Gillian A. Horne

Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma

Background:Multiple myeloma is a plasma cell disorder that is characterised by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in the blood or urine and associated organ dysfunction. It accounts for approximately 1% of cancers and 13% of haematological cancers. Myeloma arises from an asymptomatic proliferation of monoclonal plasma cells termed monoclonal gammopathy of undetermined significance (MGUS).Methods:MicroRNA expression profiling of serum samples was performed on three patient groups as well as normal controls. Validation of the nine microRNAs detected as promising biomarkers was carried out using TaqMan quantitative reverse transcription PCR. MicroRNA levels in serum were normalised using standard curves to determine the numbers of microRNAs per μl of serum.Results:Three serum microRNAs, miR-720, miR-1308 and miR-1246, were found to have potential as diagnostic biomarkers in myeloma. Use of miR-720 and miR-1308 together provides a powerful diagnostic tool for distinguishing normal healthy controls, as well as patients with unrelated illnesses, from pre-cancerous myeloma and myeloma patients. In addition, the combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients.Conclusion:We have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for multiple myeloma.

BRD4 associates with p53 in DNMT3A-mutated leukemia cells and is implicated in apoptosis by the bromodomain inhibitor JQ1

The bromodomain and extra terminal (BET) family protein bromodomain containing protein 4 (BRD4) is an epigenetic regulator recently identified as a therapeutic target for several hematological cancers, notably mixed lineage leukemia‐fusion acute myeloid leukemia (MLL‐AML). Here, we show that the BRD4 bromodomain inhibitor JQ1 is highly active against the p53‐wild‐type Ontario Cancer Institute (OCI)‐AML3 cell line which carries mutations in nucleophosmin (NPM1) and DNA methyltransferase 3 (DNMT3A) genes commonly associated with poor prognostic disease. We find that JQ1 causes caspase 3/7‐mediated apoptosis and DNA damage response in these cells. In combination studies, we show that histone deacetylase (HDAC) inhibitors, the HDM2 inhibitor Nutlin‐3, and the anthracycline daunorubicin all enhance the apoptotic response of JQ1. These compounds all induce activation of p53 suggesting that JQ1 might sensitize AML cells to p53‐mediated cell death. In further experiments, we show that BRD4 associates with acetylated p53 but that this association is not inhibited by JQ1 indicating that the protein–protein interaction does not involve bromodomain binding of acetylated lysines. Instead, we propose that JQ1 acts to prevent BRD4‐mediated recruitment of p53 to chromatin targets following its activation in OCI‐AML3 cells resulting in cell cycle arrest and apoptosis in a c‐MYC‐independent manner. Our data suggest that BET bromodomain inhibition might enhance current chemotherapy strategies in AML, notably in poor‐risk DNMT3A/NPM1‐mutated disease.

Nanog Requires BRD4 to Maintain Murine Embryonic Stem Cell Pluripotency and Is Suppressed by Bromodomain Inhibitor JQ1 Together with Lefty1

Embryonic stem cells (ESCs) are maintained in an undifferentiated state through expression of the core transcriptional factors Nanog, Oct4, and Sox2. However, the epigenetic regulation of pluripotency is poorly understood. Differentiation of ESCs is accompanied by a global reduction of panacetylation of histones H3 and H4 suggesting that histone acetylation plays an important role in maintenance of ESC pluripotency. Acetylated lysine residues on histones are read by members of the bromodomain family that includes BET (bromodomain and extraterminal domain) proteins for which highly potent and selective inhibitors have been developed. In this study we demonstrate that the pan-BET bromodomain inhibitor JQ1 induces rapid spontaneous differentiation of murine ESCs by inducing marked transcriptional downregulation of Nanog as well as the stemness markers Lefty1 and Lefty2, but not Myc, often used as a marker of BET inhibitor activity in cancer. We show that the effects of JQ1 are recapitulated by knockdown of the BET family member BRD4 implicating this protein in Nanog regulation. These data are also supported by chromatin immunoprecipitation experiments which confirm BRD4 binding at the Nanog promoter that is known to require acetylation by the histone acetyltransferase MOF for transcriptional activity. In further support of our findings, we show that JQ1 antagonizes the stem cell-promoting effects of the histone deacetylase inhibitors sodium butyrate and valproic acid. Our data suggest that BRD4 is critical for the maintenance of ESC pluripotency and that this occurs primarily through the maintenance of Nanog expression.

