Sofia Bengtzen

Prognostic DNA methylation patterns in cytogenetically normal acute myeloid leukemia are predefined by stem cell chromatin marks

Cytogenetically normal acute myeloid leukemia (CN-AML) compose between 40% and 50% of all adult acute myeloid leukemia (AML) cases. In this clinically diverse group, molecular aberrations, such as FLT3-ITD, NPM1, and CEBPA mutations, recently have added to the prognostic accuracy. Aberrant DNA methylation is a hallmark of cancer, including AML. We investigated in total 118 CN-AML samples in a test and a validation cohort for genome-wide promoter DNA methylation with Illumina Methylation Bead arrays and compared them with normal myeloid precursors and global gene expression. IDH and NPM1 mutations were associated with different methylation patterns (P = .0004 and .04, respectively). Genome-wide methylation levels were elevated in IDH-mutated samples (P = .006). We observed a negative impact of DNA methylation on transcription. Genes targeted by Polycomb group (PcG) proteins and genes associated with bivalent histone marks in stem cells showed increased aberrant methylation in AML (P < .0001). Furthermore, high methylation levels of PcG target genes were independently associated with better progression-free survival (odds ratio = 0.47, P = .01) and overall survival (odds ratio = 0.36, P = .001). In summary, genome-wide methylation patterns show preferential methylation of PcG targets with prognostic impact in CN-AML.

Effects of PRIMA-1 on chronic lymphocytic leukaemia cells with and without hemizygous p53 deletion

The tumour suppressor gene p53 is the most commonly mutated gene in solid tumours. Although less common in haematological malignancies, 10–15% of B‐cell chronic lymphocytic leukaemia (B‐CLL) cases carry a p53 mutation. Recently, the compound P53‐dependent reactivation and induction of massive apoptosis (PRIMA‐1) has been shown to induce cytotoxic effects and apoptosis in human tumour cells by restoration of the transcriptional activity of mutated p53. This is believed to be mediated by a change in the conformation of mutated p53 protein, restoring DNA binding and activation of p53 target genes. We studied the effects of PRIMA‐1 and commonly used anti‐leukaemic drugs on B‐CLL cells from 14 patients with and without hemizygous p53 deletion. Cells obtained from peripheral blood or bone marrow were exposed to PRIMA‐1 and fludarabine alone or in combination. PRIMA‐1 showed cytotoxic effects on B‐CLL cells from samples with and without hemizygous p53 deletion. Furthermore, conventional B‐CLL drugs were less effective in cell samples with hemizygous p53 deletion and the response depended on the size of the p53 deleted clone. Finally, we found evidence for synergistic and additive effects of PRIMA‐1 in combination with fludarabine.

Mutated and non-mutated TP53 as targets in the treatment of leukaemia

TP53 is mutated in 10–20% of cases of chronic lymphocytic leukaemia (CLL) and 3–8% of cases of acute myeloid leukaemia (AML). Recently, two classes of compounds that restore the function of p53 in tumours have been described. PRIMA‐1 (p53‐dependent reactivation and induction of massive apoptosis) restores the wild‐type conformation of mutant TP53, whereas RITA (reactivation of p53 and induction of tumour cell apoptosis) increases intracellular levels of p53. We evaluated the effects of RITA alone and in combination with PRIMA‐1 or conventional cytostatics on leukaemic cells isolated from AML and CLL patients. AML samples with −17, which are more resistant to daunorubicin and cytarabine compared with samples without −17, were effectively killed by PRIMA‐1. RITA, which stabilizes the function of wild‐type p53, induced apoptosis in AML cells. In contrast to that seen with PRIMA‐1, AML patient samples without −17 were significantly more sensitive to RITA. Similarly, RITA exerted dose‐dependent apoptosis and cytotoxicity in CLL cells, which was significantly more pronounced in samples without hemizygous TP53 deletion. Notably, a synergistic effect was observed in all CLL samples with RITA and fludarabine in combination. In both AML and CLL cells exposure to RITA resulted in induction of intracellular p53. We conclude that small molecules targeting p53 might be of clinical importance in the future for treating drug‐resistant leukaemia.

