.: honoraria from Incyte, Pfizer, Angelini, Novartis, Bristol-Myers Squibb, and Amgen; research grants from Incyte and Novartis. J.M.: research grant and travel support from Novartis. W.R.S.: honoraria from AbbVie, Amgen, Astellas, Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Lipomed, Novartis, Pfizer und Thermo Fisher. P.V.: research funding and honoraria from Novartis and Incyte, and honoraria from BMS/Celgene, Blueprint, Pfizer, AOP Orphan. The other authors (Y.F., D.B., C.S., I.S., L.D.-S., G.E., M.S.-G.) have not disclosed conflicts of interest., (AJCR Copyright © 2021.)

Canine mastocytomas (MCTs) are characterized by rapid proliferation of neoplastic mast cells (MCs) and clinical signs caused by MC-derived mediators. In dogs suffering from MCT, histamine receptor 1 (HR1) antagonists are frequently used to control mediator-related clinical symptoms. Previous studies have shown that the HR1 antagonists loratadine and terfenadine exert some growth-inhibitory effects on neoplastic MCs. We examined whether other HR1 antagonists used in clinical practice (desloratadine, rupatadine, cyproheptadine, dimetindene, diphenhydramine) affect proliferation and survival of neoplastic MCs. Furthermore, we analysed whether these HR1 antagonists counteract IgE-dependent histamine release from a MC line harbouring a functional IgE-receptor. HR1 antagonists were applied on two canine MC lines, C2 and NI-1, and on primary MCs obtained from three MCT samples. The HR1 antagonists desloratadine, rupatadine and cyproheptadine were found to be more potent in decreasing proliferation of C2 and NI-1 cells when compared with dimetindene and diphenhydramine. Similar effects were seen in primary neoplastic MCs, except for diphenhydramine, which exerted more potent growth-inhibitory effects than the other HR1 antagonists. Drug-induced growth-inhibition in C2 and NI-1 cells was accompanied by apoptosis. Loratadine, desloratadine and rupatadine also suppressed IgE-dependent histamine release in NI-1 cells. However, drug concentrations required to elicit substantial effects on growth or histamine release were relatively high (>10 µM). Therefore, it remains unknown whether these drugs or similar, more potent, HR1-targeting drugs can suppress growth or activation of canine neoplastic MCs in vivo., (© 2020 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

In an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38- and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38- and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38- LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38- cells variably expressed "aberrant" membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication-mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38- LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies., (© 2020 by The American Society of Hematology.)

