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

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.

Epigenetic dysregulation in chronic myeloid leukaemia: A myriad of mechanisms and therapeutic options.

The onset of global epigenetic changes in chromatin that drive tumor proliferation and heterogeneity is a hallmark of many forms of cancer. Identifying the epigenetic mechanisms that govern these changes and developing therapeutic approaches to modulate them, is a well-established avenue pursued in translational cancer medicine. Chronic myeloid leukemia (CML) arises clonally when a hematopoietic stem cell (HSC) acquires the capacity to produce the constitutively active tyrosine kinase BCR-ABL1 fusion protein which drives tumor development. Treatment with tyrosine kinase inhibitors (TKI) that target BCR-ABL1 has been transformative in CML management but it does not lead to cure in the vast majority of patients. Thus novel therapeutic approaches are required and these must target changes to biological pathways that are aberrant in CML - including those that occur when epigenetic mechanisms are altered. These changes may be due to alterations in DNA or histones, their biochemical modifications and requisite 'writer' proteins, or to dysregulation of various types of non-coding RNAs that collectively function as modulators of transcriptional control and DNA integrity. Here, we review the evidence for subverted epigenetic mechanisms in CML and how these impact on a diverse set of biological pathways, on disease progression, prognosis and drug resistance. We will also discuss recent progress towards developing epigenetic therapies that show promise to improve CML patient care and may lead to improved cure rates.

The chronic myeloid leukemia stem cell: stemming the tide of persistence.

Chronic myeloid leukemia (CML) is caused by the acquisition of the tyrosine kinase BCR-ABL1 in a hemopoietic stem cell, transforming it into a leukemic stem cell (LSC) that self-renews, proliferates, and differentiates to give rise to a myeloproliferative disease. Although tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL1 have transformed CML from a once-fatal disease to a manageable one for the vast majority of patients, only ∼10% of those who present in chronic phase (CP) can discontinue TKI treatment and maintain a therapy-free remission. Strong evidence now shows that CML LSCs are resistant to the effects of TKIs and persist in all patients on long-term therapy, where they may promote acquired TKI resistance, drive relapse or disease progression, and inevitably represent a bottleneck to cure. Since their discovery in patients almost 2 decades ago, CML LSCs have become a well-recognized exemplar of the cancer stem cell and have been characterized extensively, with the aim of developing new curative therapeutic approaches based on LSC eradication. This review summarizes our current understanding of many of the pathways and mechanisms that promote the survival of the CP CML LSCs and how they can be a source of new gene coding mutations that impact in the clinic. We also review recent preclinical approaches that show promise to eradicate the LSC, and future challenges on the path to cure.

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.

Chromatin looping defines expression of TAL1, its flanking genes, and regulation in T-ALL.

TAL1 is an important regulator of hematopoiesis and its expression is tightly controlled despite complexities in its genomic organization. It is frequently misregulated in T-cell acute lymphoblastic leukemia (T-ALL), often due to deletions between TAL1 and the neighboring STIL gene. To better understand the events that lead to TAL1 expression in hematopoiesis and in T-ALL, we studied looping interactions at the TAL1 locus. In TAL1-expressing erythroid cells, the locus adopts a looping "hub" which brings into close physical proximity all known TAL1 cis-regulatory elements including CTCF-bound insulators. Loss of GATA1 results in disassembly of the hub and loss of CTCF/RAD21 from one of its insulators. Genes flanking TAL1 are partly dependent on hub integrity for their transcriptional regulation. We identified looping patterns unique to TAL1-expressing T-ALL cells, and, intriguingly, loops occurring between the TAL1 and STIL genes at the common TAL1/STIL breakpoints found in T-ALL. These findings redefine how TAL1 and neighboring genes communicate within the nucleus, and indicate that looping facilitates both normal and aberrant TAL1 expression and may predispose to structural rearrangements in T-ALL. We also propose that GATA1-dependent looping mechanisms may facilitate the conservation of TAL1 regulation despite cis-regulatory remodeling during vertebrate evolution.

Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease.

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease - with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Leukaemia exposure alters the transcriptional profile and function of BCR::ABL1 negative macrophages in the bone marrow niche.

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages.

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Tyrosine Kinase Inhibitor Independent Gene Expression Signature in CML Offers New Targets for LSPC Eradication Therapy.

