Christer Paul

Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia.

This study was designed to analyze the effect of global and gene-specific DNA methylation patterns on the outcome of patients with acute myeloid leukemia (AML). Methylation of CDKN2B (p15), E-cadherin (CDH) and hypermethylated in cancer 1 (HIC1) promoters and global DNA methylation by luminometric methylation assay (LUMA) was analyzed in 107 AML patients and cytogenetic and molecular mutational analysis was performed. In addition, genome-wide promoter-associated methylation was assessed using the Illumina HumanMethylation27 array in a proportion of the patients. Promoter methylation was discovered in 66, 66 and 51% of the patients for p15, CDH and HIC1, respectively. In multivariate analysis, low global DNA methylation was associated with higher complete remission rate (hazard ratio (HR) 5.9, P=0.005) and p15 methylation was associated with better overall (HR 0.4, P=0.001) and disease-free survival (HR 0.4, P=0.016). CDH and HIC1 methylation were not associated with clinical outcome. Mutational status and karyotype were not significantly associated with gene-specific methylation or global methylation. Increased genome-wide promoter-associated methylation was associated with better overall and disease-free survival as well as with LUMA hypomethylation. We conclude that global and gene-specific methylation patterns are independently associated with the clinical outcome in AML patients.

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.

Selenite is a potent cytotoxic agent for human primary AML cells.

Selenite is a potent inhibitor of malignant cell growth. Although the cytotoxic effects have been extensively investigated in vitro, there are only a limited number of studies using primary tumor cells with concomitant comparison to conventional drugs. An ex vivo model with primary cells from 39 consecutive patients with acute myeloid leukemia (AML) were exposed to a panel of conventional cytotoxic drugs, and the effects on viability were compared to those of clinically achievable concentrations of selenite. Selenite at 5 microM caused the lowest mean survival of primary tumor cells in the panel of all tested drugs (28.95% CI 18.60-39.30%). The cells showed a significant (p<0.05) correlation in the resistance to all tested conventional AML drugs whereas selenite did not, indicating sensitivity to selenite also in multi drug resistant cells. Exposure to selenite also resulted in an increased mRNA expression of the antioxidant proteins TrxR1 and Grx, while staining for TrxR1 showed decreased protein levels. The results strongly suggest a great potential for selenite in the treatment of multi drug resistant AML.

Fibrinopeptide a (FPA) in patients with acute myocardial infarction.

Abstract The concentration of fibrinopeptide A (FPA) was significantly increased in 29 patients admitted to the coronary care unit due to suspicion of myocardial infarction. The increase of FPA was more pronounced in patients with a verified myocardial infarction and patients with large infarctions had higher values than patients with small infarctions. Treatment with beta-blocking agents did not diminish the rise in FPA. These findings indicate that the plasma coagulation system is activated during the acute phase of a myocardial infarction and support earlier clinical and laboratory results that these patients are in a hypercoagulable state. Determination of FPA seems, in this respect to be of value.

Association of ABCB1 polymorphisms with survival and in vitro cytotoxicty in de novo acute myeloid leukemia with normal karyotype

Overexpression of the multi-drug transporter P-glycoprotein, encoded by the ABCB1 gene, is a clinically relevant problem in acute myeloid leukemia (AML). Polymorphisms in ABCB1 might contribute to cancer risk and therapeutic response. We therefore investigated the influence of polymorphisms G1199A, C1236T, G2677T/A and C3435T on cancer susceptibility, in vitro cytotoxicity and overall survival in 100 de novo AML patients with normal karyotype. Patients with 1236C/C or 2677G/G genotypes showed poorer survival than patients with other genotypes (P=0.03 and P=0.02, respectively). Both these genotypes were significant factors for survival in multivariate analysis, along with age, NPM1 and FLT3 mutation status. In vitro cytotoxicity studies demonstrated that leukemic cells from 1236T/T and 2677T/T patients were significantly more susceptible to mitoxantrone (P=0.02), and tended to be more susceptible to etoposide and daunorubicin (P=0.07–0.09), but not to cytarabine. No significant difference in allele frequencies was found between patients and healthy volunteers (n=400).

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.

Anti-leukaemic effects induced by APR-246 are dependent on induction of oxidative stress and the NFE2L2/HMOX1 axis that can be targeted by PI3K and mTOR inhibitors in acute myeloid leukaemia cells

The small molecule APR‐246 (PRIMA‐1MET) is a novel drug that restores the activity of mutated and unfolded TP53 protein. However, the mechanisms of action and potential off‐target effects are not fully understood. Gene expression profiling in TP53 mutant KMB3 acute myeloid leukaemia (AML) cells showed that genes which protected cells from oxidative stress to be the most up‐regulated. APR‐246 exposure also induced reactive oxygen species (ROS) formation and depleted glutathione in AML cells. The genes most up‐regulated by APR‐246, confirmed by quantitative real time polymerase chain reaction, were heme oxygenase‐1 (HMOX1, also termed HO‐1), SLC7A11 and RIT1. Up‐regulation of HMOX1, a key regulator of cellular response to ROS, was independent of TP53 mutational status. NFE2L2 (also termed Nrf2), a master regulator of HMOX1 expression, showed transcriptional up‐regulation and nuclear translocation by APR‐246. Down‐regulation of NFE2L2 by siRNA in AML cells significantly increased the antitumoural effects of APR‐246. The PI3K inhibitor wortmannin and the mTOR inhibitor rapamycin inhibited APR‐246‐induced nuclear translocation of NFE2L2 and counteracted the protective cellular responses to APR‐246, resulting in synergistic cell killing together with APR‐246. In conclusion, ROS induction is important for antileukaemic activities of APR‐246 and inhibiting the protective response of the Nrf‐2/HMOX1 axis using PI3K inhibitors, enhances the antileukaemic effects.