Purpose: Ponatinib is the only approved tyrosine kinase inhibitor (TKI) suppressing BCR-ABL1 T315I -mutated cells in chronic myeloid leukemia (CML). However, due to side effects and resistance, BCR-ABL1 T315I -mutated CML remains a clinical challenge. Hydroxyurea (HU) has been used for cytoreduction in CML for decades. We found that HU suppresses or even eliminates BCR-ABL1 T315I + sub-clones in heavily pretreated CML patients. Based on this observation, we investigated the effects of HU on TKI-resistant CML cells in vitro., Methods: Viability, apoptosis and proliferation of drug-exposed primary CML cells and BCR-ABL1+ cell lines were examined by flow cytometry and 3 H-thymidine-uptake. Expression of drug targets was analyzed by qPCR and Western blotting., Findings: HU was more effective in inhibiting the proliferation of leukemic cells harboring BCR-ABL1 T315I or T315I-including compound-mutations compared to cells expressing wildtype BCR-ABL1. Moreover, HU synergized with ponatinib and ABL001 in inducing growth inhibition in CML cells. Furthermore, HU blocked cell cycle progression in leukemic cells, which was accompanied by decreased expression of CDK4 and CDK6. Palbociclib, a more specific CDK4/CDK6-inhibitor, was also found to suppress proliferation in primary CML cells and to synergize with ponatinib in producing growth inhibition in BCR-ABL1 T315I + cells, suggesting that suppression of CDK4/CDK6 may be a promising concept to overcome BCR-ABL1 T315I -associated TKI resistance., Interpretation: HU and the CDK4/CDK6-blocker palbociclib inhibit growth of CML clones expressing BCR-ABL1 T315I or complex T315I-including compound-mutations. Clinical studies are required to confirm single drug effects and the efficacy of `ponatinib+HU´ and ´ponatinib+palbociclib´ combinations in advanced CML., Funding: This project was supported by the Austrian Science Funds (FWF) projects F4701-B20, F4704-B20 and P30625., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE-dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI-1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC 50 values ranging between 0.1 and 1 μM in primary MCT cells and between 1 and 3 μM in C2 and NI-1 cells. In C2 cells, the combination "ibrutinib + midostaurin" produced synergistic growth-inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE-dependent histamine release in primary MCT cells, with IC 50 values ranging from 0.05 to 0.1 μM in NI-1 cells, and from 0.05 to 1 μM in primary MCT cells. In summary, ibrutinib exerts anti-proliferative effects in canine neoplastic MCs and counteracts IgE-dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials., (© 2019 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Mast cell leukemia (MCL) is a highly fatal malignancy characterized by devastating expansion of immature mast cells in various organs. Although considered a stem cell disease, little is known about MCL-propagating neoplastic stem cells. We here describe that leukemic stem cells (LSCs) in MCL reside within a CD34 + /CD38 - fraction of the clone. Whereas highly purified CD34 + /CD38 ─ cells engrafted NSG hSCF mice with fully manifesting MCL, no MCL was produced by CD34 + /CD38 + progenitors or the bulk of KIT + /CD34 - mast cells. CD34 + /CD38 - MCL cells invariably expressed CD13 and CD133, and often also IL-1RAP, but did not express CD25, CD26 or CLL-1. CD34 + /CD38 - MCL cells also displayed several surface targets, including CD33, which was homogenously expressed on MCL LSCs in all cases, and the D816V mutant form of KIT. Although CD34 + /CD38 - cells were resistant against single drugs, exposure to combinations of CD33-targeting and KIT-targeting drugs resulted in LSC-depletion and markedly reduced engraftment in NSG hSCF mice. Together, MCL LSCs are CD34 + /CD38 - cells that express distinct profiles of markers and target antigens. Characterization of MCL LSCs should facilitate their purification and should support the development of LSC-eradicating curative treatment approaches in this fatal type of leukemia.

In chronic myeloid leukemia (CML), resistance against second-generation tyrosine kinase inhibitors (TKI) remains a serious clinical challenge, especially in the context of multi-resistant BCR-ABL1 mutants, such as T315I. Treatment with ponatinib may suppress most of these mutants, including T315I, but is also associated with a high risk of clinically relevant side effects. We screened for alternative treatment options employing available tyrosine kinase inhibitors (TKI) in combination. Dasatinib and bosutinib are two second-generation TKI that bind to different, albeit partially overlapping, spectra of kinase targets in CML cells. This observation prompted us to explore anti-leukemic effects of the combination dasatinib + bosutinib in highly resistant primary CML cells, various CML cell lines (K562, K562R, KU812, KCL22) and Ba/F3 cells harboring various BCR-ABL1 mutant-forms. We found that bosutinib synergizes with dasatinib in inducing growth inhibition and apoptosis in all CML cell lines and in Ba/F3 cells exhibiting BCR-ABL1 T315I . Clear synergistic effects were also observed in primary CML cells in all patients tested (n = 20), including drug-resistant cells carrying BCR-ABL1 T315I . Moreover, the drug combination produced cooperative or even synergistic apoptosis-inducing effects on CD34 + /CD38 - CML stem cells. Finally, we found that the drug combination is a potent approach to block the activity of major additional CML targets, including LYN, KIT and PDGFRα. Together, bosutinib and dasatinib synergize in producing anti-leukemic effects in drug-resistant CML cells. Whether such cooperative TKI effects also occur in vivo in patients with drug-resistant CML, remains to be determined in forthcoming studies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and invasion of neoplastic stem cells in various myeloid malignancies. Although mast cells (MCs) reportedly express CD44, little is known about the regulation and function of this receptor in neoplastic cells in systemic mastocytosis (SM). We found that clonal CD34 + /CD38 - stem cells, CD34 + /CD38 + progenitor cells, and CD117 ++ /CD34 - MCs invariably express CD44 in patients with indolent SM (ISM), SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia (MCL). In addition, all human MCL-like cell lines examined (HMC-1, ROSA, and MCPV-1) displayed cytoplasmic and cell-surface CD44. We also found that expression of CD44 in neoplastic MCs depends on RAS-MEK and STAT5 signaling and increases with the aggressiveness of SM. Correspondingly, higher levels of soluble CD44 were measured in the sera of patients with advanced SM compared with ISM or cutaneous mastocytosis and were found to correlate with overall and progression-free survival. To investigate the functional role of CD44, a xenotransplantation model was employed using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and a short hairpin RNA (shRNA) against CD44. In this model, the shRNA-mediated knockdown of CD44 resulted in reduced MC expansion and tumor formation and prolonged survival in SCID mice compared with HMC-1.2 cells transduced with control shRNA. Together, our data show that CD44 is a RAS-MEK/STAT5-driven MC invasion receptor that correlates with the aggressiveness of SM. Whether CD44 can serve as therapeutic target in advanced SM remains to be determined in forthcoming studies., (© 2018 by The American Society of Hematology.)

