Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells.

In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.

High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia.

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML. (Blood. 2000;96:1517-1524)

In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2'-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene.

The deoxycytidine kinase (dck) gene encodes the enzyme responsible for the metabolic activation of the antileukemic drugs cytosine arabinoside (AraC) and 5-aza-2'-deoxycytidine (decitabine, DAC). The dck locus was analyzed at the chromosomal and the molecular level in a model of rat leukemic cell lines, in which AraC and DAC resistance was induced, that was marked by dck deficiency. At the chromosomal level, karyotype analysis of metaphase spreads revealed the presence of an aberrant 2q + chromosome in the AraC-resistant cell line and a (Xq:11q) translocation in its subclone RA/7. The DAC-resistant lines were identical to the parental RCL/O. Fluorescence in situ hybridization on normal rat fibroblast metaphase spreads localized the rat dck gene to chromosome 14q21-q22, a region that was not involved in any of the observed karyotypic aberrations. Analysis at the molecular level revealed an identical rearrangement of the dck gene in the AraC-resistant cell line RCL/A and its subclone RA/7 that resulted in the absence of dck expression, as assessed by RT-PCR. No genomic rearrangements were observed in a DAC-resistant cell line RCL/D or in its subclone RD/1. However, detection of a single-stranded conformation polymorphism (SSCP) allowed the identification of a single C to G substitution (His to Gln) in the dck cDNA of the DAC-resistant RD/1 clone. The data demonstrate that exposure to AraC and DAC induces a resistant phenotype marked by functional dck deficiency that may be the consequence of mutations occurring in the dck gene.

De novo induced mutations in the deoxycytidine kinase (dck) gene in rat leukemic clonal cell lines confer resistance to cytarabine (AraC) and 5-aza-2'-deoxycytidine (DAC).

We have investigated whether cytarabine (AraC) or decitabine (DAC) induce deficiency of deoxycytidine kinase (DCK) through different mutations of the dck gene, related to their distinct interference with DNA replication. Also, it is not known whether mutations of the dck gene are the result of selection of mutants or de novo induction. To address these issues, three subclones of a rat leukemic cell line (RCL/O), sensitive to cytotoxicity mediated by AraC and DAC, were exposed to gradually increasing concentrations (from 0.1 to 10 microM) of either AraC or DAC over a 140 days vs a 180 days period. During the course of resistance induction DCK activity was monitored. We found that all clones acquired irreversible cross-resistance, at marginally cytotoxic AraC or DAC concentrations of 0.1 to 0.4 times the IC50 for the parental clones. Furthermore, all resistant cell lines were DCK deficient and harbored different mutations in the dck gene. AraC induced both rearrangements and point mutations in the dck gene when administered over 140 days and 180 days, respectively. 140 days DAC induction yielded point mutations only. All point mutations detected were nonrandomly distributed within the dck coding region. SSCP analysis showed that in the majority of resistant clones more than one bandshift was present. The data suggest the presence of multiple resistant clones, originating from one sensitive clone and thus arguing against selection of mutants as a mechanism for the development of AraC and DAC resistance.

Cloning of the Dck gene encoding rat deoxycytidine kinase.

In order to study the mutational inactivation of deoxycytidine kinase (Dck) in a rat model for acute myeloid leukemia we have cloned the complete coding region of the rat Dck gene. Using primers chosen from the human Dck cDNA sequence, we obtained a rat-specific probe via PCR and used it to isolate two clones from a rat lymphocyte cDNA library. The ORF showed 89.7 and 92.2% nucleotide identity with the human and mouse Dck, respectively, and encodes a 260-amino-acid protein, that is 91.9 and 94.6% homologous to human and mouse Dck, respectively. Northern blot analysis of rat tissues revealed high expression of a 4.1-kb Dck transcript in the thymus, whereas spleen, liver and lung samples showed weak expression of the gene. This tissue-specific expression pattern was confirmed by cDNA-PCR analysis.

Role of deoxycytidine kinase in an in vitro model for AraC- and DAC-resistance: substrate-enzyme interactions with deoxycytidine, 1-beta-D-arabinofuranosylcytosine and 5-aza-2'-deoxycytidine.

Deoxycytidine kinase activity (dCk) was monitored in cell lines from a rat acute myeloid leukemia model of acquired resistance to cytosine arabinoside (AraC) and decitabine (DAC). In both AraC-resistant cell lines (RCL/A and its subclone RA/7), as well as in a DAC-resistant cell line (RCL/D) which we generated from the drug-sensitive RCL/0 cell line, a total deficiency of dCk activity and a cross-resistance for AraC and DAC was demonstrated. Furthermore, the metabolization of deoxycytidine (dC) was severely impaired in all these cell lines. Km values for dC (9.4 microM in RCL/0 cells) had increased 70- to 100-fold in RCL/D (Km = 673.2 microM), in RCL/A (Km = 947.2 microM) and in RA/7 (Km = 817.5 microM). Vmax values were unaltered in RCL/D and RA/7, and twofold increased in RCL/A. Addition of hydroxyurea (HU) to cell cultures stimulated dCk salvage pathway activity in RCL/0 cells for dC, AraC, and DAC by increasing Vmax values approximately 160% leaving Km constants unchanged. In all resistant cell lines, HU pre-incubation did not influence the level of dCk activity, leaving Km and Vmax values unaltered. These data indicate that deficiency of dCk activity is crucial in the mechanism of drug resistance in this model.

Increase in Ara-C sensitivity in Ara-C sensitive and -resistant leukemia by stimulation of the salvage and inhibition of the de novo pathway.

