ALBERT

Description: 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

Description: 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.

Description: 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.