The Novel Immunoproteasome Inhibitor PR-924: Anti-Leukemic Activity and Mechanisms Of Resistance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3841-3841
Author(s):  
Denise Niewerth ◽  
Johan van Meerloo ◽  
Yehuda Assaraf ◽  
Gerrit Jansen ◽  
Tessa C Hendrickx ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Line ◽  
Hematological Malignancies ◽  
Acquired Resistance ◽  
Proteasome Inhibition ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Gene Encoding ◽  
Exon 2 ◽  
Subunit Expression

Abstract Proteasome inhibition with bortezomib (BTZ) is an effective treatment for hematological malignancies and an emerging treatment strategy for acute leukemia. However, the side-effects of BTZ and emergence of BTZ-resistance call for alternative therapeutic approaches. The immunoproteasome may serve as a candidate since its expression is higher than constitutive proteasome expression in cells of hematological malignancies, including acute lymphocytic leukemia (ALL). To this end, the new immunoproteasome inhibitor PR-924 may elicit anti-leukemic activity, since it bears greater specificity for the β5i subunit of the immunoproteasome compared to other proteasome inhibitors including BTZ and carfilzomib. The aim of the current study was to examine the proteasome inhibition capacity and cell growth inhibitory impact of PR-924 in acute leukemia cells and sublines with acquired resistance to BTZ. We further determined whether PR-924 itself would be prone to resistance development and if so, to explore the underlying molecular basis. To assess the anti-leukemic activity of PR-924, its cytotoxicity was determined in two human cell lines of hematological origin: the T-ALL cell line CCRF-CEM and the acute myeloid leukemia cell line THP1, and their 150-fold BTZ-resistant sublines CEM/BTZ200, and THP1/BTZ200, respectively. Parental CEM and THP1 cells displayed similar sensitivity to PR-924 (IC50 CEM: 1.8 µM ± 0.4, THP1: 1.5 µM ± 0.2), whereas their BTZ-resistant lines displayed a moderate 10-12 fold cross-resistance to PR-924 (IC50 CEM/BTZ200: 21.1 µM ± 0.5, THP1/BTZ200: 15.6 µM ± 1.6). Flow cytometric analysis revealed that PR-924-induced cell death was mediated by induction of apoptosis. Moreover, PR-924 exposure resulted in a (up to 50%) reduction of cell surface expression of HLA Class I. To determine whether PR-924 activity relies on specific inhibition of β5i, proteasome activity inhibition experiments were performed over a range of 10 nM – 10 µM PR-924. Notably, in parental cells, β5i activity was already inhibited by 20% at 10 nM PR-924 and > 90% inhibition was achieved at 100 nM PR-924. At higher PR-924 concentrations of 1-10 µM, inhibition of both β5 and β1i activities was observed, thus indicating that PR-924 blocks the β5i activity far below concentrations that exert anti-proliferative activity (1.5 µM), consistent with earlier studies of PR-924 (Parlati et al. Blood, 2009). Next, acquired resistance to PR-924 was provoked in CEM and THP1 cells by step-wise increasing PR-924 concentrations in cell culture, starting at the IC50 concentrations. Following this process, CEM cells resistant to 20 µM PR-924 (CEM/PR20) and THP1 cells resistant to 12 mM PR-924 (THP1/PR12) exhibited IC50 values of 22.1 µM PR-924 (resistance factor 13) and 14.3 µM PR-924 (resistance factor 10), respectively. In addition, these PR-924 resistant cells displayed 10-fold cross-resistance to BTZ. To explore mechanisms of PR-924 resistance, we first sequenced exon 2/3 of the PSMB8 gene (encoding β5i), a similar functioning coding region that was previously reported to harbor PSMB5 mutations in BTZ-resistant leukemia cells (Franke et al. Leukemia, 2012). However, no mutations were found in PSMB8 exon 2/3. Remarkably, we did identify mutations in exon 2 of the PSMB5 gene (encoding the S1 pocket of β5) in both CEM/PR20 (Met45Ile) and of THP1/PR12 (Ala49Thr); these amino acid substitutions are similar to those observed in BTZ-resistant sublines. Finally, we characterized proteasome subunit expression by ProCISE analysis in the PR-924 resistant cell lines. This method is a subunit-specific active-site ELISA assay, which utilizes a purified proteasome standard curve to calculate ng of subunit per µg total protein. Expression of constitutive proteasome subunits in CEM/PR20 and THP1/PR8 cells were upregulated up to 2.5-fold, compared to their parental counterparts, whereas immunoproteasome subunit expression was moderately decreased (up to 2-fold) in CEM/PR20 and increased in THP1/PR8 (up to 1.6-fold). In conclusion, PR-924 displayed significant anti-leukemic activity. Although there was cross-resistance to BTZ, PR-924 retained activity in BTZ-resistant leukemia cells. Despite PR-924 specificity to the β5i subunit of the proteasome, its anti-leukemic effect requires concentrations that block both β5 and β5i subunits. This notion is underscored by emergence of acquired mutations in PSMB5 rather than in PSMB8. Disclosures: Kirk: Onyx Pharmaceuticals: Employment, Equity Ownership. Anderl:Onyx: Employment.