Leukemic stem cells (LSCs) are an emerging target of curative anti-leukemia therapy. In acute lymphoblastic leukemia (ALL), LSCs frequently express CD34 and often lack CD38. However, little is known about markers and targets expressed in ALL LSCs. We have examined marker- and target expression profiles in CD34 + /CD38 - LSCs in patients with Ph + ALL (n = 22) and Ph - ALL (n = 27) by multi-color flow cytometry and qPCR. ALL LSCs expressed CD19 (B4), CD44 (Pgp-1), CD123 (IL-3RA), and CD184 (CXCR4) in all patients tested. Moreover, in various subgroups of patients, LSCs also displayed CD20 (MS4A1) (10/41 = 24%), CD22 (12/20 = 60%), CD33 (Siglec-3) (20/48 = 42%), CD52 (CAMPATH-1) (17/40 = 43%), IL-1RAP (13/29 = 45%), and/or CD135 (FLT3) (4/20 = 20%). CD25 (IL-2RA) and CD26 (DPPIV) were expressed on LSCs in Ph + ALL exhibiting BCR/ABL1 p210 , whereas in Ph + ALL with BCR/ABL1 p190 , LSCs variably expressed CD25 but did not express CD26. In Ph - ALL, CD34 + /CD38 - LSCs expressed IL-1RAP in 6/18 patients (33%), but did not express CD25 or CD26. Normal stem cells stained negative for CD25, CD26 and IL-1RAP, and expressed only low amounts of CD52. In xenotransplantation experiments, CD34 + /CD38 - and CD34 + /CD38 + cells engrafted NSG mice after 12-20 weeks, and targeting with antibodies against CD33 and CD52 resulted in reduced engraftment. Together, LSCs in Ph + and Ph - ALL display unique marker- and target expression profiles. In Ph + ALL with BCR/ABL1 p210 , the LSC-phenotype closely resembles the marker-profile of CD34 + /CD38 - LSCs in chronic myeloid leukemia, confirming the close biologic relationship of these neoplasms. Targeting of LSCs with specific antibodies or related immunotherapies may facilitate LSC eradication in ALL., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Systemic mastocytosis is a complex disease defined by abnormal growth and accumulation of neoplastic mast cells in various organs. Most patients exhibit a D816V-mutated variant of KIT , which confers resistance against imatinib. Clinical problems in systemic mastocytosis arise from mediator-related symptoms and/or organ destruction caused by malignant expansion of neoplastic mast cells and/or other myeloid cells in various organ systems. DCC-2618 is a spectrum-selective pan KIT and PDGFRA inhibitor which blocks KIT D816V and multiple other kinase targets relevant to systemic mastocytosis. We found that DCC-2618 inhibits the proliferation and survival of various human mast cell lines (HMC-1, ROSA, MCPV-1) as well as primary neoplastic mast cells obtained from patients with advanced systemic mastocytosis (IC 50 <1 μM). Moreover, DCC-2618 decreased growth and survival of primary neoplastic eosinophils obtained from patients with systemic mastocytosis or eosinophilic leukemia, leukemic monocytes obtained from patients with chronic myelomonocytic leukemia with or without concomitant systemic mastocytosis, and blast cells obtained from patients with acute myeloid leukemia. Furthermore, DCC-2618 was found to suppress the proliferation of endothelial cells, suggesting additional drug effects on systemic mastocytosis-related angiogenesis. Finally, DCC-2618 was found to downregulate IgE-mediated histamine release from basophils and tryptase release from mast cells. Together, DCC-2618 inhibits growth, survival and activation of multiple cell types relevant to advanced systemic mastocytosis. Whether DCC-2618 is effective in vivo in patients with advanced systemic mastocytosis is currently under investigation in clinical trials., (Copyright © 2018 Ferrata Storti Foundation.)

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although the disease can be kept under control using BCR/ABL1 tyrosine kinase inhibitors (TKIs) in most cases, some patients relapse or have resistant disease, so there is a need to identify new therapeutic targets in this malignancy. Recent data suggest that leukemic SCs (LSCs) in CML display the stem-cell (SC)-mobilizing cell surface enzyme dipeptidyl-peptidase IV (DPPIV = CD26) in an aberrant manner. In the present study, we analyzed the effects of the DPPIV blocker vildagliptin as single agent or in combination with the BCR/ABL1 TKI imatinib or nilotinib on growth and survival of CML LSCs in vitro and on LSC engraftment in an in vivo xenotransplantation nonobese diabetic SCID-IL-2Rγ -/- (NSG) mouse model. We found that nilotinib induces apoptosis in CML LSCs and inhibits their engraftment in NSG mice. In contrast, no substantial effects were seen with imatinib or vildagliptin. Nevertheless, vildagliptin was found to reduce the "mobilization" of CML LSCs from a stroma cell layer consisting of mouse fibroblasts in an in vitro co-culture model, suggesting reduced disease expansion. However, although vildagliptin and nilotinib produced cooperative effects in individual experiments, overall, no significant effects of coadministered vildagliptin over nilotinib or imatinib treatment alone were seen on the engraftment of CML cells in NSG mice. Gliptins may be interesting drugs in the context of CML and nilotinib therapy, but our preclinical studies did not reveal a major cooperative effect of the drug-combination vildagliptin + nilotinib on engraftment of CML cells in NSG mice., (Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Acute lymphoblastic leukemia (ALL) is characterized by leukemic expansion of lymphoid blasts in hematopoietic tissues. Despite improved therapy only a subset of patients can be cured. Therefore, current research is focusing on new drug-targets. Members of the BCL-2 family and components of the PI3-kinase/mTOR pathway are critically involved in the regulation of growth and survival of ALL cells. We examined the effects of the pan-BCL-2 blocker obatoclax and the PI3-kinase/mTOR-inhibitor BEZ235 on growth and survival of ALL cells. In 3 H-thymidine uptake experiments, both drugs suppressed the in vitro proliferation of leukemic cells in all patients with Philadelphia chromosome-positive (Ph + ) ALL and Ph - ALL (obatoclax IC 50 : 0.01-5 μM; BEZ235, IC 50 : 0.01-1 μM). Both drugs were also found to produce growth-inhibitory effects in all Ph + and all Ph - cell lines tested. Moreover, obatoclax and BEZ235 induced apoptosis in ALL cells. In drug-combination experiments, obatoclax and BEZ235 exerted synergistic growth-inhibitory effects on ALL cells. Finally, we confirmed that ALL cells, including CD34 + /CD38 - stem cells and all cell lines express transcripts for PI3-kinase, mTOR, BCL-2, MCL-1, and BCL-xL. Taken together, this data shows that combined targeting of the PI3-kinase/mTOR-pathway and BCL-2 family-members is a potent approach to counteract growth and survival of ALL cells., Competing Interests: CONFLICTS OF INTEREST The authors of this manuscript have the following conflicts of interest to declare: P.V. received honoraria from Novartis and Ariad. W.R.S. received a research grant from Amgen and honoraria from Novartis, Amgen, and Ariad. G.H. received research funding from Gilead and honoraria from Novartis, Ariad, and Amgen. U.J. received honoraria from Novartis and Abbvie. A.W.H. received honoraria from Amgen, Ariad and Jazz Pharmaceuticals.