PRIMA-1 induces apoptosis in acute myeloid leukaemia cells with p53 gene deletion

The p53 tumour suppressor gene located on chromosome 17p13 is the most frequently mutated gene in human tumours. About 5–8% of cases with acute myeloid leukaemia (AML) carry the p53 mutation. Recently, the compound p53‐dependent reactivation and induction of massive apoptosis (PRIMA‐1) has been shown to induce cytotoxic effects and apoptosis in human tumour cells by restoration of the transcriptional activity of mutated p53. This is believed to be mediated by a change in the conformation of mutated p53 protein, restoring DNA binding and activation of p53 target genes. We studied the effects of PRIMA‐1 and commonly used antileukaemic drugs on AML cells from 62 patients. Cells were obtained from peripheral blood or bone marrow and were exposed to PRIMA‐1, cytarabine, daunorubicin, chlorodeoxyadenosine and fludarabine. This study showed that PRIMA‐1 had cytotoxic effects on AML cells. Conventional AML drugs were less effective in cells with hemizygous p53 deletion. Interestingly, our data indicated that PRIMA‐1 was more effective in this subgroup of patients compared with patients with normal chromosome 17. Our data suggest that the concept of restoration of p53 protein by PRIMA‐1 or a PRIMA‐1‐based new drug may increase the efficacy of AML treatment in patients with p53 mutations.

Differential methylation in CN-AML preferentially targets non-CGI regions and is dictated by DNMT3A mutational status and associated with predominant hypomethylation of HOX genes.

The extent and role of aberrant DNA methylation in promoter CpG islands (CGIs) have been extensively studied in leukemia and other malignancies. Still, CGIs represent only a small fraction of the methylome. We aimed to characterize genome-wide differential methylation of cytogenetically normal AML (CN-AML) cells compared with normal CD34+ bone marrow cells using the Illumina® 450K methylation array. Differential methylation in CN-AML was most prominent in genomic areas far from CGIs, in so called open sea regions. Furthermore, differential methylation was specifically found in genes encoding transcription factors (TFs), with WT1 being the most differentially methylated TF. Among genetic mutations in AML, DNMT3A mutations showed the most prominent association with the DNA methylation pattern, characterized by hypomethylation of CGIs (as compared with DNMT3A wild type cases). The differential methylation in DNMT3A mutant cells vs. wild type cells was predominantly found in HOX genes, which were hypomethylated. These results were confirmed and validated in an independent CN-AML cohort. In conclusion, we show that, in CN-AML, the most pronounced changes in DNA methylation occur in non-CGI regions and that DNMT3A mutations confer a pattern of global hypomethylation that specifically targets HOX genes.

Effects of arsenic trioxide (As2O3) on leukemic cells from patients with non-M3 acute myelogenous leukemia: studies of cytotoxicity, apoptosis and the pattern of resistance

Abstract: Arsenic oxide (As2O3) has recently been reported to induce remission in a high percentage of patients with acute promyelocytic leukemia (APL). The aim of this study was to investigate the effects of As2O3 at therapeutic concentrations on cell viability and apoptosis on leukemic cells from patients with non‐M3 acute myelogenous leukemia (AML) and to study the resistance profile compared to conventional AML drugs. Cells from 20 patients were exposed to therapeutic concentrations of As2O3 continuously for 96h. As2O3 reduced the viability in blast cells from all the 20 tested patients compared to unexposed controls (p‐value: 0.02 at 0.05 µM; <0.005 at 1.0 µM and higher concentrations). An increase in the apoptotic rate was also seen after incubation with As2O3. Parallel to the incubation with arsenic the in vitro sensitivity to a number of chemotherapeutic agents commonly used in AML was studied. Correlation coefficients for the in vitro sensitivity were highly significant between the conventional AML drugs except for Ara‐C. For As2O3, all the correlation coefficients were negative and ranged between −0.05 and −0.51. Furthermore, increased P‐gp expression in a multidrug resistant HL‐60 cell line did not decrease the sensitivity to As2O3 as compared to the parental cell line. Neither did a P‐gp‐transfected variant of the K562 cell line show decreased sensitivity to As2O3. We conclude that As2O3 at therapeutic concentrations induces apoptosis and cytotoxic effects in blast cells from patients with non‐M3 AML, and that As2O3 differs from conventional AML drugs with respect to the mechanisms that confer resistance to the drugs.