scholarly journals Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 665
Author(s):  
Margot S.F. Roeten ◽  
Johan van Meerloo ◽  
Zinia J. Kwidama ◽  
Giovanna ter Huizen ◽  
Wouter H. Segerink ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Proteasome Subunit

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

Human Plasmacytoid Dendritic Cell like Leukemia Cell Line (PMDC05) Established from a Patient with CD4+CD56+ Acute Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4456-4456
Author(s):  
Miwako Narita ◽  
Nozomi Tochiki ◽  
Norihiro Watanabe ◽  
Anri Saitoh ◽  
Shigeo Hashimoto ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cell ◽  
Acute Leukemia ◽  
Cell Line ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Type I ◽  
Gm Csf ◽  
Antigen Presenting

Abstract Human dendritic cell precursors are commonly divided into two distinct subsets: myeloid DC and Plasmacytoid DC (pDC). The pDC, which show plasma cell like morphology, have been defined as the population that produce a large amount of type I interferon in response to viruses. The surface phenotypes of human pDCs are defined as CD4+, DC11c−, CD45RA+, IL3Rα (CD123)+, CD1c (BDCA-1)−, CD303 ((BDCA-2)+ and lineage negative. On the other hand, leukemia/lymphoma cells in CD4+CD56+ leukemia/lymphoma have been proposed to be of pDC lineage. CD4+CD56+ pDC leukemia/lymphoma are a rare hematological malignancy, totally only about 100 cases in the world by the literatures. We established a pDC like leukemia cell line (PMDC05) from leukemia cells of a patient with CD4+CD56+ acute leukemia. PMDC05 showed a complex hypoploid chromosomal abnormalities (44, XY) including add(5)(q22), add(15)(q26) and del(15)(q11q15), which is identical to original leukemia cells. Abnormalities including 5q and 15q are reported as the frequent aberrations in CD4+CD56+ pDC leukemia/lymphoma. PMDC05, which morphology was similar to plasma cells, was positive for CD4, CD56, CD123, CD33, CD86, HLA-ABC, HLA-DR, CD1a, CD40, and CD83 but negative for linage markers. Cytokine receptors for GM-CSF, IL3Rα and IL-6Rα were positive on PMDC05. The expression of Trail and Flt-3L was positive. By the culture with IL-3, CPG-A/B, GM-CSF, molecules associated with antigen presentation such as CD1a and CD40 were up-regulated. Besides, the addition of LPS increased the expression of CD40, CD80 and CD83 on PMDC05. PMDC05 by itself possessed a potent antigen presenting ability to naïve T cells and the treatment of PMDC05 with IL-3, CPG-A/B, or GM-CSF enhanced the antigen presenting ability to naïve T cells. TLR7, TLR 8 and TLR 9 as well as TLR1, TLR2, TLR4 were demonstrated to be expressed on PMDC05 by RT-PCR and RQ-PCR showed that the expression of TLR7 and TLR9 was most characteristic. λ-like 14.1 and preTα was also demonstrated to be expressed on PMDC05 by RT/RQ-PCR. PMDC05 possessed an ability to uptake the antigens like FITC-dextran and lucifer yellow. Although IFN-α was not identified to be secreted from PMDC05 by the stimulation of influenza virus, IFN-γ and TNF-α was demonstrated to be secreted to the similar level in pDC, which was examined simultaneously with PMDC05 by CBA assay. These data demonstrated that newly established leukemia cell line PMDC05 is involved in pDC lineage and PMDC05 provides invaluable tools not only for the elucidation of pathophysiology of CD4+CD56+ leukemia/lymphoma but also for the investigation of differntiation and regulation of pDC. In addition, PMDC05 could be applied for generating tumor-specific CTL clone, which may be used for anti-tumor cellular immunotherapy.

