Acute Lymphoblastic Leukemia Patient Samples Research Articles

Methylation Profiling of Apoptotic Genes in Leukemia Cell Lines and Patients.

^equal contribution Apoptosis is a morphologically and biochemically distinct form of eukaryotic cell death that occurs under a variety of physiological and pathological conditions. Apoptosis is executed via 2 main pathways that lead to the activation of caspases: the death receptor (DR) pathway and the mitochondrial pathway. Defects in apoptotic mechanisms play an important role in enabling neoplastic cells to survive by ignoring death inducing signals leading to protection from the lack of survival factors. DNA methylation is a reversible mechanism of gene inactivation that might be responsible for impaired apoptosis in leukemia cells. Methylation profiling of apoptotic genes can identify possible gene targets for epigenetic therapy using demethylating agents like 5-aza-deoxycytidine and azacytidine. It can also be used for classification and prognosis. We performed a comprehensive DNA methylation analysis of apoptosis pathway genes with CpG islands in promoter/exon 1 regions in 5 leukemic cell lines (TF1, K562, Raji, HL60 and Jurkat), 20 patients with acute myelogenous leukemia (AML), 20 patients with acute lymphoblastic leukemia (ALL), and 6 control samples (normal peripheral blood cells). We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Our analysis covered a total of 25 genes: 11 in the intrinsic pathway (BAX, BAK, HRK, CYC1, DIABLO, ATM, APAF1, MAP3K5, CHEK2,PAK1, and BNIP 3), 4 in the extrinsic pathway (TRADD,PYCARD, CARD 10 and BID), 5 genes of the Caspase family (Caspase 2, 3, 6, 7, 9) in addition to 4 of the programmed cell death genes (PDCD 2, 4, 6, and 11). By using a 15% cutoff level to call a gene methylated, methylation was observed in 7 genes in at least one cell line: BNIP3 (Raji), PYCARD (K562), HRK (Tf1, Raji and Jurkat), CARD10 (Raji and HL-60), BID (Raji), PAK1 (Raji) and Casp 6 (Raji). We then performed methylation analysis of 4 genes (BNIP3, PYCARD, PAK1 and CARD 10) in AML and ALL patient samples. Methylation frequencies over a 15% cutoff are summarized in Table 1. We conclude that DNA methylation-associated epigenetic silencing is likely to play a role in modulating apoptosis in a subset of AML and ALL patients, with PYCARD being the most frequent target.Table 1GenesMethylation in AML patientsMethylation in ALL patientsPYCARD6/204/20BNIP31/203/20CARD100/202/20PAK10/201/20

Arsenic Trioxide Sensitizes Glucocorticoid-Resistant T-Cell Acute Lymphoblastic Leukemia to Dexamethasone through Inactivation of AKT and Downregulation of XIAP.

Arsenic trioxide (ATO) is a very effective agent for the treatment of acute promyelocytic leukemia (APL). Because of its pro-apoptotic activity already at low dosage, ATO may be beneficial for combination treatment of drug-resistant disease to augment the effect of other chemotherapeutic agents. In childhood acute lymphoblastic leukemia (ALL), de novo resistance to glucocorticoids (GC) is markedly associated with poor outcome. As a mechanism of action, GCs induce apoptosis in steroid-sensitive leukemic cells. Recent evidence suggests that GC-resistance is associated with altered levels of pro- and anti-apoptotic regulatory proteins. We therefore evaluated the potential of ATO as a chemo-sensitizer in a model of GC-resistant ALL. As expected, ATO effectively induced cell death in ALL cell lines and in primary ALL patient samples at clinically relevant concentrations (IC50 values between 0.4–1.2 uM). Interestingly, ATO re-sensitized the GC-resistant ALL lines CEM C1-15, MOLT-4 and Jurkat to dexamethasone at doses that do not affect cell survival as single agent. The same effect was observed using primary cells from two patients with GC-resistant T-cell ALL. The sensitizing effect of ATO could not be observed in combination with the standard ALL chemotherapeutic agents daunorubicin, asparaginase and vincristine, suggesting that a mechanism specifically relevant for GC-resistance is involved. ATO, alone or at lower dose in combination with dexamethasone, induced cell death by activating caspase-3 and −8, which resulted in PARP cleavage. We then investigated the MAPK and PI3K/AKT pathways as candidate mechanisms of action with key apoptosis regulators such as Bad and Bcl-2 as targets. The MEK1/2 inhibitor PD98059 only marginally augmented the response to dexamethasone, suggesting that this pathway would not play a major role for GC sensitization. In contrast, we found that low dose ATO treatment resulted in a rapid decrease of AKT phosphorylation at the activating serine 473 phosphorylation site, while AKT levels remained unaffected. Concomitantly, the AKT targets Bad and XIAP appeared to be modulated. The pro-apoptotic regulator Bad was induced, and its phosphorylation was decreased. Downregulation of Bad mediated by siRNA partly rescued cells from ATO-induced cell death indicating that Bad might have a sensitizing role in ATO-mediated apoptosis. Moreover, ATO induced downregulation of the NF-kB target XIAP. The protein levels of other NF-kB targets such as Bcl-2 and Bcl-xL were unchanged. Downregulation of XIAP could be due to decreased protein stability resulting from reduced XIAP phosphorylation by AKT. The effects of low dose ATO on AKT phosphorylation as well as Bad and XIAP levels were more pronounced when cells were treated in combination with dexamethasone. Ongoing experiments will further define the role of XIAP and NF-kB as specific therapeutic targets in this model. These results indicate that resistance to GCs is - at least in part - mediated through apoptosis regulators in ALL. Our data provide evidence that ATO may be beneficial as sensitizing agent in defined subgroups of ALL patients, next to its role in APL and multiple myeloma treatment.