Tyrosine kinase inhibitors (TKI) have revolutionised the treatment of CML. However, TKI do not eliminate the leukaemia stem cells (LSC), which can re-initiate the disease. Thus, finding new therapeutic targets in CML LSC is key to finding a curative treatment. Using microarray datasets, we defined a list of 227 genes that were differentially expressed in CML LSC compared to the healthy controls but were not affected by TKI in vitro. Two of them, CD33 and PPIF, are targeted by gemtuzumab-ozogamicin and cyclosporin A, respectively. We treated CML and the control CD34+ cells with either drug with or without imatinib to investigate the therapeutic potential of the TKI-independent gene expression programme. Cyclosporine A, in combination with imatinib, reduced the number of CML CFC compared with non-CML controls, but only at supra-therapeutic concentrations. Gemtuzumab-ozogamicin showed an EC50 of 146 ng/mL, below the plasma peak concentration of 630 ng/mL observed in the AML patients and below the EC50 of 3247 ng/mL observed in the non-CML cells. Interestingly, gemtuzumab-ozogamicin seems to promote cell cycle progression in CML CD34+ cells and demonstrated activation of the RUNX1 pathway in an RNAseq experiment. This suggests that targeting the TKI-independent genes in CML LSC could be exploited for the development of new therapies in CML.

The Spliceosome: A New Therapeutic Target in Chronic Myeloid Leukaemia.

RNA splicing factors are frequently altered in cancer and can act as both oncoproteins and tumour suppressors. They have been found mutated or deregulated, justifying the growing interest in the targeting of splicing catalysis, splicing regulatory proteins, and/or specific, key altered splicing events. We recently showed that the DNA methylation alterations of CD34+CD15- chronic myeloid leukaemia (CML) cells affect, among others, alternative splicing genes, suggesting that spliceosome actors might be altered in chronic-phase (CP)-CML. We investigated the expression of 12 spliceosome genes known to be oncogenes or tumour suppressor genes in primary CP-CML CD34+ cells at diagnosis (n = 15). We found that CP-CML CD34+ cells had a distinct splicing signature profile as compared with healthy donor CD34+ cells or whole CP-CML cells, suggesting: (i) a spliceosome deregulation from the diagnosis time and (ii) an intraclonal heterogeneity. We could identify three profile types, but there was no relationship with a patient's characteristics. By incubating cells with TKI and/or a spliceosome-targeted drug (TG003), we showed that CP-CML CD34+ cells are both BCR::ABL and spliceosome dependent, with the combination of the two drugs showing an additive effect while sparing healthy donors cells. Our results suggest that the spliceosome may be a new potential target for the treatment of CML.

Autosomal-dominant microtia linked to five tandem copies of a copy-number-variable region at chromosome 4p16.

Recently, large-scale benign copy-number variations (CNVs)--encompassing over 12% of the genome and containing genes considered to be dosage tolerant for human development--were uncovered in the human population. Here we present a family with a novel autosomal-dominantly inherited syndrome characterized by microtia, eye coloboma, and imperforation of the nasolacrimal duct. This phenotype is linked to a cytogenetically visible alteration at 4pter consisting of five copies of a copy-number-variable region, encompassing a low-copy repeat (LCR)-rich sequence. We demonstrate that the approximately 750 kb amplicon occurs in exact tandem copies. This is the first example of an amplified CNV associated with a Mendelian disorder, a discovery that implies that genome screens for genetic disorders should include the analysis of so-called benign CNVs and LCRs.

A KDM4A-PAF1-mediated epigenomic network is essential for acute myeloid leukemia cell self-renewal and survival.

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

ULK1 inhibition promotes oxidative stress-induced differentiation and sensitizes leukemic stem cells to targeted therapy.

Inhibition of autophagy has been proposed as a potential therapy for individuals with cancer. However, current lysosomotropic autophagy inhibitors have demonstrated limited efficacy in clinical trials. Therefore, validation of novel specific autophagy inhibitors using robust preclinical models is critical. In chronic myeloid leukemia (CML), minimal residual disease is maintained by persistent leukemic stem cells (LSCs), which drive tyrosine kinase inhibitor (TKI) resistance and patient relapse. Here, we show that deletion of autophagy-inducing kinase ULK1 (unc-51­like autophagy activating kinase 1) reduces growth of cell line and patient-derived xenografted CML cells in mouse models. Using primitive cells, isolated from individuals with CML, we demonstrate that pharmacological inhibition of ULK1 selectively targets CML LSCs ex vivo and in vivo, when combined with TKI treatment. The enhanced TKI sensitivity after ULK1-mediated autophagy inhibition is driven by increased mitochondrial respiration and loss of quiescence and points to oxidative stress­induced differentiation of CML LSCs, proposing an alternative strategy for treating patients with CML.

The leukaemia stem cell: similarities, differences and clinical prospects in CML and AML.