Rapid detection of DNMT3A R882 codon mutations allows early identification of poor risk patients with acute myeloid leukemia

Since being characterized by Ley et al . in 2010 [1], mutations in the de novo methyltransferase 3A (DNMT3A) gene have been increasingly recognized as an independent adverse prognostic factor for patients diagnosed with acute myeloid leukemia (AML) [1 – 7]. Moreover, a number of recent studies have presented data to suggest that patients with DNMT3Amutated AML show improved outcomes with certain induction chemotherapy regimens, further highlighting the clinical importance of this gene [3,8,9]. However, as yet, no rapid screening test for DNMT3A mutations suitable for standard hospital laboratories has been described that can realistically infl uence initial patient management. We present here a novel method that, using simple techniques, can detect all mutations occurring at the R882 codon hotspot ( ~ 60% of all DNMT3A coding mutations) within a few hours of bone marrow aspiration. DNMT3A mutations are found in 22% of all patients with AML [1]. Th ey occur with increased frequency in cytogenetically normal profi les (29 – 34%) [1 – 3] and rarely, if ever, in combination with favorable cytogenetic profi les such as t(8;21), t(15;17) or inv(16) [6]. Th ey are also signifi cantly associated with the French – American – British subtypes M4/M5, older age, high white blood cell count and with nucleophosmin 1 (NPM1) mutations [4,5]. Importantly, the presence of these mutations has consistently been shown to confer a signifi cantly poorer outcome in terms of overall survival and disease-free survival, independent of other prognostic factors [1 – 7]. Recently, Patel et al . have reported that patients with DNMT3A-mutated AML show a signifi cantly improved survival with dose-intensifi ed daunorubicin (90 mg/m 2 ) compared to standard-dose daunorubicin (45 mg/m 2 ) [3], although this fi nding clearly needs to be confi rmed in prospective randomized trials. Other studies have reported superior responses for patients with DNMT3Amutated AML with high-dose idarubicin [8] and also with hypomethylating agents such as decitabine [9]. Thus, immediate determination of DNMT3A mutational status could potentially provide the clinician with the necessary information to recommend the use of nonstandard induction regimens as well as providing important prognostic information for the patient. In addition, if mutated DNMT3A could be unambiguously and rapidly demonstrated, it would arguably obviate the need for more expensive cytogenetic analysis because favorable-risk translocations are highly unlikely to be present [6]. Indeed, it might be argued that mutated DNMT3A, along with partial tandem duplication mutations of MLL (MLL-PTD), is the pre-eminent adverse genetic lesion in normal cytogenetic AML [10], as its prognostic impact appears to override that of other mutated genes including NPM1, with which it is commonly associated, IDH1/2, CEPBA and even FLT3 [1,11]. In this regard, it is interesting from the article by Patel et al . that patients with mutated DNMT3A, MLL-PTD and NPM1 all appeared to benefi t from high-dose daunorubicin, suggesting that prognostically adverse AML warrants more intensive treatment. In addition to diagnosis, detection of mutated DNMT3A might also provide a useful biomarker for assessing response to treatment and for monitoring minimal residual disease post-treatment [6,12]. Current methods for detection of DNMT3A mutations involve direct Sanger sequencing of polymerase chain reaction (PCR)-amplifi ed DNA, denaturing high-performance liquid chromatography (dHPLC, e.g. WAVE TM system) or high resolution melting point (HRM) analysis [13,14]. Th ese methods are reported to achieve sensitivities of 5 – 10% of mutant to wild-type gene copy number [13]. However, such technologies are not generally available in standard hospital laboratories, and therefore rapid detection of the mutations is not practical prior to commencing chemotherapy. Here, we present an eff ective strategy for rapid detection of mutations specifi cally involving codon R882 in exon 23 of the DNMT3A gene. Our method allows pre-treatment identifi cation of a subset of patients (estimated at 13% of all AML cases) who are predicted to carry this particular mutation and may potentially benefi t from receiving intensifi ed L eu k L ym ph om a D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y N yu M ed ic al C en te r on 0 5/ 22 /1 3

DNMT3A mutations at R882 hotspot are only found in major clones of acute myeloid leukemia

DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily refl ect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientifi c review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the fi nal print and fi nal online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication. Just Accepted by Leukemia & Lymphoma

Genomic landscapes and clonality of de novo AML

We agree that it is important to understand whether mutations are responsible for the initiation of AML or cooperate with initiating mutations to cause disease progression or relapse.1,2 Whereas initiating mutations may be more likely to appear in founding clones, cooperating mutations might appear either in founding clones or subclones derived from founding clones. In our study, it was not possible to define the clonal architecture for all samples, both because AML genomes harbor a comparatively small number of mutations and because for 150 of 200 samples, only exome sequencing was performed. Nevertheless, we have used the data in Table S6 (available with the full text of our article at NEJM.org) to identify variants in significantly mutated genes that can be assigned with high confidence to either a founding clone or a subclone. Mutations in some genes appear almost exclusively in founding clones, which suggests that they are disease initiators. These genes include RUNX1 (9 of 9 mutations in founding clones), NPM1 (3 of 3), U2AF1 (5 of 5), DNMT3A (38 of 40), IDH2 (13 of 14), IDH1 (15 of 17), and KIT(5 of 6). In contrast, mutations in NRAS (1 of 12 in founding clones), TET2 (13 of 18), KRAS(4 of 6), CEBPA (3 of 5), WT1 (3 of 6), PTPN11 (4 of 8), and FLT3 (6 of 13), are often found in subclones, suggesting that they are often cooperating mutations. Many additional genomes will need to be tested to make these tentative assignments more definitive.