Sensitivity of Pediatric Acute Leukemia Cells to Bortezomib and Epoxyketone-Based Proteasome Inhibitors: Correlations with Proteasome Subunit Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1513-1513
Author(s):  
Denise Niewerth ◽  
Niels Franke ◽  
Gerrit Jansen ◽  
Johan van Meerloo ◽  
Yehuda Assaraf ◽  
...  
Keyword(s):  
Second Generation ◽  
Lymphoblastic Leukemia ◽  
Proteasome Inhibitors ◽  
Differential Sensitivity ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Pediatric Leukemia ◽  
Proteasome Subunit ◽  
Subunit Expression ◽  
Proteasome Subunits

Abstract Abstract 1513 Good response to glucocorticoids (GC) has favorable prognostic value for the survival of children with acute lymphoblastic leukemia (ALL). Hence, GC-resistant and relapsed ALL patients may benefit from GC-sensitization strategies. For this purpose, the reversible proteasome inhibitor (PI) Bortezomib (BTZ) is currently being evaluated in clinical trials in combination with Dexamethasone (DEX) and other drugs. Despite the encouraging results of BTZ in several hematological malignancies, emergence of resistance to BTZ may be a limiting factor to its efficacy. Therefore, the aim of our study was to examine the differential sensitivity of pediatric leukemia cells to BTZ and DEX, as compared to second generation PIs designed to overcome BTZ resistance. These include the epoxyketone-based irreversibly binding PIs Carfilzomib (CFZ), its orally bioavailable analog ONX 0912, and the immunoproteasome inhibitor ONX 0914. The drug concentration required for 50% cell death (LC50) was determined in pediatric patient samples (29 ALL and 12 AML) after 4 days drug exposure using the MTT cytotoxicity assay. Furthermore, the sensitivity to PIs was correlated with protein expression levels of the constitutive proteasome subunits beta5, beta1 and beta2, and the (immuno) proteasome subunits beta5i and beta1i. ALL cells were significantly more sensitive for BTZ than AML cells (median LC50: 6.0 nM vs 14.2 nM, respectively, p=0.002), and also markedly more sensitive to Dex (median LC50: 23.0 nM vs. >600 nM, p<0.001). Sensitivity profiles for the PIs CFZ, ONX 0912 and ONX 0914 are presented in Table 1. Collectively, ALL cells were significantly more sensitive than AML cells for all these 3 PIs with irreversible binding properties. LC50 concentrations for CFZ were comparable to those of BTZ. In descending order, ONX 0912 and ONX 0914 displayed lower potencies than BTZ/CFZ, but LC50 concentrations were still in the low nanomolar range.Table 1.Difference in in vitro sensitivity to proteasome inhibitors and proteasome subunit expression between pediatric ALL and AML patientsAcute Lymphoblastic LeukemiaAcute Myeloid LeukemiaANOVA p-valueNMedian LC50 (nM)RangeNMedian LC50 (nM)RangeDrugs    BTZ296.03.0–46.11114.210.1–61.00.002    CFZ274.10.08–8.71020.86.0–30.80.000    ONX 09122719.27.6–80.91093.755.7–3940.000    ONX 09142744.68.4–1171024889.2–6780.000    DEX2723.00.50–>60012600.0164–>6000.000Subunit expressionRatio*Ratio*    beta5280.760.00–30.0106.02.2–23.90.080    beta5i2762.58.5–3661055.010.6–3400.714    beta1282.40.00–28.11011.70.92–26.10.029    beta1i2835.15.42–1061017.77.2–49.50.032    beta2284.80.38–23.41020.47.4–39.10.000    beta2iN.D.N.D.N.D.N.D.N.D.N.D.N.D.N.D.: Not Determined. * Ratio proteasome subunit / β-actin based on loading of 15 ug total protein (Western blot analysis) For ALL, LC50 concentrations for CFZ and ONX 0912 were significantly correlated (r=0.449, p=0.019). Interestingly, for AML, a significant correlation was observed between BTZ and CFZ LC50 concentrations (r=0.900, p=0.001), suggestive for overlapping activities. Expression of constitutive proteasome subunits is higher in AML cells than ALL cells. Within ALL samples, constitutive proteasome subunit expression did not correlate with LC50 concentrations for each of the PIs. Within AML patients, however, beta 5 expression significantly correlated with BTZ LC50 (r=0.980, p<0.001). A trend towards a significant correlation was observed for BTZ LC50 and beta 1 (r=0.550, p=0.125) and beta 2 expression (r=0.500, p=0.17). Next, LC50 concentrations of CFZ correlated significantly with beta 5 (r=0.783, p=0.013) and beta 1 (r=0.817, p=0.007) expression. Finally, both in ALL and AML samples, no correlations were revealed for immunoproteasome subunits expression and LC50 concentrations for BTZ, CFZ, ONX 0912 and ONX 0914. In conclusion, ALL cells were more sensitive to PIs than AML, which may be due lower constitutive proteasome unit expression. Pediatric leukemia cells display marked sensitivity to BTZ and second generation PIs, but lack cross-resistance between BTZ and several second generation PIs. Together, for second generation PIs, these data may hold promise for circumvention of BTZ resistance and further exploration of efficacy assessments in combination with other drugs, in particular GCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Establishment of a FDC-P1 murine cell line with human KIT N822K gene overexpression

