Supplementary Table S6. ChIP-sequencing datasets used in the present study.

Supplementary Table S1. Genome-wide analysis of mRNA expression levels in HPCs, HSCs, LSCs and LPCs (E-MTAB-2581, Affymetrix HuGe 1.0 ST).

Supplementary Table S2. mRNA expression changes in CML and normal CD34+ cells resulting from treatment of EZHi (GSK343) (EMTAB-2893, MTAB-3552; Affymetrix HuGe 1.0 ST).

Supplementary Table S3. Pathway analysis of mRNA expression changes resulting from treatment of CML CD34+ cells with EZH2i (GSK343).

Supplementary Table S4. Comparison of the efficacy of EZH2i with inhibitors of other novel CML drug targets (4-15) in like-for-like assays.

Supplementary Figure S1. mRNA levels of PRC2 components in CML and normal samples used in this study. Supplementary Figure S2. Effect of TKI treatment on mRNA levels of PRC2 components on CML cells in vitro. Supplementary Figure S3. Analysis of promoters reprogrammed for H3K27me3 levels at promoters. Supplementary Figure S4. Relationship between changes in levels of H3K27me3 and changes in mRNA expression at H3K27me3 targets. Supplementary Figure S5. EZH2i treatment of CML and normal CD34+ cells. Supplementary Figure S6. EZH2i treatment of CML CD34+CD38- cells (LSCs). Supplementary Figure S7. Validation of EZH2 shRNAs in a CML cell line. Supplementary Figure S8. The effect of treatment of CML or normal CD34+ cells with EZH2i (GSK343; 1000 nM) on the global mRNA levels of H3K27me3 and H3K27me1 targets. Supplementary Figure S9. Both imatinib and EZH2i selectively target upregulation of H3K27me3 target genes. Supplementary Figure S10. Nilotinib, dasatinib and EZH2i selectively target upregulation of H3K27me3 target genes. Supplementary Figure S11. mRNA expression levels for H3K27me3 target genes in HSC and LSCs and their behaviour after TKI treatment. Supplementary Figure S12. mRNA expression levels for H3K27me3 target genes in HPC and LPCs (â‰^ CML CD34+) and their behaviour after EZH2i treatment. Supplementary Figure S13. Primary CML cells engrafted in NSG mice treated in vivo with EZH2i and TKI, alone and in combination. Supplementary Figure S14. Validation of mRNA expression changes determined by Affymetrix GeneChip analysis (E-MTAB-2581, Affymetrix HuGe 1.0 ST). Supplementary Figure S15. PRC2 mis-regulation in the three CML patient samples used for Affymetrix GeneChIP and ChIP-sequencing in this study. Supplementary Figure S16. Determining thresholds for identifying H3K27me3 target genes. Supplementary Figure S17. Validation of seqMiner(3) promoter classifications.

Supplementary Table S5. Primary CML and normal samples that were used in this study.

The role that genetic background may play in the responsiveness of organisms to interventions such as caloric restriction (CR) is underappreciated but potentially important. We investigated the impact of genetic background on a suite of metabolic parameters in female recombinant inbred ILSXISS mouse strains previously reported to show divergent lifespan responses to 40% CR (TejJ89-lifespan extension; TejJ48-lifespan unaffected; TejJ114-lifespan shortening). Body mass was reduced across all strains following 10 months of 40% CR, although this loss (relative to ad libitum controls) was greater in TejJ114 relative to the other strains. Gonadal white adipose tissue (gWAT) mass was similarly reduced across all strains following 40% CR, but brown adipose tissue (BAT) mass increased only in strains TejJ89 and TejJ48. Surprisingly, glucose tolerance was improved most notably by CR in TejJ114, while both strains TejJ89 and TejJ114 were hyperinsulinemic following CR relative to their AL controls. We subsequently undertook an unbiased metabolomic approach in gWAT and BAT tissue derived from strains TejJ89 and TejJ114 mice under AL and 40% CR. In gWAT from TejJ89 a significant reduction in several long chain unsaturated fatty acids was observed following 40% CR, but gWAT from TejJ114 appeared relatively unresponsive to CR with far fewer metabolites changing. Phosphatidylethanoloamine lipids within the BAT were typically elevated in TejJ89 following CR, while some phosphatidylglycerol lipids were decreased. However, BAT from strain TejJ114 again appeared unresponsive to CR. These data highlight strain-specific metabolic differences exist in ILSXISS mice following 40% CR. We suggest that precisely how different fat depots respond dynamically to CR may be an important factor in the variable longevity under 40% CR reported in these mice.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsamir183, the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication.