In this study the hypothesis that inhibition of the de novo pathway results in stimulation of salvage pathway activity was tested. The key enzyme in the balance between these two pathways is ribonucleotide reductase (RR), which can be inhibited by hydroxyurea (HU). The metabolism of 1-beta-D-arabinofuranosylcytosine and 5-Aza-2 deoxycytidine (Aza-dC), which are activated via the salvage pathway, was evaluated in cells from Ara-C-sensitive and -resistant myelocytic leukemia cell line (BNML-Cl/0 and BNML-Cl/Ara-C). The combination of HU and Ara-C caused as much as a threefold increase of Ara-CTP; it significantly increased the incorporation of Ara-C into DNA and induced synergistic cytotoxicity, as evaluated in a colony assay. Even in the deoxycytidine (CdR) kinase-deficient Ara-C-resistant cell line, HU was partially able to restore sensitivity to Ara-C and Aza-dC. dCTP levels are reduced during the first 10 h after incubation with HU, but this effect vanishes at the time when phosphorylation is maximal. Increased CdR kinase activity in cell-free extracts could explain the enhanced synthetic salvage pathway activity, which is likely due to the fact that more enzyme is present (Vmax has increased by Km unchanged). RR inhibition combined with Ara-C might provide a means of eliminating leukemic cells with suboptimal anabolic salvage pathway activity, which otherwise survive Ara-C chemotherapy.

Substrate-specific deoxycytidine kinase deficiency in 1-beta-D-arabinofuranosylcytosine-resistant leukemic cells.

In this study we describe the establishment of a leukemic cell line (BNML-CL/ara-C), originating from the 1-beta-D-arabinofuranosylcytosine (ara-C)-resistant brown Norway rat myelocytic leukemia model (BNML/ara-C), that retains the in vivo generated ara-C resistance. Its biological and biochemical characteristics have been compared with a cell line, derived from the ara-C-sensitive BNML model (BNML-CL/O). Resistance to ara-C was attributed to a decrease in phosphorylation of ara-C. Deoxycytidine (dCyd) kinase activity in crude cell extracts with dCyd as substrate showed similar enzyme activities in both cell lines, whereas with ara-C as substrate no dCyd kinase activity was detectable in the ara-C-resistant cell line. Two isoenzymes of dCyd kinase with different substrate specificities have been described (Cheng, Y.C., Domin, B., and Lee, L.S. Biochim. Biophys. Acta, 481: 481-492, 1977), cytoplasmic (dCyd kinase I, substrates: dCyd and ara-C) and mitochondrial (dCyd kinase II, substrates: dCyd and thymidine). In the ara-C-sensitive BNML model, thymidine induced a reduction of dCyd kinase activity when dCyd was used as substrate. However, thymidine did not affect kinase activity with ara-C was used as substrate. In the BNML-CL/ara-C, thymidine even induces a dCyd kinase inhibition of 85% with dCyd as substrate. It is likely that the ara-C-specific dCyd kinase deficiency in BNML-CL/ara-C cells was due to a selective loss of dCyd kinase I, whereas dCyd kinase II activity remained intact.

Cell kinetics after high dose cytosine arabinoside in patients with acute myelocytic leukemia.

In this study 10 patients with acute myelocytic leukemia (AML) each received a rapid intravenous injection of high dose cytosine arabinoside (HD Ara-C; 1 g/m2). Bone marrow aspirates were obtained before and after Ara-C administration to determine the percentage of cells in S-phase measured by flow cytometry. In 5 out of 10 cases synchronization of the leukemic cells in S-phase of the cell cycle was observed. However, the time of maximum synchronization turned out to be difficult to predict. Therefore, the strong correlation between percentage of cells in S-phase at diagnosis and the time of maximal accumulation of S-phase cells after Ara-C administration, as observed by others in childhood AML, could not be confirmed for adult AML patients. Although synchronization of AML cells after in vivo Ara-C administration could be demonstrated in at least half of the patients, the practical consequences are such that clinical application was hampered.

Deoxycytidine kinase, thymidine kinase and cytidine deaminase and the formation of Ara-CTP in leukemic cells in different phases of the cell cycle.

In this study we investigated the Ara-CTP-forming capacity of leukemic cells in different phases of the cell cycle. Cells from two leukemic cell lines and leukemic bone marrow cells from patients and rats (BNML model) with acute myelocytic leukemia were separated according to cell cycle phase by means of an albumin density gradient in a specially designed sedimentation chamber. We found that the activity of CdR kinase and Cyt deaminase is much less influenced by cell-cycle phase progression than TdR kinase activity. For the leukemic cell lines HL-60 and BNML-CL/O CdR kinase activity is even independent of cell-cycle phase. In addition, Ara-CTP formation is not restricted to cells in S-phase. Cell cycle phase-independent Ara-CTP formation creates a situation in which cells which are not in S-phase during exposure to Ara-C might undergo the cytotoxic effects of Ara-C as soon as they enter S-phase.

Deoxycytidine kinase and deoxycytidine deaminase values correspond closely to clinical response to cytosine arabinoside remission induction therapy in patients with acute myelogenous leukemia.

In this study, it has been shown that in 21 patients with AML the dCyd kinase and dCyd deaminase activities correspond closely to the clinical response to ara-C remission induction therapy. Patients with primary disease were treated with a conventional-dose ara-C regimen whereas nonresponders and relapsed patients followed an ID ara-C regimen (1 g/m2 X 12). Of these 21 patients (11 with primary disease and ten relapsed), seven had ara-C resistant disease (three primary and four relapsed patients). Five of the seven patients had a very low dCyd kinase and normal dCyd deaminase activity, whereas the other two had a normal dCyd kinase and an increased dCyd deaminase activity.