ALBERT

Beschreibung: Cancer is caused by accumulated genomic and epigenetic abnormalities during the development of an individual, particularly during the neonatal period, when developmental plasticity is actively occurring. Myeloid-specific deletion of pten in embryos or after 3 weeks of age causes acute monocytic or myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) following a transient myeloproliferative neoplasm (MPN) in adult mice, which can mimic the human diseases to varying degrees. However, it is not clear how the timing of genomic and epigenetic abnormalities contributes to the disease phenotype in a mouse that is of an age comparable to human children. We hypothesized that during the development/aging process, the timing of when the genomic abnormality or “hit” occurs, such as loss of Pten or Nf1, is a critical determinant of the disease phenotype. We tested this by investigating the effect of somatic deletion of Pten at an age of 8 days, one of the most vulnerable stages for malignancy development in mice with or without a germline mutant Nf1. Through crossbreeding, we generated mice with Ptenfl/flNf1Fcr/+Mx1-Cre+ on a C57BL6/129 genetic background, and conditionally deleted Pten in a myeloid-specific manner by intraperitoneal injection of Poly(I:C). Mice with a pten deletion and mutant Nf1 (ptenkoNf1mut, hereafter referred as double mutant) showed signs of sickness at the end of the 2nd week of life, and all died by age 3-5 weeks (equivalent to 1-3 years old in humans). The natural survival in double mutant mice (n=10) was significantly shorter than those with wild type pten and Nf1 (ptenwt; Nf1wt, hereafter referred as WT, n=6, median 0.9 vs 〉14 months, p

Beschreibung: Germline mutations in GATA2, a gene that encodes for transcription factors involved in hematopoiesis and vascular development, have recently been described in MonoMAC syndrome, Emberger syndrome and in select cases of mild chronic neutropenia. These disorders are unified by their predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Patients with MonoMAC syndrome have also been noted to display monosomy 7 in their bone marrows in up to 50% of cases. Overexpression of GATA2 due to somatic mutations in cases of de novo pediatric AML, has also been shown to be a negative predictor of outcome. Juvenile myelomonocytic leukemia is a rare childhood malignancy with overlapping features of MDS and myeloproliferative neoplasm (MPN) that can transform to AML and is characterized by hyperactive RAS signaling. Mutations in NF1, NRAS, KRAS, PTPN11, and CBL are found in 85-90% of newly diagnosed patients, and monosomy 7 is the most common recurrent karyotypic abnormality seen in JMML. We therefore hypothesized that mutations in GATA2 may play a role in the development of JMML. Samples from 57 patients with JMML were screened for GATA2 mutations. Patient samples and clinical data were collected from the Children's Oncology Group (COG) trial AAML0122. DNA was extracted as per previous protocols from peripheral blood or bone marrow and whole genome amplified using Qiagen REPLI-g kit according to manufacturer specifications. We performed bidirectional Sanger sequencing (Beckman Coulter Genomics) of the entire coding region of GATA2 (NM_001145661.1) and aligned the sequences using CLC Workbench software (CLC Bio, Aarhus, Denmark). Only missense, splice site or nonsense mutations were evaluated using SIFT (Sorting Tolerant From Intolerant) to predict the impact on the structure and function of identified mutations on the protein. Patient J384 was found to have a nonsense point mutation at c.988C〉T (R330X) in the N-terminal region of the zinc finger portion of the protein (Figure 1a). This hotspot mutation has been reported in several patients with mild chronic neutropenia who displayed a predisposition to developing MDS and AML. The patient was also found to have a missense point mutation at c.962T〉G (L321R) predicted to be damaging by SIFT. Subcloning of the gene using a TA cloning kit with pCR 2.1 vector (Invitrogen), followed by direct sequencing of individual colony picks, revealed that the two sequence variants only occurred in a trans configuration. Out of 40 amplicons sequenced, 20 were found to have the c.988C〉T transition, 16 were found to be have the c.962T〉G variant, and four were found to be wild type. We therefore hypothesize that the c.988C〉T was inherited as a germline event and that c.962T〉G was somatically acquired in the majority of the remaining wild type alleles. No other point mutations or insertions/deletions were discovered in this cohort.Figure 1Identification of 2 distinct GATA2 mutations in patient J384.Figure 1. Identification of 2 distinct GATA2 mutations in patient J384. This patient was previously identified to have a KRAS G12D mutation (c.35G〉A) as well as monosomy 7. This patient died prior to undergoing transplant within months of diagnosis. While the patient technically met criteria for the diagnosis of