Acta Naturae ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 51-55
Author(s):  
Elmira R. Vagapova ◽  
T. D. Lebedev ◽  
V. I. Popenko ◽  
O. G. Leonova ◽  
P. V. Spirin ◽  
...  
Keyword(s):  
Cell Line ◽  
Cellular Response ◽  
Ectopic Expression ◽  
Model Systems ◽  
Leukemia Cells ◽  
Gene Encoding ◽  
Murine Cell Line ◽  
Therapeutic Drugs ◽  
Kit Gene ◽  
Stromal Cell Line

The mechanism of resistance of leukemia cells to chemotherapeutic drugs remains poorly understood. New model systems for studying the processes of malignant transformation of hematopoietic cells are needed. Based on cytokine-dependent murine acute myeloid leukemia (AML) FDC-P1 cells, we generated a new cell line with ectopic expression of the KIT gene encoding mutant human receptor tyrosine kinase (N822K). We investigated the role played by overexpression of the mutant KIT in the survival of leukemia cells and their sensitivity to therapeutic drugs. We also generated a co-culture system consisting of FDC-P1 murine leukemia cells and a HS-5 human stromal cell line. Our data can be used for a further comprehensive analysis of the role of KIT N822K mutation in the cellular response to anti-leukemic drugs, growth factors, and cytokines. These data are of interest in the development of new effective therapeutic approaches to the treatment of acute leukemia.

scholarly journals Marcks Marks Resistance to Proteasome Inhibitors: Exocytosis of Polyubiquitinated Proteins in Bortezomib-Resistant Leukemia Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3712-3712
Author(s):  
Niels Franke ◽  
Gertjan J.L. Kaspers ◽  
Yehuda G Assaraf ◽  
Johan Van Meerloo ◽  
Denise Niewerth ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Acquired Resistance ◽  
Proteasome Inhibitors ◽  
Leukemia Cells ◽  
Advisory Committees ◽  
Resistance Level ◽  
Proteolytic Stress ◽  
Resistant Cells

Abstract Despite the proven efficacy of proteasome inhibitors like bortezomib (BTZ) in multiple myeloma, mantle cell lymphoma, and in an experimental setting in pediatric acute leukemia, development of drug resistance remains a primary hindrance. To further understand the molecular basis underlying this chemoresistance phenomenon, various leukemia cell line models with acquired resistance to BTZ were developed and characterized. One common characteristic was that acquisition of point mutations in PSMB5 and upregulation of the β5-subunit of the proteasome were key determinants of BTZ-resistance in vitro. However, it remains unclear how these drug resistance modalities translate to the overcoming of proteolytic stress imposed by proteasome inhibition. From this perspective, we here undertook a multi-modality (DNA, mRNA, miRNA) array-based analyses of human CCRF-CEM acute leukemia cells and two BTZ-resistant subclones [one with a low resistance level [(10-fold, CEM/BTZ7) and another subline with a high resistance level (140-fold, CEM/BTZ200)] to determine whether or not complementary mechanisms contribute to BTZ resistance. Gene expression profiling studies revealed markedly reduced proteolytic stress induction in drug resistant cells over a broad BTZ concentration range. Moreover, several genes involved in cytoskeleton regulation and vesicle migration were increased in resistant cells. Of all genes, myristoylated alanine-rich C-kinase substrate (MARCKS) was the most differentially overexpressed gene with 25- to 42-fold upregulation in CEM/BTZ7 and CEM/BTZ200 cells, respectively. These observations were corroborated at the protein level and solely included unphosphorylated MARCKS rather than phosphorylated MARCKS, which was marginally expressed in CEM/BTZ cells. Interestingly, MARCKS upregulation was also observed in other BTZ-resistant and leukemia cells (CEM and THP1) with acquired resistance to the proteasome inhibitor salinosporamide A and the immunoproteasome inhibitor PR924. Given the overexpression of MARCKS in proteasome inhibitor-resistant leukemia cells, we further explored whether or not MARCKS overexpression may serve as a predictive marker of BTZ resistance in clinical samples of acute leukemia patients. To this end, we examined primary patient specimens from a phase II childhood refractory/relapsed ALL trial in which BTZ is administered in two intensive re-induction regimens containing vincristine, prednisone, PEG-asparaginase, doxorubicin or cyclophosphamide and etoposide followed by methotrexate treatment. MARCKS expression was demonstrated in 64% of therapy-refractory pediatric leukemia specimens (n=44) wherein higher MARCKS expression trended (p=0.09) towards a dismal response to BTZ-containing chemotherapy. Finally, from a mechanistic perspective, we showed a concentration-dependent association of MARCKS protein with the emergence of ubiquitin-containing vesicles in the resistant cells. This association with MARCKS protein was lost upon exocytosis of these vesicles, which were found to be extruded and taken up in co-cultures with recipient HeLa cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance through vesicular exocytosis of ubiquitinated proteins in BTZ-resistant cells to overcome proteolytic stress over a broad range of cytotoxic BTZ concentrations. Disclosures Kaspers: Janssen-Cilag: Research Funding. Smeets:Novartis: Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cloos:Takeda: Honoraria.