Distinct DNA Methylation Patterns of HOX Genes in Leukemia.

The HOX subset of homeobox gene family comprises 39 genes organized in 4 clusters A-D on 4 different chromosomes and in 13 paralog groups defined across the clusters. HOX genes are important regulators of development and tissue differentiation. Dysregulated expression disturbs hematopoiesis and HOX genes are frequent partners in chromosomal translocations involved in leukemia. DNA methylation of promoter-associated CpG islands is an epigenetic modification resulting in transcriptional silencing of affected genes. Methylation pattern is faithfully copied during cell replication to subsequent cell generations and hypermethylation thus represents a permanent mark functionally equivalent to a loss-of-function mutation. Epigenetic silencing of HOX genes may disrupt normal development of blood cells and thus be involved in the development of leukemia, as was recently reported for HOXA5. We performed a comprehensive DNA methylation analysis of 22 HOX genes with CpG islands in promoter to exon 1 regions: HOXA1, A4, A5, A7, A9, A10, A11; B1, B4, B5, B7, B8, B9, B13; C4, C5, C8, C9, C10, C13; D1 and D10 in 16 leukemic cell lines, 24 samples from patients with acute myelogenous leukemia (AML), 20 samples from patients with acute lymphoblastic leukemia (ALL), and 15 control samples of normal blood cells. We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Overall, lymphoblastic leukemic cell lines (TALL, Raji, ALL1, JTAg, CEM, BJAB, Jurkat) showed the highest degree of HOX genes methylation (average 60%), followed by myeloid leukemic cell lines (HL60, KG1a, ML1, HEL, TF-1, OCI-AML3, K562, TF1i, KG1, average methylation 40%), p=0.000. Analysis of ALL and AML patient samples revealed significant differences in methylation levels of several HOX genes. Consistently with their reported overexpression in leukemia, HOX A9, B4, and also HOXB5 and HOXD10 were seldom methylated in AML patients (2/24, 0/24, 1/12, 7/24, respectively), while hypermethylation over a 10% cutoff value was observed frequently in ALL patients (10/20, 6/19, 6/17, 9/19, respectively). The differences in methylation were statistically significant (p<0.05). On the other hand, HOXA4 showed significantly higher methylation densities in AML (median 74%, range 35–95%) and ALL (median 49%, range 12–91%) than in normal controls (median 34%, range 17–59%), p<0.01. HOXC4 was more frequently methylated in normal controls (11/13) and AML (21/24) than in ALL (12/20 patients). ALL patients also showed significantly lower methylation densities of HOXC4 (median 12%, range 5–39%) than AML patients (median 32%, range 4–54%, p=0.001), underscoring the importance of HOXC4 for lymphoid cells. Unsupervised hierarchical clustering of methylation densities of all analyzed HOX genes clearly separated AML from ALL patients. We propose that epigenetic silencing of HOX genes by DNA methylation plays a role in the process of leukemic transformation of hematopoietic cells. Further analyses will help to gain a better insight into the role of HOX gene dysregulation in leukemia.