Throughout the lifespan of an individual, the immune system undergoes complex changes while facing novel and chronic infections. Helminths, which infect over one billion people and impose heavy livestock productivity losses, typically cause chronic infections by avoiding and suppressing host immunity. Yet, how age affects immune responses to lifelong parasitic infection is poorly understood. To disentangle the processes involved, we employed supervised statistical learning techniques to identify which factors among haematopoietic stem and progenitor cells (HSPC), and both innate and adaptive responses regulate parasite burdens and how they are affected by host age. Older mice harboured greater numbers of the parasites’ offspring than younger mice. Protective immune responses that did not vary with age were dominated by HSPC, while ageing specifically eroded adaptive immunity, with reduced numbers of naïve T cells, poor T cell responsiveness to parasites, and impaired antibody production. We identified immune factors consistent with previously-reported immune responses to helminths, and also revealed novel interactions between helminths and HSPC maturation. Our approach thus allowed disentangling the concurrent effects of ageing and infection across the full maturation cycle of the immune response and highlights the potential of such approaches to improve understanding of the immune system within the whole organism.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPC) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPC maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase dependent pathway mediated by the up-regulation of hsa-mir183, the down-regulation of its direct target EGR1 and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPC. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPC, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication. For personal use only. on February 19, 2018. by guest www.bloodjournal.org From

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Abstract A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)–persistent leukemic stem cells (LSC). These are BCR–ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specific inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused significant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in significant loss of LSCs compared to TKI alone, in vitro, and in long-term bone marrow murine xenografts. Our findings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs. Significance: In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a significant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248–57. ©2016 AACR. See related article by Xie et al., p. 1237. This article is highlighted in the In This Issue feature, p. 1197

The mechanistic target of rapamycin (mTOR) signalling pathway plays a highly conserved role in aging; mice lacking ribosomal protein S6 kinase 1 (S6K1-/-) have extended lifespan and healthspan relative to wild type (WT) controls. Exactly how reduced mTOR signalling induces such effects is unclear, although preservation of stem cell function may be important. We show, using gene expression analyses, that there was a reduction in expression of cell cycle genes in young (12 week) and aged (80 week) S6K1-/- BM-derived c-Kit+ cells when compared to age-matched WT mice, suggesting that these cells are more quiescent in S6K1-/- mice. In addition, we investigated hematopoietic stem cell (HSC) frequency and function in young and aged S6K1-/-and WT mice. Young, but not aged, S6K1-/-mice had more LSK (lineage-, c-Kit+, Sca-1+) cells (% of bone marrow (BM)), including the most primitive long-term repopulating HSCs (LT-HSC) relative to WT controls. Donor-derived engraftment of LT-HSCs in recipient mice was unaffected by genotype in young mice, but was enhanced in transplants using LT-HSCs derived from aged S6K1-/- mice. Our results are the first to provide evidence that age-associated HSC functional decline is ameliorated in a long-lived mTOR mutant mouse.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y- and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y+ and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal.

We are currently in the midst of a revolution in ageing research, with several dietary, genetic and pharmacological interventions now known to modulate ageing in model organisms. Excitingly, these interventions also appear to have beneficial effects on late-life health. For example, dietary restriction (DR) has been shown to slow the incidence of age-associated cardiovascular disease, metabolic disease, cancer and brain ageing in non-human primates and has been shown to improve a range of health indices in humans. While the idea that DR's ability to extend lifespan is often thought of as being universal, studies in a range of organisms, including yeast, mice and monkeys, suggest that this may not actually be the case. The precise reasons underlying these differential effects of DR on lifespan are currently unclear, but genetic background may be an important factor in how an individual responds to DR. Similarly, recent findings also suggest that the responsiveness of mice to specific genetic or pharmacological interventions that modulate ageing may again be influenced by genetic background. Consequently, while there is a clear driver to develop interventions to improve late-life health and vitality, understanding precisely how these act in response to particular genotypes is critical if we are to translate these findings to humans. We will consider of the role of genetic background in the efficacy of various lifespan interventions and discuss potential routes of utilising genetic heterogeneity to further understand how particular interventions modulate lifespan and healthspan.

Reduced signalling through the insulin/insulin-like growth factor-1 signalling (IIS) pathway is a highly conserved lifespan determinant in model organisms. The precise mechanism underlying the effects of the IIS on lifespan and health is currently unclear, although cellular stress resistance may be important. We have previously demonstrated that mice globally lacking insulin receptor substrate 1 (Irs1(-/-) ) are long-lived and enjoy a greater period of their life free from age-related pathology compared with wild-type (WT) controls. In this study, we show that primary dermal fibroblasts and primary myoblasts derived from Irs1(-/-) mice are no more resistant to a range of oxidant and nonoxidant chemical stressors than cells derived from WT mice.

Chronic myeloid leukaemia (CML) is a hematological malignancy resulting from the transformation of a primitive hematopoietic progenitor by the fusion oncogene BCR-ABL, a constitutively active tyrosine kinase. In recent years major advances have been made in the treatment of CML with the development of tyrosine kinase inhibitors (TKIs), resulting in high rates of remission in CML chronic phase (CP) patients. However, relapse is driven by quiescent and self-renewing BCR-ABL+ CML stem cells (LSCs) that are resistant to TKIs. Consequently, identification of novel proteins or pathways which can be drug-targeted to eliminate the LSCs is a primary goal of current CML research.