scholarly journals Establishment and Characterization of an Irinotecan-Resistant Human Colon Cancer Cell Line

2021 ◽  
Vol 10 ◽  
Author(s):  
Zhuo-Xun Wu ◽  
Yuqi Yang ◽  
Leli Zeng ◽  
Harsh Patel ◽  
Letao Bo ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Drug Resistance ◽  
Cell Line ◽  
Acquired Resistance ◽  
Human Colon ◽  
Cross Resistance ◽  
Human Colon Cancer ◽  
Active Efflux

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. Irinotecan is widely used as a chemotherapeutic drug to treat CRC. However, the mechanisms of acquired resistance to irinotecan in CRC remain inconclusive. In the present study, we established a novel irinotecan-resistant human colon cell line to investigate the underlying mechanism(s) of irinotecan resistance, particularly the overexpression of ABC transporters. The irinotecan-resistant S1-IR20 cell line was established by exposing irinotecan to human S1 colon cancer cells. MTT cytotoxicity assay was carried out to determine the drug resistance profile of S1-IR20 cells. The drug-resistant cells showed about 47-fold resistance to irinotecan and cross-resistance to ABCG2 substrates in comparison with S1 cells. By Western blot analysis, S1-IR20 cells showed significant increase of ABCG2, but not ABCB1 or ABCC1 in protein expression level as compared to that of parental S1 cells. The immunofluorescence assay showed that the overexpressed ABCG2 transporter is localized on the cell membrane of S1-IR20 cells, suggesting an active efflux function of the ABCG2 transporter. This finding was further confirmed by reversal studies that inhibiting efflux function of ABCG2 was able to completely abolish drug resistance to irinotecan as well as other ABCG2 substrates in S1-IR20 cells. In conclusion, our work established an in vitro model of irinotecan resistance in CRC and suggested ABCG2 overexpression as one of the underlying mechanisms of acquired resistance to irinotecan. This novel resistant cell line may enable future studies to overcome drug resistance in vitro and improve CRC treatment in vivo.

scholarly journals ESAM Is a Novel Human Hematopoietic Stem Cell Marker Associated with a Subset of Human Leukemias

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1154-1154
Author(s):  
Tomohiko Ishibashi ◽  
Takafumi Yokota ◽  
Hirokazu Tanaka ◽  
Michiko Ichii ◽  
Takao Sudo ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Line ◽  
Research Funding ◽  
Leukemia Cell ◽  
Scid Mice ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Significant Advance ◽  
Hematopoietic Stem ◽  
Human Hscs