Glucocorticoid-induced glucocorticoid-receptor expression and promoter usage is not linked to glucocorticoid resistance in childhood ALL

AbstractGlucocorticoid (GC) resistance is an adverse prognostic factor in childhood acute lymphoblastic leukemia (ALL), but little is known about causes of GC resistance. Up-regulation of the glucocorticoid receptor (GR) has been suggested as an essential step to the induction of apoptosis in leukemic cells. In this study we investigated whether baseline mRNA expression levels of the 5 different GR promoter transcripts (1A1, 1A2, 1A3, 1B, and 1C) or differences in the degree of regulation of the GR or GR promoter transcripts upon GC exposure are related to GC resistance. Therefore, mRNA levels of the 5 GR promoter transcripts and of the GR were measured by quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR; Taqman) technology in primary ALL cells prior to and after 3, 8, and 24 hours of prednisolone exposure. GR expression is induced upon GC exposure in primary ALL patient samples, which is opposite to what is found in tissues in which GCs do not induce apoptosis. GC resistance in childhood ALL cannot be attributed to an inability of resistant cells to up-regulate the expression of the GR upon GC exposure, nor to differences in GR promoter usage (at baseline and upon GC exposure).

Ectopic Expression of the Proto-oncogene Mer in Pediatric T-Cell Acute Lymphoblastic Leukemia

The Mer receptor tyrosine kinase, cloned from a B-lymphoblastoid library, is the mammalian orthologue of the chicken retroviral oncogene v-eyk and sends antiapoptotic and transforming signals when activated. To determine if Mer expression is ectopic in T-cell acute lymphoblastic leukemia (ALL) and potentially important in leukemogenesis, we analyzed Mer expression in normal human thymocytes and lymphocytes and in pediatric ALL patient samples. Reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to determine expression of Mer in sorted human thymocyte populations, lymphocytes, and lymphocytes activated by phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. Mer expression in 34 T-cell ALL (T-ALL) patient samples was evaluated by reverse transcription-PCR, and Mer protein expression in a separate cohort of 16 patient samples was assayed by flow cytometry and Western blot. Mer expression was absent in normal thymocytes or lymphocytes, and in T cells activated with phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. In contrast, Jurkat cells and T-ALL patient samples expressed unique 180 to 185 kDa Mer protein glycoforms. Substantial Mer RNA levels were principally observed in a subset of T-ALL patient samples that expressed B220 (P = 0.004) but lacked surface expression of CD3 (P = 0.02) and CD4 (P = 0.006), a phenotypic profile consistent with immature lymphoblasts. In addition, 8 of 16 T-ALL patient samples had Mer protein detected by flow cytometry and Western blot. Transforming Mer signals may contribute to T-cell leukemogenesis, and abnormal Mer expression may be a novel therapeutic target in pediatric ALL therapy.

T-Cell activation by t(9;22) acute lymphoblastic leukemia-derived dendritic-like cells is associated with increased tapasin expression.

Dendritic-like cells from t(9;22) acute lymphoblastic leukemia (ALL) blasts can activate T cells, while the original unmodified leukemic blasts cannot. To determine whether these functional differences were associated with differences in antigen-processing machinery (APM) component expression, we have measured the level of APM component expression in unmodified blasts and ALL-derived dendritic-like cells. Seven t(9;22) ALL patient samples and one cell line were studied for APM component expression utilizing a unique panel of recently developed monoclonal antibodies and a recently developed intracellular staining technique. In addition, the HLA class I antigen cell surface expression was measured. HLA class I antigens were similarly expressed on the unmodified blasts and on the autologous dendritic-like cells. Intracellular HLA class I antigen and tapasin expression (P=0.03 for both) were upregulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. These results provide a potential mechanism for the ability of t(9;22) ALL-derived dendritic-like cells to induce T-cell activation and, suggest that tapasin upregulation may serve as a marker to standardize and monitor the quality of the dendritic-like cells used in immunotherapy.

A New Genotyping Chip for Childhood ALL: Using a Novel Multiplex Allele-Specific Primer Extension Strategy To Detect Twelve Polymorphisms in Six Genes Encoding for Drug-Metabolizing Enzymes.