APR‐246 exhibits anti‐leukemic activity and synergism with conventional chemotherapeutic drugs in acute myeloid leukemia cells

Background:  APR‐246 belongs to a new generation of the compounds that restore normal p53 function in cells with mutated or wild type p53. The purpose of this study was to examine the effects of APR‐246 alone and in combination with other drugs in acute myeloid leukemia (AML) cells.

Chromosomal aberrations in 17p predict in vitro drug resistance and short overall survival in acute myeloid leukemia.

Chromosomal aberrations are important prognostic parameters in acute myeloid leukemia (AML). Indicators of poor prognosis include del(5q)/−5, del(7q)/−7, abnormal 3q or complex karyotype. In recent years, it has become clear that aberrations in 17p represent one of the indicators of poor prognosis in haematological malignancies. In AML, deletions in 17p have been shown to indicate a dismal prognosis; genetic aberrations in 9p have also been discussed as influencing long-term survival in AML. In this study, we correlated genetic abnormalities in chromosomes 9 and 17 in patients with de novo AML to in vitro cytotoxicity of conventional anti-leukemic drugs, and long-term overall survival. Blast cells were isolated from 387 patients diagnosed with AML. Chromosomal analysis was successful in 336 cases. All samples were tested for in vitro cytotoxicity against fludarabine, amsacrine, mitoxantrone, etoposide, daunorubicin and Ara-C after being cultured for 4 days, using an ATP assay. Among the 336 patients, five main groups were identified. Abnormal chromosome 17 (n = 22), abnormal 9p (n = 13), monosomy 7 or deletion 7q (n = 35), complex karyotype (n = 52) and normal karyotype (n = 132). Patients with abnormalities of chromosome 17 showed significantly greater resistance to all drugs tested and significantly shorter overall survival compared with patients with normal and complex karyotypes (p = 0.0001 and 0.041, respectively). All patients with abnormalities of chromosome 17 died within 11 months of diagnosis. A tendency towards shorter overall survival and greater drug resistance was also noted when comparing chromosome 17 abnormalities with del(7q)/−7, but the differences did not reach statistical significance. Patients with abnormal 9p showed significantly shorter overall survival but did not differ significantly as regards in vitro drug resistance compared with patients presenting with a normal karyotype. Chromosomal abnormalities affecting the p53 pathway have a significant impact on cytostatic drug resistance and survival in AML. Developing new drugs targeting the p53 pathway could be a way to improve treatment of AML.

microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.

Effects of retinoids on cell toxicity and apoptosis in leukemic blast cells from patients with non-M3 AML

All-trans retinoic acid (ATRA) induces complete remission in acute promyelocytic leukemia (APL or M3). In this study we measured the effect of retinoids alone and in combination with daunorubicin (DNR) on cell growth and apoptosis in blast cells from patients with non-M3 AML. Cells from 21 patients were incubated in 0.2 microM daunorubicin for 1 h or in 1 microM ATRA or 9-cis-RA continuously and in the combinations of DNR with both retinoids. Cell toxicity and apoptosis were analyzed after 96 h. Both ATRA and 9-cis-RA reduced the viability significantly to 86 and 84%, respectively (P = 0.003 for ATRA and 0.02 for 9-cis-RA). The expression of CD34 correlated to a higher sensitivity to ATRA (P = 0.003). When retinoids were added to DNR the mean decrease in viability was 11 percentage points with ATRA (P = 0.003) and nine percentage points with 9-cis-RA (P = 0.02). Apoptosis was induced by both retinoids and the percentage of apoptotic cells was increased from 16% in the controls to 24% with ATRA (P = 0.03) and to 26% with 9-cis-RA (P = 0.04). When the retinoids were added to DNR the apoptotic rate increased from 41% with DNR alone to 51% with ATRA (P = 0.01) and to 49% with 9-cis-RA (P = 0.03). We conclude that ATRA and RA exert a slight but clear cytotoxic and apoptotic effect on AML blast cells after 96 h incubation and that retinoids can have an additive or synergistic effects on cell toxicity when added to daunorubicin.