Abstract Murine hematopoietic stem cells (HSCs) can be isolated with high efficiency as Lineage- Sca-1+ c-kitHigh (LSK) CD34-/Low CD150+ CD48- cells. In humans, however, the same method is not useful because of critical differences between murine and human HSC phenotypes. Such discrepancy has hampered the translation of findings in mice into a human preclinical or clinical context. Therefore, the identification of common HSC antigens between the two species would be a significant advance with respect to translational studies of HSC biology. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel maker for HSCs in mice (Blood, 2009). We also found that ESAM is functionally important for murine HSCs to reconstitute hematopoiesis after 5-FU treatment (J Immunol, 2012). In the present study, we have extended our research of ESAM to human HSCs and leukemia. We first examined whether ESAM expression showed potential as a marker of human HSCs. In addition to adult BM, the majority of CD34+ CD38- cells in cord blood (CB) and G-CSF mobilized peripheral blood expressed ESAM. The addition of anti-CD90 and CD45RA antibodies divides the adult BM CD34+ CD38- fraction into three subpopulations, namely HSCs, multipotent progenitors (MPPs), and multi-lymphoid progenitors (MLPs). We found that HSCs expressed high levels of ESAM whereas MPPs expressed lower levels and many MLPs lost ESAM expression. Functional assessment for ESAM-/Low and ESAMHigh cells in the CD34+ CD38- fractionconfirmed that high ESAM expression distinguishes progenitors that are more primitive and multipotent. We also identified a subset of CD34+ CD38- cells in adult BM and CB that expressed extremely high levels of ESAM, namely ESAMBright cells. Gene expression profiles of the CD34+ CD38- ESAMHigh and CD34+ CD38- ESAMBright populations showed that the former cells expressed HSC-related genes whereas the latter showed more endothelial-related profiles. Indeed, the CD34+ CD38- ESAMBright cells produced CD31+ endothelial cells, but not CD45+ hematopoietic cells, in co-culture with MS5 stromal cells. These results suggest that the CD34+ CD38- fraction, which is conventionally considered the human HSC fraction, also contains a substantial number of non-hematopoietic progenitors. Thus, the inclusion of ESAM provides a more accurate estimation of HSC numbers. Since some of HSC-related antigens are useful for determining leukemia lineage and have utility as prognostic indicators, we determined whether ESAM might also be a valuable addition to this antigen panel. First, we examined human leukemia cell lines. Tested myeloid leukemia lines including KG-1a, HL60, THP1, U937 and Kasumi were uniformly negative for ESAM expression. Jurkat and MOLT4, lymphoid lineage lines were also negative. On the other hand, HEL, an erythroid leukemia cell line, and CMK, a megakaryocytic leukemia cell line, exhibited high expression of ESAM. Additionally, K562 cells, which originated from CML that subsequently transformed into acute erythro-leukemia, also express ESAM. We then evaluated ESAM expression on primary acute leukemia cells, which were isolated from patients upon diagnosis. Interestingly, while all of ALL cases were virtually negative for ESAM, more than half of AML cases were ESAM-positive. Notably, the ESAM expression pattern on AML cases substantially differs even in the same FAB classification. We inferred that AML cells might change their ESAM expression levels according to cell intrinsic features and/or the surrounding environment in vivo. Therefore, we inoculated ESAM- KG-1a cells into NOD/SCID mice and harvested reconstituted KG-1a (rKG-1a) cells after the inoculation. They were then cultured in vitro and inoculated again into NOD/SCID mice. FACS analyses revealed that, although parental KG-1a cells were ESAM-negative, rKG-1a cells expressed a substantial amount of ESAM. Notably, rKG-1a cells were more aggressive and killed the recipient mice in a shorter period. This observation indicates that leukemia cells change their surface phenotype according to the environment, and that ESAM expression may be related to the acquisition of a more aggressive phenotype. In conclusion, we demonstrate that ESAM is a reliable marker of HSCs in humans as well as in mice. Additionally, ESAM is expressed on some of human acute leukemia cells and might be useful for lineage determination and as prognostic indicator. Disclosures Yokota: SHIONOGI & CO., LTD.: Research Funding. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Aurora-A Kinase: A Novel Target for the Immunotherapy Against Human Leukemias.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1796-1796 ◽  
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Kohichiro Suemori ◽  
Taichi Azuma ◽  
Kiyotaka Kuzushima ◽  
...  
Keyword(s):  
Cell Line ◽  
Hematological Malignancies ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Normal Cells ◽  
Target Peptide ◽  
Primary Leukemia

