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

MTSS1 is a critical epigenetically regulated tumor suppressor in CML

Chronic myeloid leukemia (CML) is driven by malignant stem cells that can persist despite therapy. We have identified Metastasis suppressor 1 (Mtss1/MIM) to be downregulated in hematopoietic stem and progenitor cells from leukemic transgenic SCLtTA/Bcr-Abl mice and in patients with CML at diagnosis, and Mtss1 was restored when patients achieved complete remission. Forced expression of Mtss1 decreased clonogenic capacity and motility of murine myeloid progenitor cells and reduced tumor growth. Viral transduction of Mtss1 into lineage depleted SCLtTA/Bcr-Abl bone marrow cells decreased leukemic cell burden in recipients, and leukemogenesis was reduced upon injection of Mtss1 overexpressing murine myeloid 32D cells. Tyrosine kinase inhibitor (TKI) therapy and reversion of Bcr-Abl expression increased Mtss1 expression but failed to restore it to control levels. CML patient samples revealed higher DNA methylation of specific Mtss1 promoter CpG sites that contain binding sites for Kaiso and Rest transcription factors. In summary, we identified a novel tumor suppressor in CML stem cells that is downregulated by both Bcr-Abl kinase-dependent and -independent mechanisms. Restored Mtss1 expression markedly inhibits primitive leukemic cell biology in vivo, providing a therapeutic rationale for the Bcr-Abl-Mtss1 axis to target TKI resistant CML stem cells in patients

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

Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1−/− bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1−/− in MLL—AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia

PIWIL4 Acts as a piRNA Binding, Epigenetically Active and Growth Regulatory Protein in Human Acute Myeloid Leukemia

Piwi proteins are critically important for maintaining the self-renewing stem cell population in lower organisms through epigenetic silencing of transposable elements via DNA methylation and H3K9me3 marks, in close interaction with a novel class of non-coding RNA called piwi interacting RNA (piRNA)

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

Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance

Feasibility of Azacitidine Added to Standard Chemotherapy in Older Patients with Acute Myeloid Leukemia — A Randomised SAL Pilot Study

Introduction: Older patients with acute myeloid leukemia (AML) experience short survival despite intensive chemotherapy. Azacitidine has promising activity in patients with low proliferating AML. The aim of this dose-finding part of this trial was to evaluate feasibility and safety of azacitidine combined with a cytarabine- and daunorubicin-based chemotherapy in older patients with AML. Trial Design: Prospective, randomised, open, phase II trial with parallel group design and fixed sample size. Patients and Methods: Patients aged 61 years or older, with untreated acute myeloid leukemia with a leukocyte count of ,20,000/ml at the time of study entry and adequate organ function were eligible. Patients were randomised to receive azacitidine either 37.5 (dose level 1) or 75 mg/sqm (dose level 2) for five days before each cycle of induction (7+3 cytarabine plus daunorubicine) and consolidation (intermediate-dose cytarabine) therapy. Dose-limiting toxicity was the primary endpoint. Results: Six patients each were randomised into each dose level and evaluable for analysis. No dose-limiting toxicity occurred in either dose level. Nine serious adverse events occurred in five patients (three in the 37.5 mg, two in the 75 mg arm) with two fatal outcomes. Two patients at the 37.5 mg/sqm dose level and four patients at the 75 mg/sqm level achieved a complete remission after induction therapy. Median overall survival was 266 days and median event-free survival 215 days after a median follow up of 616 days. Conclusions: The combination of azacitidine 75 mg/sqm with standard induction therapy is feasible in older patients with AML and was selected as an investigational arm in the randomised controlled part of this phase-II study, which is currently halted due to an increased cardiac toxicity observed in the experimental arm. Trial Registration: This trial is registered at clinical trials.gov (identifier: NCT00915252)

The Inhibitor of Growth Protein 5 (ING5) Depends on INCA1 as a Co-Factor for Its Antiproliferative Effects

The proteins of the Inhibitor of Growth (ING) family are involved in multiple cellular functions such as cell cycle regulation, apoptosis, and chromatin remodeling. For ING5, its actual role in growth suppression and the necessary partners are not known. In a yeast-two-hybrid approach with human bone marrow derived cDNA, we identified ING5 as well as several other proteins as interaction partners of Inhibitor of cyclin A1 (INCA1) that we previously characterized as a novel interaction partner of cyclin A1/CDK2. ING5 expression in leukemic AML blasts was severely reduced compared to normal bone marrow. In line, ING5 inhibited bone marrow colony formation upon retroviral transduction. However, Inca1−/− bone marrow colony formation was not suppressed by ING5. In murine embryonic fibroblast (MEF) cells from Inca1+/+ and Inca1−/− mice, overexpression of ING5 suppressed cell proliferation only in the presence of INCA1, while ING5 had no effect in Inca1−/− MEFs. ING5 overexpression induced a delay in S-phase progression, which required INCA1. Finally, ING5 overexpression enhanced Fas-induced apoptosis in Inca1+/+ MEFs, while Inca1−/− MEFs were protected from Fas antibody-induced apoptosis. Taken together, these results indicate that ING5 is a growth suppressor with suppressed expression in AML whose functions depend on its interaction with INCA1

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