Background/Aim Despite great improvements in the treatment of childhood Acute Lymphoblastic Leukemia (ALL), resistant to treatment still remains a leading cause of cancer-related death in children. Although the underlying mechanism is not well understood, studies have shown that polymorphisms in those genes involved in drug metabolism may not only modify the susceptibility to the cancer but also influence the risk of relapse. Knowledge of patient's pharmacogenetic make-up thus becomes important as we will move into a new era of individualizing therapy within the next decade. With the completion of the Human Genome Project, there is huge information on the SNPs which are involved in the drug metabolism. Hence, a highly efficient genotyping strategy is required to study multiple candidate genes on a single platform. This will provide timely reports for oncologists to tailor treatments for patients based on their pharmacogenetic constitution. Therefore, we aimed to develop an integrative genotyping platform, a DNA chip capable of detecting several mutations simultaneously. We plan to use this new tool to investigate the roles of 12 polymorphisms in 6 important drug-metabolizing genes (Table 1) in the risk of ALL as well as their impact on the treatment outcome in our Southeast Asian population, where only a few pharmacogenetic studies have been reported.Methods Two allele-specific primers are designed to interrogate each polymorphism and all primers for wild-type or mutant are pooled to obtain 2 mixtures. Eleven fragments spanning 12 polymorphic alleles are amplified in 3 multiplex PCR and pooled, followed by multiplex allele-specific primer extensions using 2 primer mixtures respectively. Only fully matched primers will be extended with TAMRA-labelled ddNTP. The products are subsequently loaded on DNA chip and the extended primers are captured by corresponding complementary oligo tags. The fluorescence will then disclose the genotype in imaging system. Currently we have used 40 DNA control samples to validate this platform.Results Homozygosity displayed fluorescence from only 1 allele while signals from both indicated heterozygosity (Fig 1). We have performed PCR-RFLP tests on NQO1, MTHFR and GSTP1, and used those results to verify the accuracy of the chip. 96.7% accuracy has been achieved.Conclusion Our preliminary results have demonstrated the validity of this novel genotyping strategy which has great potential to become a simple and reliable method for pharmacogenetics study.Future Plan Further verification and optimization are in progress. We will finally apply this genotyping strategy to screen childhood ALL patient samples to study the influence of these polymorphic genes on the disease.The 6 polymorphic genes involved in drug metabolism [Display omitted] GenePolymorphism(s)Drugs affectedTPMT*3A (G460A, A719G), *6 (A539T)6-mercaptopurine, 6-thioguanineNQO1*2 (C609T)AlkylatorsCYP1A1*2A (T6235C), *2B (A4889G, T6235C), *4 (C4887A)othersCYP2D6*3 (A2637 deletion), *4 (G1934A)othersGSTP1A105GGlucocorticoidsMTHFRC677T, A1298CMethotrexate

Ectopic Expression of Mer Receptor Tyrosine Kinase in Childhood T Cell Acute Lymphoblastic Leukemia.

Tyrosine kinases play an important role in normal cellular growth and differentiation. Deregulation of tyrosine kinase activity can result in cellular transformation leading to the development of human cancer. The Mer receptor tyrosine kinase, initially cloned from a human B lymphoblastoid cell line, is expressed in a spectrum of hematopoetic, epithelial, and mesenchymal cell lines. Interestingly, while the RNA transcript of Mer is detected in numerous T and B lymphoblastic cell lines, Mer RNA is not found in normal human thymocytes, lymphocytes or in PMA/PHA stimulated lymphocytes. We have developed an anti-human Mer monoclonal antibody to further study the ectopic Mer expression in lymphoblasts. The antibody detects several forms of the Mer protein including a fully glycosylated 205 kD Mer protein in monocytes and a less glycosylated 170–180 kD Mer protein in acute lymphoblastic leukemia (ALL) cell lines. We analyzed lymphoblasts from 16 T cell ALL patients diagnosed between July 1995 and July 2004 at The Children's Hospital, Denver for Mer protein surface expression. Of the T cell ALL patient samples, we found that 9/16 (56%) were positive for Mer protein surface expression. Although we were not able to statistically evaluate the clinical outcomes relative to Mer expression due to the study sample number, there was a statistically significant association between positive expression of Mer and lack of surface expression of CD3 (p = 0.035). We found that 7/9 (78%) of the Mer positive lymphoblasts lacked CD3 surface expression, while only 1/6 (17%) of the Mer negative samples lacked CD3 surface expression (see figure). Lymphoblasts that lack surface expression of CD3 represent an immature phenotype and have been associated with a decreased event free survival as compared with surface positive CD3 lymphoblasts. Further investigation of the ectopic expression of Mer in lymphoblasts may reveal the use of this novel Mer glycosylated protein as a prognostic marker and possibly a future therapeutic target in the treatment of childhood leukemia. [Display omitted]

Differential Methotrexate Resistance in Childhood T- Versus Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay

Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P =.46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P =.001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = -.58, P <.001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Differential Methotrexate Resistance in Childhood T- Versus Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay

AbstractMethotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P = .46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P = .001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = −.58, P < .001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Tyrosine Phosphorylation of CRKL in Philadelphia+ Leukemia

The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia.

Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia

The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia.