Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia

BACKGROUND. In severe congenital neutropenia the maturation of myeloid progenitor cells is arrested. The myelodysplastic syndrome and acute myeloid leukemia develop in some patients with severe congenital neutropenia. Abnormalities in the signal-transduction pathways for granulocyte colony-stimulating factor (G-CSF) may play a part in the progression to acute myeloid leukemia. METHODS. We isolated genomic DNA and RNA from hematopoietic cells obtained from two patients with acute myeloid leukemia and histories of severe congenital neutropenia. The nucleotide sequences encoding the cytoplasmic domain of the G-CSF receptor were amplified by means of the polymerase chain reaction and sequenced. Murine myeloid 32D.C10 cells were transfected with complementary DNA encoding the wild-type or mutant G-CSF receptors and tested for their responses to G-CSF. RESULTS. Point mutations in the gene for the G-CSF receptor were identified in both patients. The mutations, a substitution of thymine for cytosine at the codon for glutamine at position 718 (Gln718) in one patient and at the codon for glutamine at position 731(Gln731) in the other, caused a truncation of the C-terminal cytoplasmic region of the receptor. Both mutant and wild-type genes for the G-CSF receptor were present in leukemic cells from the two patients. In one patient, the mutation was also found in the neutropenic stage, before the progression to acute myeloid leukemia. The 32D.C10 cells expressing mutant receptors had abnormally high proliferative responses but failed to mature when cultured in G-CSF. The mutant G-CSF receptors also interfered with terminal maturation mediated by the wild-type G-CSF receptor in the 32D.C10 cells that coexpressed the wild-type and mutant receptors. CONCLUSIONS. Mutations in the gene for the G-CSF receptor that interrupt signals required for the maturation of myeloid cells are involved in the pathogenesis of severe congenital neutropenia and associated with the progression to acute myeloid leukemia

A Limited Role for the Cell Cycle Regulator Cyclin A1 in Murine Leukemogenesis

The quest for novel therapeutic targets in acute myeloid leukemia (AML) is still ongoing. One of such targets, cyclin A1, was shown to be overexpressed in AML including AML stem cells. However, the function of cyclin A1 in AML is largely unknown, and the data on its impact on patients´ survival remain controversial. Therefore, we developed a transgenic mouse model of stem cell-directed inducible cyclin A1 overexpression and crossed these mice with PML-RARα-knockin mice, which develop an AML M3-like phenotype. To observe the effects of cyclin A1 loss-of-function, we also crossed PML-RARα-knockin mice to cyclin A1-knockout mice. Neither overexpression nor loss of cyclin A1 significantly altered leukemogenesis in PML-RARα-knockin mice. These findings imply that upregulation of cyclin A1 is not essential for leukemogenesis. Our data suggest that cyclin A1 does not represent a suitable target for AML therapy

Serotonin transporter gene hypomethylation predicts impaired antidepressant treatment response

Variation in the serotonin transporter gene (5-HTT; SERT; SLC6A4) has been suggested to pharmacogenetically drive interindividual differences in antidepressant treatment response. In the present analysis, a 'pharmaco-epigenetic' approach was applied by investigating the influence of DNA methylation patterns in the 5-HTT transcriptional control region on antidepressant treatment response. Ninety-four patients of Caucasian descent with major depressive disorder (MDD) (f = 61) were analysed for DNA methylation status at nine CpG sites in the 5-HTT transcriptional control region upstream of exon 1A via direct sequencing of sodium bisulfite treated DNA extracted from blood cells. Patients were also genotyped for the functional 5-HTTLPR/rs25531 polymorphisms. Clinical response to treatment with escitalopram was assessed by intra-individual changes of HAM-D-21 scores after 6 wk of treatment. Lower average 5-HTT methylation across all nine CpGs was found to be associated with impaired antidepressant treatment response after 6 wk (p = 0.005). This effect was particularly conferred by one individual 5-HTT CpG site (CpG2 (GRCh37 build, NC_000017.10 28.563.102; p = 0.002). 5-HTTLPR/rs25531 haplotype was neither associated with 5-HTT DNA methylation nor treatment response. This analysis suggests that DNA hypomethylation of the 5-HTT transcriptional control region - possibly via increased serotonin transporter expression and consecutively decreased serotonin availability - might impair antidepressant treatment response in Caucasian patients with MDD. This pharmaco-epigenetic approach could eventually aid in establishing epigenetic biomarkers of treatment response and thereby a more personalized treatment of MDD.Katharina Domschke, Nicola Tidow, Kathrin Schwarte, Jürgen Deckert, Klaus-Peter Lesch, Volker Arolt, Peter Zwanzger and Bernhard T. Baun

Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants

Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 315 phosphorylation was necessary for the activation of topoisomerase I-dependent HR by p53. Moreover, overexpression of cyclin A1, which mimics the situation in tumors, inappropriately stimulated DSB-induced HR in the presence of oncogenic p53 mutants (not Wtp53). This effect required cyclin A1/cdk-mediated phosphorylation for stable complex formation with topoisomerase I. We conclude that p53 mutants have lost the balance between activation and repression of HR, which results in a net increase of potentially mutagenic DNA rearrangements. Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability

Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival

Recent evidence suggests CML stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, following re-transplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off, were able to persist in vivo and re-initiate leukemia in secondary recipients upon Bcr-Abl re-expression. Bcr-Abl knockdown in human CD34+ CML cells cultured for 12 days in physiological growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%, however the surviving fraction was enriched for primitive leukemic cells capable of growth in long-term culture initiating cell assay and expansion upon removal of dasatinib and addition of growth factors. Together these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance

Comprehensive genomic analysis of refractory multiple myeloma reveals a complex mutational landscape associated with drug resistance and novel therapeutic vulnerabilities

The outcomes of patients with multiple myeloma (MM) refractory to immunomodulatory agents (IMiDs) and proteasome inhibitors (PIs) remain poor. In this study, we performed whole genome and transcriptome sequencing of 39 heavily pretreated relapsed/refractory MM (RRMM) patients to identify mechanisms of resistance and potential therapeutic targets. We observed a high mutational load and indications of increased genomic instability. Recurrently mutated genes in RRMM, which had not been previously reported or only observed at a lower frequency in newly diagnosed MM, included NRAS, BRAF, TP53, SLC4A7, MLLT4, EWSR1, HCFC2, and COPS3. We found multiple genomic regions with bi-allelic events affecting tumor suppressor genes and demonstrated a significant adverse impact of bi-allelic TP53 alterations on survival. With regard to potentially resistance conferring mutations, recurrently mutated gene networks included genes with relevance for PI and IMiD activity; the latter particularly affecting members of the Cereblon and the COP9 signalosome complex. We observed a major impact of signatures associated with exposure to melphalan or impaired DNA double-strand break homologous recombination repair in RRMM. The latter coincided with mutations in genes associated with PARP inhibitor sensitivity in 49% of RRMM patients; a finding with potential therapeutic implications. In conclusion, this comprehensive genomic characterization revealed a complex mutational and structural landscape in RRMM and highlights potential implications for therapeutic strategies

Cyclin A1 promoter hypermethylation in human papillomavirus-associated cervical cancer

BACKGROUND: The aim of this study was to evaluate epigenetic status of cyclin A1 in human papillomavirus-associated cervical cancer. Y. Tokumaru et al., Cancer Res 64, 5982-7 (Sep 1, 2004)demonstrated in head and neck squamous-cell cancer an inverse correlation between cyclin A1 promoter hypermethylation and TP53 mutation. Human papillomavirus-associated cervical cancer, however, is deprived of TP53 function by a different mechanism. Therefore, it was of interest to investigate the epigenetic alterations during multistep cervical cancer development. METHODS: In this study, we performed duplex methylation-specific PCR and reverse transcriptase PCR on several cervical cancer cell lines and microdissected cervical cancers. Furthermore, the incidence of cyclin A1 methylation was studied in 43 samples of white blood cells, 25 normal cervices, and 24, 5 and 30 human papillomavirus-associated premalignant, microinvasive and invasive cervical lesions, respectively. RESULTS: We demonstrated cyclin A1 methylation to be commonly found in cervical cancer, both in vitro and in vivo, with its physiological role being to decrease gene expression. More important, this study demonstrated that not only is cyclin A1 promoter hypermethylation strikingly common in cervical cancer, but is also specific to the invasive phenotype in comparison with other histopathological stages during multistep carcinogenesis. None of the normal cells and low-grade squamous intraepithelial lesions exhibited methylation. In contrast, 36.6%, 60% and 93.3% of high-grade squamous intraepithelial lesions, microinvasive and invasive cancers, respectively, showed methylation. CONCLUSION: This methylation study indicated that cyclin A1 is a potential tumor marker for early diagnosis of invasive cervical cancer