Abstract Aurora-A kinase (Aurora-A) is one of the serine/threonine kinase families, which is located on the long arm of chromosome 20q13, is mainly expressed in G2/M phase of cell-cycle and regulates mitotic cell division process in normal cells. In normal tissues, Aurora-A is exclusively expressed in testis, but uncertain in CD34+ hematopoietic progenitor cells. Overexpression of Aurora-A is observed in various human solid tumors and hematological malignancies, gene-transfer of Aurora-A resulted in tumor-transformation, in vitro. And, we previously reported that Aurora-A was overexpressed in lymphoma cells and was implicated in the proliferation. These lines of evidence suggested that Aurora-A was one of cancer-testis antigens, might directly be correlated with tumorigenesis, and postulated that Aurora-A simultaneously might be an immunotherapeutic candidate antigen for patients with hematological malignancies. [Purpose and methods] In this study, we set out to answer the question whether Aurora-A could be a target of immunotherapy for leukemia or not. Firstly we started to explore candidate epitopes derived from Aurora-A for the induction of epitope-specific CTL. 9-mer peptides of Aurora-A which were algorithmically predicted to bind to HLA-A*0201 molecules were synthesized and assessed their binding affinites by HLA-A*0201 stabilization assay with T2 cells. Aurora-A derived-peptide-specific CTL were generated by repetitive stimulations of the peripheral blood mononuclear cells (PBMC) obtained from HLA-A*0201+ healthy individual. Target-specific cytotoxicity was defined by standard 51Cr release assay as described previously. HLA-class I restriction was confirmed by blocking test with anti-HLA class I antibody (w6/32) or anti-HLA-DR antibody (L243). The expression of Aurora-A mRNA and Aurora-A protein in cell line cells and primary leukemia cells were assessed by semi-quantitative real-time PCR (RQ-PCR) and western blot (WB). Circulating Aurora-A-specific CTL precursors in PBMC of patients with leukemia in remission who received chemotherapy or allogeneic stem cell transplantation (allo-SCT) was measured by ELISPOT assay and tetramer assay. [ Results] We succeeded in establishing a CD8+ CTL which could recognize a 9-mer peptide (aa207-215;YLILEYAPL) derived from Aurora-A in the context of HLA-A*0201, the most common HLA class I type in Caucasian. Those CTL lysed target-peptide loaded autologous EBV-immortalized B cell line cells (LCL), but not those without target-peptide or target-peptide loaded HLA-A*0201-negative LCL. Both expression level of Aurora-A mRNA and protein in leukemia cells were high, especially in primary CML cells, but very low in normal PBMCs. The Aurora-A peptide (aa207-215;YLILEYAPL)-specific CTL could lyse HLA-A*0201+ and Aurora-A mRNA expressing leukemia cell lines and primary leukemia cells, but not normal cells. Furthermore, the degree of target-cell lysis was in concordance with the expression level of Aurora-A mRNA. Finally, the Aurora-A-epitope specific CTL precursors were detected in PBMC of remission state from an AML patient who received allo-SCT and an ALL patient treated solely by chemotherapy. [Conclusion] We identified a novel epitope derived from Aurora-A (aa207-215) which exerted CTL activity against leukemias. Our data suggest that this newly defined epitope of Aurora-A may be naturally expressed by primary leukemias in HLA-A*0201 restriction, and Aurora-A can be a potential target of immunotherapy for leukemias.

Human Plasmacytoid Dendritic Cell Line (PMDC05) Established from a Patient with CD4+CD56+ Acute Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4330-4330
Author(s):  
Miwako Narita ◽  
Nozomi Tochiki ◽  
Shigeo Hashimoto ◽  
Asuka Sekiguchi ◽  
Norihiro Watanabe ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Acute Leukemia ◽  
Cell Line ◽  
Plasma Cell ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Type I ◽  
Leukocyte Culture ◽  
Antigen Presenting

Abstract Human dendritic cell (DCs) precursors are commonly divided into two distinct subsets: myeloid DCs (mDC) and Plasmacytoid DCs (pDCs). The pDCs, which show plasma cell like morphology, have been defined as the population that produce a large amount of type I interferon in response to viruses. The surface phenotypes of human pDCs are defined as CD4+, DC11c−, CD45RA+, IL3Rα (CD123)+ and lineage negative. But the developmental pathways and the regulations of pDCs have not been fully understood. On the other hand, CD4+CD56+ malignant cells in leukemia/lymphoma have been proposed to be of pDC lineage. CD4+CD56+ pDC leukemia/lymphoma are a rare hematological malignancy, totally only about 100 cases in the world by the literatures. In the recent report, these newly described CD4+CD56+ leukemic pDCs share common phenotypic and functional features with their normal counterparts. We encountered a patient with CD4+CD56+ acute leukemia in December 2004. The leukemia cells have been cultured in IMDM with 10 % FBS and a leukemia cell-derived cell line (PMDC05) was established. The effects of various cytokines on the differentiation and the function of PMDC05 were assayed by using IL-3, IL-4, IL-6, GM-CSF and CD40L alone or in combination. To evaluate the effects of CD40L, PMDC05 were cultured over adherent cell layer of 90 Gy irradiated CD40L cDNA-transfected NIH-3T3 cells at cell ration of 5:1 for 2 days. For investigation of the response of PMDC05 against the danger signals through toll like receptors, inactivated influenza viruses (A/H1N1 and A/H3N2) and GpG ODN 2006 were used. Antigen presenting ability of PMDC05 was evaluated by mixed leukocyte culture consisting of 50 Gy irradiated-PMDC05 cultured with various cytokines as stimulator cells and normal peripheral blood non-adherent cells and naïve cells as responder cells. PMDC05 maintained plasma cell like morphology with abundant cytoplasm and some cells showed small dendrites. PMDC05 showed a complex hypoploid chromosomal abnormalities (44, XY) including add(5)(q22), add(15)(q26) and del(15)(q11q15), which are identical to original leukemia cells. Abnormalities including 5q and 15q are reported as the frequent aberrations in CD4+CD56+ pDC leukemia/lymphoma. The surface phenotypes of PMDC05 were negative for CD3, CD14, CD16, CD19 and CD11c and highly positive for CD4, CD45RA, CD56, CD123, CD86, and HLA-DR. Moreover BDCA4 that is specific antigen of human blood pDC is markedly expressed on PMDC05. No TCR or IgH gene rearrangement was detected. Stimulation of PMDC05 with IL-3/CD40L, virus RNA or CpG, which are known as the potent exogenous signals for maturation of normal pDCs, showed to induce the high expression of the maturation markers such as CD83/CD40 and the production of INF-α. PMDC05 were demonstrated to possess a potent antigen presenting ability to allogeneic CD4+ cells in mixed leukocyte culture. The antigen presenting ability was remarkably enhanced in PMDC05 cultured with IL-3/CD40L for 2 days. These data demonstrated that newly established leukemia cell line PMDC05 is involved in pDC lineage and PMDC05 provides invaluable tools not only for the elucidation of pathophysiology and innovation of therapy in CD4+CD56+ leukemia/lymphoma but for the investigation of human pDCs.