Multiple myeloma (MM) is a malignancy characterized by monoclonal paraproteinemia and tissue plasmocytosis. In advanced MM cytopenia and osteopathy may occur. Although several effective treatment strategies have been developed in recent years, there is still a need to identify new drug targets and to develop more effective therapies for patients with advanced MM. We examined the effects of 15 targeted drugs on growth and survival of primary MM cells and 5 MM cell lines (MM.1S, NCI-H929, OPM-2, RPMI-8226, U-266). The PI3-kinase blocker BEZ235, the pan-BCL-2 inhibitor obatoclax, the Hsp90-targeting drug 17AAG, and the Polo-like kinase-1 inhibitor BI2536, were found to exert major growth-inhibitory effects in all 5 MM cell lines tested. Moreover, these drugs suppressed the in vitro proliferation of primary bone marrow-derived MM cells and induced apoptosis at pharmacologic drug concentrations. Apoptosis-inducing effects were not only seen in the bulk of MM cells but also in MM stem cell-containing CD138-/CD20+/CD27+ memory B-cell fractions. Synergistic growth-inhibitory effects were observed in MM cell lines using various drug combinations, including 17AAG+BI2536 in MM.1S, OPM-2, RPMI-8226, and U-266 cells, 17AAG+BEZ235 in MM.1S, OPM-2, RPMI-8226, and U-266 cells, 17AAG+obatoclax in MM.1S, NCI-H929, OPM-2, and RPMI-8226 cells, BI2536+BEZ235 in MM.1S, NCI-H929, OPM-2, and RPMI-8226 cells, BI2536+obatoclax in MM.1S, OPM-2 and RPMI-8226 cells, and BEZ235+obatoclax in MM.1S and RPMI-8226 cells. Together, our data show that various targeted drugs induce profound and often synergistic anti-neoplastic effects in MM cells which may have clinical implications and may contribute to the development of novel treatment strategies in advanced MM.

Histamine dihydrochloride (HDC) plus IL-2 has been proposed as a novel maintenance-immunotherapy in acute myeloid leukemia (AML). We analyzed the immunophenotype and function of natural killer (NK) cells in blood of AML patients treated after chemotherapy with HDC plus IL-2. The treatment caused a striking expansion of CD56brightCD16neg and CD56brightCD16low NK cell subpopulations. A reduced NK cell fraction recovered and high proportions of cells expressed the activating receptors NKG2D, NKp30, and NKp46. Concomitantly, KIR-expressing NK cells were reduced and NK cells with inhibitory NKG2A/CD94 receptors increased beyond normal levels. In addition, the immunotherapy-induced NK cells exhibited high capacity to produce IFN-γ and to degranulate. Furthermore, we provide evidence from subsequent in vitro studies that this is caused in part by direct effects of IL-2 on the CD56bright cells. IL-2 specifically induced proliferation of both CD56bright subpopulations, but not of CD56dim cells. It further preserved the expression of activating receptors and the capacity to produce IFN-γ and to degranulate. These data suggest that therapy with HDC plus IL-2 supports the reconstitution of a deficient NK cell fraction through the specific amplification of CD56bright NK cells giving rise to a functional NK cell compartment with high potential to combat leukemic disease., Competing Interests: Research grants for unrelated work within the last three years were received by P.V. from Novartis and Ariad and by W.R.S. from Meda and Thermo Fisher. Personal honoraria were obtained by P.V. from Pfizer, Ariad, Celgene and by W.R.S. from Novartis, Ariad, Celgene, Meda and Amgen. All other authors did not receive any support from commercial entities and declare no conflicts of interest.

Purpose: In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs., Experimental Design: CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812., Results: In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib., Conclusions: CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML., (©2015 American Association for Cancer Research.)

The Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin lymphoma and anaplastic large-cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MCs) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MCs in advanced SM, and whether this surface structure may serve as therapeutic target in SM. As assessed by flow cytometry, CD30 was found to be expressed on the surface of neoplastic MCs in 3 of 25 patients (12%) with indolent SM, 4 of 7 patients (57%) with aggressive SM, and 4 of 7 patients (57%) with MC leukemia. The immature RAS-transformed human MC line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed MC line HMC-1.2 expressed no detectable CD30. The CD30-targeting antibody-conjugate brentuximab-vedotin inhibited proliferation in neoplastic MCs, with lower IC50 values obtained in CD30(+) MCPV-1.1 cells (10 µg/mL) compared with CD30(-) HMC-1.2 cells (>50 µg/mL). In addition, brentuximab-vedotin suppressed the engraftment of MCPV-1.1 cells in NSG mice. Moreover, brentuximab-vedotin produced apoptosis in all CD30(+) MC lines tested as well as in primary neoplastic MCs in patients with CD30(+) SM, but did not induce apoptosis in neoplastic MCs in patients with CD30(-) SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE-induced histamine release in CD30(+) MCs. Finally, brentuximab-vedotin and the KIT D816V-targeting drug PKC412 produced synergistic growth-inhibitory effects in MCPV-1.1 cells. Together, CD30 is a promising new drug target for patients with CD30(+) advanced SM., (© 2015 by The American Society of Hematology.)

Although mast cells (MC) play an important role in allergic reactions, their physiologic role remains unknown. In mice, several models of MC-deficiency have been developed. However, no comparable human model is available. We examined the in vitro- and in vivo effects of the KIT-targeting drug imatinib on growth and development of human MC. Imatinib was found to inhibit stem cell factor (SCF)-induced differentiation of MC in long-term suspension cultures (IC50: 0.01 µM). Correspondingly, long-term treatment of chronic myeloid leukemia (CML) patients with imatinib (400 mg/day) resulted in a marked decrease in MC. In patients with continuous complete molecular response during therapy, bone marrow MC decreased to less than 5% of pre-treatment values, and also serum tryptase concentrations decreased significantly (pre-treatment: 32.0 ± 11.1 ng/ml; post-therapy: 3.4 ± 1.8, p<0.01). Other myeloid lineages, known to develop independently of KIT, were not affected by imatinib-therapy. Imatinib also produced a substantial decrease in MC-development in mice. However, no clinical syndrome attributable to drug-induced MC-deficiency was recorded in our CML patients. Together, imatinib suppresses MC production in vitro and in vivo. However, drug-induced MC depletion is not accompanied by adverse clinical events, suggesting that MC are less relevant to homeostasis in healthy tissues than we assumed so far.

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34(+)/CD38(─) CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1(+) cells, whereas CD26(─) SC from the same patients produced multilineage BCR/ABL1(-) engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1(+) cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy., (© 2014 by The American Society of Hematology.)

In chronic eosinophilic leukemia, the transforming oncoprotein FIP1L1-PDGFRA is a major target of therapy. In most patients, the tyrosine kinase inhibitor (TKI) imatinib induces complete remission. For patients who are intolerant or resistant, novel TKIs have been proposed. We examined the in vitro effects of 14 kinase blockers on growth and function of EOL-1 cells, a FIP1L1-PDGFRA(+) eosinophil cell line. Major growth-inhibitory effects were seen with all PDGFR-blocking agents, with IC50 values in the low nanomolar range: ponatinib, 0.1-0.2 nmol/L; sorafenib, 0.1-0.2 nmol/L; masitinib, 0.2-0.5 nmol/L; nilotinib, 0.2-1.0 nmol/L; dasatinib, 0.5-2.0 nmol/L; sunitinib, 1-2 nmol/L; midostaurin, 5-10 nmol/L. These drugs were also found to block activation of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. All effective TKIs produced apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI, and caspase-3 staining. In addition, PDGFR-targeting TKIs were found to inhibit cytokine-induced migration of EOL-1 cells. In all bioassays used, ponatinib was found to be the most potent compound in EOL-1 cells. In addition, ponatinib was found to downregulate expression of the activation-linked surface antigen CD63 on EOL-1 cells and to suppress the growth of primary neoplastic eosinophils. We also examined drug effects on Ba/F3 cells expressing two clinically relevant, imatinib-resistant, mutant forms of FIP1L1-PDGFRA, namely T674I and D842V. Strong inhibitory effects on both mutants were seen only with ponatinib. In summary, novel PDGFR-targeting TKIs may be alternative agents for the treatment of patients with imatinib-resistant chronic eosinophilic leukemia. Although several different PDGFR-targeting agents are effective, the most potent drug appears to be ponatinib., (Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in oncology. We have shown that Hsp32, also known as heme oxygenase-1 (HO-1), serves as survival factor and potential target in Ph+ chronic myeloid leukemia. We here report that primary cells and cell lines derived from patients with acute lymphoblastic leukemia (ALL) express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. Highly enriched CD34+/CD38- ALL stem cells also expressed Hsp32. Two Hsp32-targeting drugs, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), induced apoptosis and growth arrest in the BCR/ABL1+ cell lines, in Ph- lymphoblastic cell lines and in primary Ph+ and Ph- ALL cells. The effects of PEG-ZnPP and SMA-ZnPP on growth of leukemic cells were dose-dependent. In Ph+ ALL, major growth-inhibitory effects of the Hsp32-targeting drugs were observed in imatinib-sensitive and imatinib-resistant cells. Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells. A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells. In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