JMML, it should be noted there were several atypical features, including older age at diagnosis (4 years and 10 months), and absence of hypersensitivity in myeloid progenitor cells to the cytokine granulocyte–macrophage colony stimulating factor (GM-CSF) in colony assay. This raises the possibility that patient J384 actually had MonoMAC syndrome with MDS and not JMML. This represents the first description of a GATA2 mutation in a patient suspected of having JMML. To our knowledge, this is the first report of a biallelic mutation in GATA2, combining a germline mutation with somatic acquisition. In addition, MonoMAC syndrome has not been reported to be associated with KRAS mutations to date. GATA2 mutations should therefore be considered in patients with atypical features of MDS or JMML. Panel (a) Bidirectional sequencing of patient sample J384 revealed two distinct sequence variants in both the forward (shown here) and reverse strands. Panel (b) Sequencing of 40 individual colony picks revealed that each sequence variant occurred in a trans configuration (CP 9 and CP13 are shown here as examples). In addition, 10% of colony picks (i.e. CP 32) revealed a wild type sequence, indicating that at least one of the two variants was a somatic event. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 2558 Juvenile myelomonocytic leukemia (JMML) is a rare disease of early childhood with a predilection for the monocyte/macrophage lineage. The pathogenesis of JMML is linked to dysregulated signal transduction through the NF1/RAS signaling pathway that is partially caused by genetic mutation of Ras, PTPN11, and c-CBL, or loss-of heterozygosity of Nf1. The hallmark of JMML is that JMML cells are selectively hypersensitive to GM-CSF in vitro. We previously reported that protein deficiencies of PTEN, CREB, and Egr-1 were frequently observed in JMML (67–87%). Recent research indicated that CREB was regulated by miR-34b, and Egr-1 was targeted by miR-183. We hypothesized that microRNAs may play an important role in contributing to the deficiency of these proteins. Using relative-quantitative real-time PCR, we evaluated the expression levels of miR-34b and miR-183 in mononuclear cells from 47 JMML patients. We found that the median level of miR-183 was significantly higher in JMML in comparison to normal controls (median=13.8 vs 4.2, p0.05). This suggests that miR-34b does not play a significant role in JMML. Since extreme monocyte accumulation is one of the critical characteristics of JMML, we analyzed the correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood. Strikingly, there was a significant correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood from 34 patients who had available data (p

Beschreibung: Abstract 2504 Cytokine independence is a common event in leukemia. The mechanism is often linked to deregulation of Ras/PI3K, Ras/MAP, and JAK/STAT pathways. PTEN, a major negative regulator of the Ras pathway, was initially isolated as a tumor suppressor in a variety of malignancies. We recently reported that PTEN is deficient in 67% of juvenile myelomonocytic leukemia (JMML) patients. Other groups have also found alterations of PTEN, PI3K, or AKT in 47.7% of T-cell acute lymphoblastic leukemia. Mice who are rendered pten-deficient develop myeloproliferative disorders and acute myeloid and lymphoid leukemias, and the leukemia-initiating stem cell in these mouse models could be distinguished from the normal hematopoietic stem cell by a differential response to rapamycin. The transcription factor, Egr-1, has been known to be a positive regulator of PTEN expression. It plays an important role in controlling proliferation as well as mobilization of hematopoietic stem cells. It is unknown whether PTEN had a feedback loop on regulating Egr-1 expression. Since poorly differentiated progenitors are often mobilized to the peripheral blood in leukemia patients, we hypothesized that PTEN deficiency alters the feedback loop of Egr-1 in response to cytokine stimulation. In this manner, PTEN deficiency might contribute to the cytokine independence observed in many types of leukemia. In order to evaluate this hypothesis we investigated the role of PTEN in cells responding to GM-CSF stimulation in the leukemia cell line, TF-1a. TF-1a is PTEN deficient as well as cytokine independent, and displays a constitutively high expression of Egr-1. We restored PTEN expression in TF-1a cells. This was accomplished by packaging the coding sequence of wild-type PTEN in a retrovirus vector, pBMN-GFP, that carried a gene encoding green fluorescent protein (GFP). TF-1a cells were transfected with pBMN-PTEN-GFP or pBMN-GFP by spinoculation. The pure population of transfected cells was sorted by FACS, followed by limiting dilution. The transfected single colonies were expanded for further evaluation of PTEN expression and biological function. Data from these experiments showed: 1) over-expression PTEN in TF-1a down-regulated the expression of Egr-1 and restored the responsiveness of Egr-1 to GM-CSF stimulation, 2) PTEN restored the responsiveness of TF-1a to GM-CSF and IL-3, and 3) these events are CREB-independent. In conclusion, our data demonstrates that PTEN contributes to the cell responsiveness to GM-CSF and IL-3, and has a negative feedback role to regulate Egr-1 expression in TF-1a cells. In addition to Egr-1 regulating PTEN in a positive manner, our data indicates the presence of a feedback loop from PTEN back to Egr-1. This appears to be an event independent of CREB. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Macrophages (MΦ) are professional phagocytes in the innate immune system. They are not only involved in regulation of various immune functions and inflammation, but also exhibit plasticity in modulation of tissue regeneration and repair after being polarized into M1 and M2 MΦ by different inflammatory cytokines. In addition, several recent studies show that MΦ are a new constituent of the hematopoietic stem cells (HSCs) niche and play a role in regulation of HSCs maintenance and mobilization in bone marrow (BM). However, it is not known whether MΦ can regulate HSCs self-renewal and whether the effects of MΦ on HSCs can be influenced by differential MΦ polarization. This was investigated using an ex vivo HSCs expansion model consisting of mouse bone marrow LSK (Lin-sca-1+c-Kit+) cells cultured with or without MΦ in a mouse HSCs expansion medium (StemSpanTM serum-free medium supplemented with 20ng/ml of stem cell factor [SCF] and thrombopoietin [TPO]). We found that LSK cells were expanded about 20-, 15-, and 30-fold after 6 days of co-culture with MΦ harvested from mouse BM, spleen, and peritoneal cavity, respectively, whereas there was no significant expansion after culture without MΦ or with BM Gr-1high or Gr-1low monocytes. In addition, we found that M1-MΦ polarized by INFγ were more effective than IL4-polarized M2-MΦ in promoting LSK cells expansion ex vivo (45-fold vs. 15-fold). However, the promotion of LSK cells expansion by M1-MΦ resulted in about 88% reduction in HSCs as judged by 5-week cobblestone area forming cell (CAFC) assay. In contrast, M2-MΦ significantly promoted HSCs expansion. A greater expansion of HSCs was achieved after LSK cells were co-cultured with M2-MΦ for 9 days than for 6 days (20-fold vs. 6-fold). These findings suggest that M1-MΦ are more effective than M2-MΦ in promoting LSK cells or hematopoietic progenitor cells (HPCs) expansion, at the expense of HSCs self-renewal, whereas M2-MΦ can promote HPCs expansion as well as HSCs self-renewal. This suggestion is supported by results of serial transplantation and competitive repopulation unit (CRU) assays. CRU assay showed that LT-HSCs (e.g. 4-month CRU) were increased about 13 folds relative to the starting numbers of CRU in the input after LSK cells were co-cultured with M2-MΦ for 9 days, but were barely detectable after the cells were cultured without MΦ or with M1-MΦ. The inhibitory effect of M1-MΦ on HSCs self-renewal and expansion was attenuated by inhibition of inducible nitric oxide synthase (iNOS) activity with an inhibitor or knockout iNOS. Inhibition of arginase and/or cyclooxygenase activities with an inhibitor attenuated the promotion of HSCs self-renewal and expansion by M2-MΦ. More importantly, we found that human CD34+ cells, 8-week CAFC, and SCID mice repopulating cells (SRCs) were increased 42±14, 8±2.1, and 4 folds over the input values, respectively, after human cord blood CD34+ cells were co-cultured with M2-MΦ generated from human cord blood CD34- cells for 7 days in a human HSCs expansion medium (StemSpanTM serum-free medium supplemented with 50 ng/ml of SCF, TPO, and FLT-3 ligand). These findings demonstrate that M1-MΦ and M2-MΦ have opposite effects on HSCs self-renewal, which may be important for regulation of hematopoiesis under various pathological conditions in which MΦ are differentially polarized to M1 or M2 by diverse inflammatory cytokines. In addition, M2-MΦ may be used to promote human cord blood HSCs ex vivo expansion to make human cord blood transplantation available to more patients. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD). It occurs in infancy and young children with a progressive course leading to death within one year after diagnosis. This disease is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. Mutations or other abnormalities in RAS, NF1, PTPN11, and CBL have been linked to be responsible for the pathogenesis of JMML in up to 85% of cases. Treatment is very difficult in JMML, and only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28-55%), with 5-year disease-free survival rates of 25-40%. JMML occurs in an age-range when genes are actively being turned on or off in children in adaption to the oxygenized environment after birth. Epigenetics plays a key role in this developmental plasticity. We previously reported hypermethylation on the promoter of PTEN in 77% of JMML patients, and