A Phase I Dose Escalation Study of the Triple Angiokinase Inhibitor Nintedanib Combined with Low-Dose Cytarabine in Elderly Patients with Acute Myeloid Leukemia

Nintedanib (BIBF 1120), a potent multikinase inhibitor of VEGFR-1/-2/-3, FGFR-1/-2/-3 and PDGFR-α/-β, exerts growth inhibitory and pro-apoptotic effects in myeloid leukemic cells, especially when used in combination with cytarabine. This phase I study evaluated nintedanib in combination with low-dose cytarabine (LDAC) in elderly patients with untreated or relapsed/refractory acute myeloid leukemia (AML) ineligible for intensive chemotherapy in a 3+3 design. Nintedanib (dose levels 100, 150, and 200 mg orally twice daily) and LDAC (20 mg subcutaneous injection twice daily for 10 days) were administered in 28-day cycles. Dose-limiting toxicity (DLT) was defined as non-hematological severe adverse reaction CTC grade ≥ 4 with possible or definite relationship to nintedanib. Between April 2012 and October 2013, 13 patients (median age 73 [range: 62–86] years) were enrolled. One patient did not receive study medication and was replaced. Nine (69%) patients had relapsed or refractory disease and 6 (46%) patients had unfavorable cytogenetics. The most frequently reported treatment-related adverse events (AE) were gastrointestinal events. Twelve SAEs irrespective of relatedness were reported. Two SUSARs were observed, one fatal hypercalcemia and one fatal gastrointestinal infection. Two patients (17%) with relapsed AML achieved a complete remission (one CR, one CRi) and bone marrow blast reductions without fulfilling PR criteria were observed in 3 patients (25%). One-year overall survival was 33%. Nintedanib combined with LDAC shows an adequate safety profile and survival data are promising in a difficult-to-treat patient population. Continuation of this trial with a phase II recommended dose of 2 x 200 mg nintedanib in a randomized, placebo-controlled phase II study is planned. The trial is registered to EudraCT as 2011-001086-41

Long non-coding RNAs defining major subtypes of B cell precursor acute lymphoblastic leukemia

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a novel class of RNA due to its diverse mechanism in cancer development and progression. However, the role and expression pattern of lncRNAs in molecular subtypes of B cell acute lymphoblastic leukemia (BCP-ALL) have not yet been investigated. Here, we assess to what extent lncRNA expression and DNA methylation is driving the progression of relapsed BCP-ALL subtypes and we determine if the expression and DNA methylation profile of lncRNAs correlates with established BCP-ALL subtypes. METHODS: We performed RNA sequencing and DNA methylation (Illumina Infinium microarray) of 40 diagnosis and 42 relapse samples from 45 BCP-ALL patients in a German cohort and quantified lncRNA expression. Unsupervised clustering was applied to ascertain and confirm that the lncRNA-based classification of the BCP-ALL molecular subtypes is present in both our cohort and an independent validation cohort of 47 patients. A differential expression and differential methylation analysis was applied to determine the subtype-specific, relapse-specific, and differentially methylated lncRNAs. Potential functions of subtype-specific lncRNAs were determined by using co-expression-based analysis on nearby (cis) and distally (trans) located protein-coding genes. RESULTS: Using an integrative Bioinformatics analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific and 942 relapse-specific lncRNAs and the methylation profile of three subtypes of BCP-ALL. The 1235 subtype-specific lncRNA signature represented a similar classification of the molecular subtypes of BCP-ALL in the independent validation cohort. We identified a strong correlation between the DUX4-specific lncRNAs and genes involved in the activation of TGF-β and Hippo signaling pathways. Similarly, Ph-like-specific lncRNAs were correlated with genes involved in the activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we found 23 promoter methylated lncRNAs epigenetically facilitating their expression levels. CONCLUSION: Here, we describe a set of subtype-specific and relapse-specific lncRNAs from three major BCP-ALL subtypes and define their potential functions and epigenetic regulation. The subtype-specific lncRNAs are reproducible and can effectively stratify BCP-ALL subtypes. Our data uncover the diverse mechanism of action of lncRNAs in BCP-ALL subtypes defining which lncRNAs are involved in the pathogenesis of disease and are relevant for the stratification of BCP-ALL subtypes