Advanced SM is an incurable neoplasm with short survival time. So far, no effective therapy is available for these patients. We and others have shown recently that neoplastic MC in ASM and MCL express antiapoptotic Mcl-1, Bcl-2, and Bcl-xL. In this study, we examined the effects of the pan-Bcl-2 family blocker obatoclax (GX015-070) on primary neoplastic MC, the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Obatoclax was found to inhibit proliferation in primary human neoplastic MC (IC₅₀: 0.057 μM), in HMC-1.2 cells expressing KIT D816V (IC₅₀: 0.72 μM), and in HMC-1.1 cells lacking KIT D816V (IC₅₀: 0.09 μM), as well as in C2 cells (IC₅₀: 0.74 μM). The growth-inhibitory effects of obatoclax in HMC-1 cells were accompanied by an increase in expression of Puma, Noxa, and Bim mRNA, as well as by apoptosis, as evidenced by microscopy, TUNEL assay, and caspase cleavage. Viral-mediated overexpression of Mcl-1, Bcl-xL, or Bcl-2 in HMC-1 cells was found to introduce partial resistance against apoptosis-inducing effects of obatoclax. We were also able to show that obatoclax synergizes with several other antineoplastic drugs, including dasatinib, midostaurin, and bortezomib, in producing apoptosis and/or growth arrest in neoplastic MC. Together, obatoclax exerts major growth-inhibitory effects on neoplastic MC and potentiates the antineoplastic activity of other targeted drugs. Whether these drug effects can be translated to application in patients with advanced SM remains to be determined.

Resveratrol is a naturally occurring anticancer compound present in grapes and wine that undergoes pronounced metabolism in human intestine and liver. In order to determine whether resveratrol is also bio-transformed in human breast carcinoma, metabolism experiments were conducted in breast tumor and adjacent non-tumorous specimens from 13 patients. Resveratrol was metabolized in cytosolic tissue fractions to resveratrol-3-O-sulfate: the formation rates were up to 33.5-fold higher in cancer samples than in peritumoral tissue. Further quantitative real-time RT-PCR analysis revealed similar expression of sulfotransferases SULT1A2, 1A3, and 1E1 in the paired control and tumor tissues. Sulfotransferase SULT1A1 expression was below the detection limit in all samples. Interestingly, mRNA expression of steroid sulfatase STS, but not of arylsulfatases ARS-A and ARS-B, was significantly higher (p<0.0017) in non-malignant specimens than in tumor tissue samples, which might explain the higher resveratrol-3-O-sulfate concentrations in breast cancer specimens. Cellular localization of SULT1A3 and STS was also assessed by indirect immunofluorescence on paraffin-embedded sections from control and malignant breast tissue clearly showing a correlation of qRT-PCR data with protein expression of these two enzymes. Our data elucidate the metabolism of resveratrol in malignant and non-malignant breast tissue, which must be considered in humans after oral uptake of dietary resveratrol as a chemopreventive agent., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)