decitabine, a DNA-hypomethylating reagent, significantly inhibited colony formation (CFU-GM) in JMML cells in vitro. In addition, other groups found that aberrant DNA methylation on promoters of BMP4, CALCA, CDKN2B, and RARB is significantly associated with poor prognosis in JMML. Taking together, these data suggest that epigenetic mechanisms may contribute to the pathogenesis of JMML. MicroRNAs (miRNAs) have been reported to play an important role in myeloid differentiation and activation. miRNA function is highly dependent on the cell type. Recently, we reported that miR-183 is overexpressed in JMML. Other groups have reported aberrant expression of miR-29a in acute myeloid leukemia and other cancers. Both miR-183 and miR-29a are located on chromosome 7q32 in humans, which is frequently disrupted in JMML. We hypothesized that miR-29a may be deregulated in JMML, and contribute to the aberrant epigenetic regulation in JMML. In order to test our hypothesis, we collected peripheral blood or bone marrow from 41 JMML patients and 14 normal individuals. Total RNAs were extracted from mononuclear cells (MNCs) using Trizol. We first evaluated the expression levels of miR-29a by using relative-quantitative real-time RT-PCR (qRT-PCR). We found that the expression levels (RQ) of miR-29a in patients are significantly lower than that in normal individuals (median 0.45 vs. 1.11, p〈 0.001). By analysis of the expression levels of miR-29a together with previous data on miR-183 in these JMML patients and normal controls, we found that the RQ of miR-29a is inversely correlated with RQ of miR-183 (Spearman’s r=-51, p

Beschreibung: Juvenile Myelomonocytic Leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood with a 5-year event free survival of 52% after hematopoietic stem cell transplantation (HSCT). A hallmark of JMML is aberrant Ras pathway activation due to mutations in NF1, NRAS, KRAS, PTPN11 and CBL. However, robust predictors of response are lacking, as individual mutations are not reliably associated with outcome, and relapse remains the most common reason for treatment failure. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders, including JMML (Piazza et al Nat Genet 2012, Makishima et al Nat Genet 2013, Sakaguchi et al Nat Genet, 2013). These mutations had a lower allelic frequency compared to Ras pathway mutations, but were associated with poor prognosis. These and other data suggested that SETBP1 mutations contribute to disease progression rather than initiation. We identified several patients with JMML who had clonal SETBP1 mutations detected at relapse. Analysis of mononuclear cell extracted DNA from serial samples of two patients who relapsed revealed an increase in the SETBP1 mutant allele frequency over time (Figure 1). Similarly, analysis of colonies plated in methylcellulose from serial time points indicated that the percentage of individual myeloid progenitor colonies that were heterozygous or homozygous for the SETBP1 mutation increased with each sequential sample despite intensive treatment. Based on these data, we tested the hypothesis that rare SETBP1 mutant clones exist at diagnosis in many patients who relapse, and that these rare cells undergo positive selection during treatment. Using a droplet digital PCR (ddPCR) technology with a detection threshold as low as 0.001% of mutant DNA, we identified SETBP1 mutations in 16/53 (30%) of diagnostic JMML specimens from children treated on Children's Oncology Group trial AAML0122. Of these mutations, 12 were subclonal and 4 were clonal. Event free survival (EFS) at 4 years in patients with SETBP1 mutations was 19% ± 10% compared to 51% ± 8% in those with wild type SETBP1 (p=0.006). While samples of patients who relapsed on the AAML0122 trial were not available for analysis, one patient recently undergoing treatment who had a subclonal SETBP1 mutation (0.45% allelic fraction) detected at diagnosis by ddPCR, demonstrated an overt SETBP1 mutation at relapse. Finally, we isolated and analyzed hematopoietic stem (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), and granulocyte-monocyte progenitor (GMP) populations from a relapsed sample with a SETBP1 mutation. Sanger sequencing demonstrated that all four progenitor compartments were affected by the mutation. Analysis of additional samples is underway. We conclude that the presence of a subclonal mutation in SETBP1 is a novel biomarker of adverse outcome in JMML. Understanding the mechanisms underpinning SETBP1-mediated resistance and relapse, and further identifying therapeutic vulnerabilities of HSCs expressing these mutant proteins will be critical to improve outcomes for patients with JMML and other myeloid malignancies. Furthermore, the presence of a subclonal SETBP1 mutation at diagnosis might identify JMML patients who will benefit from more intensive conditioning prior to HSCT or from novel therapeutic strategies. Figure 1 Figure 1. Disclosures Troup: Bio-Rad Laboratories: Employment.