For two decades, leukaemia stem cells (LSCs) in chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) have been advanced paradigms for the cancer stem cell field. In CML, the acquisition of the fusion tyrosine kinase BCR-ABL1 in a haematopoietic stem cell drives its transformation to become a LSC. In AML, LSCs can arise from multiple cell types through the activity of a number of oncogenic drivers and pre-leukaemic events, adding further layers of context and genetic and cellular heterogeneity to AML LSCs not observed in most cases of CML. Furthermore, LSCs from both AML and CML can be refractory to standard-of-care therapies and persist in patients, diversify clonally and serve as reservoirs to drive relapse, recurrence or progression to more aggressive forms. Despite these complexities, LSCs in both diseases share biological features, making them distinct from other CML or AML progenitor cells and from normal haematopoietic stem cells. These features may represent Achilles' heels against which novel therapies can be developed. Here, we review many of the similarities and differences that exist between LSCs in CML and AML and examine the therapeutic strategies that could be used to eradicate them.

Epigenetic Reprogramming and Emerging Epigenetic Therapies in CML.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by BCR-ABL1, an oncogenic fusion gene arising from the Philadelphia chromosome. The development of tyrosine kinase inhibitors (TKIs) to overcome the constitutive tyrosine kinase activity of the BCR-ABL protein has dramatically improved disease management and patient outcomes over the past 20 years. However, the majority of patients are not cured and developing novel therapeutic strategies that target epigenetic processes are a promising avenue to improve cure rates. A number of epigenetic mechanisms are altered or reprogrammed during the development and progression of CML, resulting in alterations in histone modifications, DNA methylation and dysregulation of the transcriptional machinery. In this review these epigenetic alterations are examined and the potential of epigenetic therapies are discussed as a means of eradicating residual disease and offering a potential cure for CML in combination with current therapies.

Gene expression profiling in polycythemia vera using cDNA microarray technology.

Polycythemia vera (PV) is a myeloproliferative disorder characterized by an increased proliferation of all three myeloid lineages. The molecular pathogenesis of PV is unknown. Using cDNA microarrays comprising 6000 human genes, we studied the gene expression profile of granulocytes obtained from 11 PV patients compared with granulocytes obtained from healthy individuals. We found that 147 genes were up-regulated by >/==" BORDER="0">2.5 fold in the majority of PV patients. Eleven of these 147 genes were up-regulated in all PV patients studied and may represent a molecular signature for this disorder. An increase in the expression of several protease inhibitors with affinity for proteases that promote apoptosis in neutrophils (e.g., cystatin F, secretory leukocyte protease inhibitor), as well as the up-regulation of a number of antiapoptotic and survival factors was found (e.g., adrenomedullin, p38 mitogen-activated protein kinase). We speculate that the deregulation of these factors may inhibit normal apoptosis and promote cell survival in the granulocytes of patients with PV. These PV-specific expression changes are likely to be biologically important in the pathophysiology of this disorder.

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

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.

Identification of genetic aberrations on chromosome 22 outside the NF2 locus in schwannomatosis and neurofibromatosis type 2.

Schwannomatosis is characterized by multiple peripheral and cranial nerve schwannomas that occur in the absence of bilateral 8th cranial nerve schwannomas. The latter is the main diagnostic criterion of neurofibromatosis type 2 (NF2), which is a related but distinct disorder. The genetic factors underlying the differences between schwannomatosis and NF2 are poorly understood, although available evidence implicates chromosome 22 as the primary location of the gene(s) of interest. To investigate this, we comprehensively profiled the DNA copy number in samples from sporadic and familial schwannomatosis, NF2, and a large cohort of normal controls. Using a tiling-path chromosome 22 genomic array, we identified two candidate regions of copy number variation, which were further characterized by a PCR-based array with higher resolution. The latter approach allows the detection of minute alterations in total genomic DNA, with as little as 1.5 kb per measurement point of nonredundant sequence on the array. In DNA derived from peripheral blood from a schwannomatosis patient and a sporadic schwannoma sample, we detected rearrangements of the immunoglobulin lambda (IGL) locus, which is unlikely to be due to a B-cell specific somatic recombination of IGL. Analysis of normal controls indicated that these IGL rearrangements were restricted to schwannomatosis/schwannoma samples. In the second candidate region spanning GSTT1 and CABIN1 genes, we observed a frequent copy number polymorphism at the GSTT1 locus. We further describe missense mutations in the CABIN1 gene that are specific to samples from schwannomatosis and NF2 and make this gene a plausible candidate for contributing to the pathogenesis of these disorders.