Epigenetic programming defines stem cell identity and entry into the proliferative compartment in chronic myeloid leukaemia (CML)

Chronic myeloid leukaemia (CML) is a haematological malignancy that is identified by the presence of a fusion oncogene, BCR-ABL1, which is a constitutive tyrosine kinase. The discovery of tyrosine kinase inhibitors (TKIs) over that past decade has resulted in significantly improved survival rates and disease management in CML patients. However, a subpopulation of BCR-ABL1+ cells in the niche are found which exhibit stem cell-like features, such as self-renewal and quiescence. These CML stem cells (LSCs) are shown to be insensitive to TKI treatment and are capable of deriving the disease during the relapse. Consequently, the elimination of LSCs is a primary goal of current research. Therefore, the aim of this thesis was to obtain a global view of the cellular processes that maintain stem cell identity in CD34+ CD38- LSCs as well as identify those processes which initiate the transition to proliferative CD34+ CD38+ CML progenitor cells (LPCs). A combined approach was exploited to investigate genome-wide gene expression profiles and histone modification signatures of normal HSCs and committed progenitors (HPCs), and their LSC and LPC counterparts. Despite having increased activity in pathways involved in cell division and proliferation, expression levels of the pathways involved in stem cell identity were not significantly different in LSCs to those found in HSCs. These pathways included Wnt, TGF-β signalling, and several novel neurotransmitter signalling pathways. By examining genome-wide histone modification patterns using ChIP-sequencing it was shown that the stem cell identities of HSCs and LSCs are programmed at the epigenetic level. All of the pathways which confer stem cell identity to both HSCs and LSCs are significantly enriched for bivalent gene promoters having both the H3K4me3 and H3K27me3 marks. These similarities were most evident in neurotransmitter signalling and it was demonstrated that these pathways are capable of promoting LSC maintenance in vitro. Intriguingly, although the stem cell entry into the proliferative state occurs through the repression of many of the same stem cell identity pathways in both HSCs and LSCs, it was shown that epigenetic reprogramming in CML mediates this repression via a different mechanism than in normal HSCs. Furthermore, abnormalities in levels of several chromatin enzymes were identified that are likely to be responsible for the epigenetic reprogramming of CML cells. The work presented in this thesis defines the chromatin landscape of a cancer stem cell for the first time and provides new therapeutic targets for the eradication of TKI resistant CML stem cells

Follicular lymphoma, a B cell malignancy addicted to epigenetic mutations

K Korfi, S Ali, J Heward and J Fitzgibbon are supported by Cancer Research UK Programme Grant [C15966/A15968] and Bloodwise Programme Grant [15002]. S Ali is also a recipient of Cancer Research UK Clinical Careers Committee research bursary [C56515/A21397]

Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition.

UNLABELLED: 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. 1237This article is highlighted in the In This Issue feature, p. 1197

Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells

e Glasgow and Manchester Experimental Cancer Medicine Centres (ECMC), which are funded by CR-UK and the Chief Scientist’s Office (Scotland). We acknowledge the funders who have contributed to this work: MRC stratified medicine infrastructure award (A.D.W.), CR-UK C11074/A11008 (F.P., L.E.M.H., T.L.H., A.D.W.); LLR08071 (S.A.A., E.C.); LLR11017 (M.C.); SCD/04 (M.C.); LLR13035 (S.A.A., K.D., A.D.W., and A.P.); LLR14005 (M.T.S., D.V.); KKL690 (L.E.P.); KKL698 (P.B.); LLR08004 (A.D.W., A.P. and A.J.W.); MRC CiC (M.E.D.); The Howat Foundation (FACS support); Friends of Paul O’Gorman (K.D. and FACS support); ELF 67954 (S.A.A.); BSH start up fund (S.A.A.); MR/K014854/1 (K.D.)

CML cells actively evade host immune surveillance through cytokine-mediated downregulation of MHC-II expression.