Genetic profiling in acute myeloid leukemia

To the Editor: In their article on the prognostic relevance of integrated genetic profiling in patients with acute myeloid leukemia (AML), Patel et al. (March 22 issue)1 propose an elaborate riskstratification system for refining prognosis for patients with intermediate-risk AML. This stratification is based on mutational analysis by DNA sequencing of 10 individual leukemia genes in addition to standard karyotyping. However, even ignoring the impracticality of such an analysis, we consider this risk stratification to be overly complicated and unjustified. Instead, on the basis of the report’s supplementary data, we believe that only two genes are worthy of mutational screening, DNMT3A and MLL. Mutations in either of these genes predict adverse outcomes independent of other mutations, including internal tandem duplication in FLT3 (FLT3-ITD), as reported previously.2 Moreover, DNMT3A and MLL mutations define a biologic subgroup of AML patients typically presenting with myelomonocytic or blastic morphology and marked leukocytosis3 who may benefit from escalation of induction chemotherapy with dose-intensified daunorubicin.4 We propose that rapid identification of unfavorable mutations in DNMT3A and partial tandem duplication in MLL (MLL-PTD) alone is required for guiding optimal treatment in patients with newly diagnosed AML.

Novel drug therapies in myeloid leukemia: a patent review

Both acute myeloid leukemia and chronic myeloid leukemia are thought to arise from a subpopulation of primitive cells, termed leukemic stem cells that share properties with somatic stem cells. Leukemic stem cells are capable of continued self-renewal, and are resistant to conventional chemotherapy and are considered to be responsible for disease relapse. In recent years, improved understanding of the underlying mechanisms of myeloid leukemia biology has led to the development of novel and targeted therapies. This review focuses on clinically relevant patent applications and their relevance within the known literature in two areas of prevailing therapeutic interest, namely monoclonal antibody therapy and small molecule inhibitors in disease-relevant signaling pathways.

Approaches for targeting self-renewal pathways in cancer stem cells: implications for hematological treatments

ABSTRACT Introduction: Self-renewal is considered a defining property of stem cells. Self-renewal is essential in embryogenesis and normal tissue repair and homeostasis. However, in cancer, self-renewal pathways, e.g. WNT, NOTCH, Hedgehog and BMP, frequently become de-regulated in stem cells, or more mature progenitor cells acquire self-renewal properties, resulting in abnormal tissue growth and tumorigenesis. Areas covered: This review considers the conserved embryonic self-renewal pathways, including WNT, NOTCH, Hedgehog and BMP. The article describes recent advances in our understanding of these pathways in leukemia and, more specifically, leukemia stem cells (LSC), how these pathways cross-talk and interact with the LSC microenvironment, and discusses the clinical implications and potential therapeutic strategies, both in preclinical and in clinical trials for hematological malignancies. Expert opinion: The conserved embryonic self-renewal pathways are frequently de-regulated in cancer stem cells (CSC), including LSCs. There is significant cross-talk between self-renewal pathways, and their downstream targets, and the microenvironment. Effective targeting of these pathways is challenging due to cross-talk, and importantly, because these pathways are important for normal stem cells as well as CSC, adverse effects on normal tissues may mean a therapeutic window cannot be identified. Nonetheless, several agents targeting these pathways are currently in clinical trials in hematological malignancies.

Stem Cell Guardians – Old and New Perspectives in LSC Biology

The introduction of tyrosine kinase inhibitors in chronic myeloid leukaemia (CML) has revolutionised disease outcome. However, despite this, progression to blast phase disease is high in those that do not achieve complete cytogenetic and major molecular response on standard therapy. As well as BCR-ABL-dependent mechanisms, disease persistence has been shown to play a key role. Disease persistence suggests that, despite a targeted therapeutic approach, BCR-ABL-independent mechanisms are being exploited to sustain the survival of a small population of cells termed leukaemic stem cells (LSCs). Increasing evidence highlights the importance of self-renewal and survival pathways in this process. This review will focus on the role of stem-cell restricted self-renewal pathways, namely Hedgehog, Notch, and Bone Morphogenic Pathway (BMP). Wingless-Int/β-Catenin (Wnt/β-Catenin) signalling will be discussed within a further review in this series in view of its regulatory role in GSK3β. Further to this, we will highlight the role of key transcriptional regulators, namely p53 and c- MYC, in targeting wider deregulated networks.