The Overexpression of ANKHD1 in Acute Leukemia Cells and Its Association with SIVA-1, a Proapoptotic Protein.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4290-4290
Author(s):  
Fabiola Traina ◽  
Patricia M.B. Favaro ◽  
Adriana S.S. Duarte ◽  
Sheila M.B. Winnschofe ◽  
Fernando F. Costa ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Protein Interactions ◽  
Jurkat Cell ◽  
Expression Patterns ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Leukemia Cells

Abstract Ankyrin-repeat-containing proteins regulate multiple cellular functions including transcriptional and cell-cycle regulation, ion channel, cell survival and cell signaling and participate in protein-protein interactions via their repeat motifs. Ankyrin Repeat and KH Domain Containing 1-ANKHD1, was first identified in LNCaP, a human prostate cancer cell line. However, protein expression patterns or protein interactions of ANKHD1 have not yet been described. The aim of this studied was to characterize the mRNA and protein expression patterns of ANKHD1 in normal hematopoietic cells and acute leukemia cells, and also to identify new proteins that interact with ANKHD1 in hematopoietic cells. The National Ethical Committee Board approved the study and informed-written consent was obtained from all subjects. Real Time RT-PCR was performed and increased levels of ANKHD1 mRNA were detected in acute leukemia cell lines; KG-1, HEL, K562, NB4, HL-60 Jurkat, MOLT4, Raji, Daudi and Namalwa (up to 10-fold increase), compared to normal bone marrow cells. Additionally, ANKHD1 mRNA expression was significantly higher in samples from patients with a diagnosis of acute myeloid leukemia (n = 30) (medians: 1.97 versus 1.05, P = 0.004) when compared to normal hematopoietic cells (n = 9), and in samples from patients with a diagnosis of acute lymphoblastic leukemia (n = 7) (medians: 2.53 versus 1.05, P = 0.005) when compared to normal hematopoietic cells (n = 9). Regarding protein expression, immunoblot analysis detected a very low expression of ANKHD1 in Peripheral Blood Mononuclear Cells (PBMC) of normal donors. Conversely, a high expression of the protein was detected in the human acute leukemia cell lines, KG-1, HEL, K562, NB4, HL-60 Jurkat, MOLT4, Raji, Daudi and Namalwa. Laser confocal analysis of K562 and Jurkat cell lines and PBMC showed that ANKHD1 is located in the cytoplasm of leukemia and normal hematopoietic cells. This cytoplasmatic localization of ANKHD1 was further confirmed by immunoblotting performed with anti-ANKHD1 antibody and tissue fractions of the Jurkat cell line. In an attempt to identify the proteins associated with ANKHD1 in hematopoietic cells, yeast two-hybrid screening was used and AH109 was transformed with PGBKT7-ANKHD1 (amino acids 1130–1243) and used to screen a Matchmaker pACT2-cDNA library prepared from normal human bone marrow (Clontech). The screening identified a new protein interaction between ANKHD1 and SIVA-1. This interaction was further confirmed by immunoprecipitation and Western Blotting analysis in the Jurkat cell line. The finding that ANKHD1 interacts with SIVA-1, which has been recently identified as a proapoptotic protein up regulated in leukemia cells, as well as the high expression of ANKHD1 in acute leukemia, described in our study, gives rise to the hypothesis that ANKHD1 protein may be involved in leukemogenesis. The identification of new disease-specific targets for acute myeloid leukemia immunotherapy expands treatment options and increases our chances of successfully treating this heterogeneous disease and lowering the unacceptably high mortality rate.

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