Targeting the fusion oncoprotein BCR-ABL with tyrosine kinase inhibitors has significantly affected chronic myeloid leukemia (CML) treatment, transforming the life expectancy of patients; however the risk for relapse remains, due to persistence of leukemic stem cells (LSCs). Therefore it is imperative to explore the mechanisms that result in LSC survival and develop new therapeutic approaches. We now show that major histocompatibility complex (MHC)-II and its master regulator class II transactivator (CIITA) are downregulated in CML compared with non-CML stem/progenitor cells in a BCR-ABL kinase-independent manner. Interferon γ (IFN-γ) stimulation resulted in an upregulation of CIITA and MHC-II in CML stem/progenitor cells; however, the extent of IFN-γ-induced MHC-II upregulation was significantly lower than when compared with non-CML CD34+ cells. Interestingly, the expression levels of CIITA and MHC-II significantly increased when CML stem/progenitor cells were treated with the JAK1/2 inhibitor ruxolitinib (RUX). Moreover, mixed lymphocyte reactions revealed that exposure of CD34+ CML cells to IFN-γ or RUX significantly enhanced proliferation of the responder CD4+CD69+ T cells. Taken together, these data suggest that cytokine-driven JAK-mediated signals, provided by CML cells and/or the microenvironment, antagonize MHC-II expression, highlighting the potential for developing novel immunomodulatory-based therapies to enable host-mediated immunity to assist in the detection and eradication of CML stem/progenitor cells.This study was funded by project grants from Leuka and Tenovus-Scotland (Ref. S12/21). This study was supported by the Glasgow Experimental Cancer Medicine Centre, which is funded by Cancer Research UK and the Chief Scientist’s Office, Scotland. Cell sorting facilities were funded by the Kay Kendall Leukaemia Fund (KKL501) and the Howat Foundation. A.T. was funded by a Bloodwise project grant (13012). P.G. was funded by a Medical Research Council (MRC) UK clinical research training fellowship grant (G1000288). H.G.J. was funded by the Friends of Paul O’Gorman Leukemia Research Centre. F.P., L.E.M.H., and T.L.H. were supported by Cancer Research UK Programme grant (C11074/A11008). D.V. was funded by LLR project grant (14005). A.M.M. was supported by an MRC project grant (MR/K014854/1)

Longitudinal expression profiling identifies a poor risk subset of patients with ABC-type Diffuse Large B Cell Lymphoma

Despite the effectiveness of immuno-chemotherapy, 40\cell lymphoma (DLBCL) experience relapse or refractory disease. Longitudinal studies have previously focused on the mutational landscape of relapse but fell short of providing a consistent relapse-specific genetic signature. In our study, we have focussed attention on the changes in gene expression profile accompanying DLBCL relapse using archival paired diagnostic/relapse specimens from 38 de novo DLBCL patients. Cell of origin remained stable from diagnosis to relapse in 80\ with only a single patient showing COO switching from ABC to GCB. Analysis of the transcriptomic changes that occur following relapse suggest ABC and GCB relapses are mediated via different mechanisms. We developed a 30-gene discriminator for ABC-DLBCLs derived from relapse-associated genes, that defined clinically distinct high and low risk subgroups in ABC-DLBCLs at diagnosis in datasets comprising both population-based and clinical trial cohorts. This signature also identified a population of \lt;60-year-old patients with superior PFS and OS treated with Ibrutinib-R-CHOP as part of the PHOENIX trial. Altogether this new signature adds to the existing toolkit of putative genetic predictors now available in DLBCL that can be readily assessed as part of prospective clinical trials

Misregulation of the PRC2 Complex in CML Stem Cells Confers Sensitivity to an EZH2 Inhibitor

No abstract available

Misregulation of the PRC2 Complex in CML Stem Cells Confers Sensitivity to an EZH2 Inhibitor

No abstract available

Genomic profiling reveals spatial intra-tumor heterogeneity in follicular lymphoma (vol 32, pg 1258, 2018)

International audienc