Ken I. Mills

Inhibition of the LSD1 (KDM1A) demethylase reactivates the all- trans -retinoic acid differentiation pathway in acute myeloid leukemia

Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non-APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine-specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4me2) across the genome, but it did increase H3K4me2 and expression of myeloid-differentiation–associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML.

Clinical Utility of Microarray-Based Gene Expression Profiling in the Diagnosis and Subclassification of Leukemia: Report From the International Microarray Innovations in Leukemia Study Group

PURPOSE The Microarray Innovations in Leukemia study assessed the clinical utility of gene expression profiling as a single test to subtype leukemias into conventional categories of myeloid and lymphoid malignancies. METHODS The investigation was performed in 11 laboratories across three continents and included 3,334 patients. An exploratory retrospective stage I study was designed for biomarker discovery and generated whole-genome expression profiles from 2,143 patients with leukemias and myelodysplastic syndromes. The gene expression profiling-based diagnostic accuracy was further validated in a prospective second study stage of an independent cohort of 1,191 patients. RESULTS On the basis of 2,096 samples, the stage I study achieved 92.2% classification accuracy for all 18 distinct classes investigated (median specificity of 99.7%). In a second cohort of 1,152 prospectively collected patients, a classification scheme reached 95.6% median sensitivity and 99.8% median specificity for 14 standard subtypes of acute leukemia (eight acute lymphoblastic leukemia and six acute myeloid leukemia classes, n = 693). In 29 (57%) of 51 discrepant cases, the microarray results had outperformed routine diagnostic methods. CONCLUSION Gene expression profiling is a robust technology for the diagnosis of hematologic malignancies with high accuracy. It may complement current diagnostic algorithms and could offer a reliable platform for patients who lack access to todays state-of-the-art diagnostic work-up. Our comprehensive gene expression data set will be submitted to the public domain to foster research focusing on the molecular understanding of leukemias.

CD200 as a prognostic factor in acute myeloid leukaemia.

The CD200 gene (aka MOX-2, OX-2) is located on chromosome 3 and encodes a type-1 membrane glycoprotein.1 This protein belongs to the immunoglobulin superfamily and is expressed on many different cell types including T and B lymphocytes and dendritic cells.2 CD200 binds multiple membrane receptor isoforms (CD200R) which have a more tissue restricted expression.3, 4 The best characterized isoform, CD200R1, has a longer cytoplasmic tail containing three conserved tyrosine residues that can be phosphorylated.1 These phosphorylated residues interact with signalling adaptor molecules such as Shc, suggesting that the CD200R1 can signal upon binding CD200 ligand providing a more localized response than that provided by cytokines.5 Although CD200(-/-)-deficient mice appear grossly normal and live a normal lifespan, they are susceptible to tissue-specific autoimmunity, suggesting that the function of this protein is to induce immune suppression through the CD200R.6 CD200 also appears to have an alternative role in regulating osteoclast development.7 In leukaemia, CD200 has been shown to be upregulated in B cells from patients with chronic lymphocytic leukemia.8 More recently, CD200 has been shown to be an independent prognostic factor for patients with multiple myeloma.9 In this latter study, expression of CD200 was associated with a bad prognosis. Here we report that in acute myeloid leukaemia (AML), there is a correlation between CD200 expression and the presence of the core binding factor (CBF) associated abnormalities, t(8;21) and inv(16). However, at the same time CD200 expression was linked to worse overall survival in other AML subsets. These data indicate that CD200 is also of prognostic value in AML.

An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5‐d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r2 correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra‐laboratory reproducibility and with comparable quality and reliability.

Elevated Bcr-Abl expression levels are sufficient for a haematopoietic cell line to acquire a drug-resistant phenotype.

A characteristic feature of chronic myeloid leukaemia (CML) is the inevitable advancement from a treatable chronic phase to a fatal, drug-resistant stage referred to as blast crisis. The molecular mechanisms responsible for this disease transition remain unknown. As increased expression of Bcr-Abl has been associated with blast crisis CML, we have established transfectants in 32D cells that express low and high levels of Bcr-Abl, and assessed their drug sensitivity. Cells with high Bcr-Abl expression levels are resistant to conventional cytotoxic drugs, and also require higher levels of STI571 (an inhibitor of Bcr-Abl), to induce cell death. Co-treatment with cytotoxic drugs and STI571 increased the sensitivity of the drug-resistant cells. Despite the drug-resistant phenotype, high Bcr-Abl levels concomitantly increased the expression of p53, p21, Bax and down-regulated Bcl-2. These cells maintain a survival advantage irrespective of a reduced proportion of cycling cells and the pro-apoptotic shift in gene expression. In addition, the level of Bcr-Abl expression (high or low) does not alter the growth factor independence and elevated Bcl-xL expression observed. Our study indicates that drug resistance can be primarily attained by increased Bcr-Abl expression, and highlights the potential of therapy which combines STI571 with conventional cytotoxic drugs.

Validation of Next Generation Sequencing Technologies in Comparison to Current Diagnostic Gold Standards for BRAF, EGFR and KRAS Mutational Analysis

Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use.

Effects of the aurora kinase inhibitors AZD1152-HQPA and ZM447439 on growth arrest and polyploidy in acute myeloid leukemia cell lines and primary blasts

Aurora kinase inhibitors are being considered for treatment of acute myeloid leukemia. Findings of this study suggest that two such inhibitors are effective apoptosis-inducing agents in primary myeloid leukemia cells. Background Aurora kinases play an essential role in the orchestration of chromosome separation and cytokinesis during mitosis. Small-molecule inhibition of the aurora kinases has been shown to result in inhibition of cell division, phosphorylation of histone H3 and the induction of apoptosis in a number of cell systems. These characteristics have led aurora kinase inhibitors to be considered as potential therapeutic agents. Design and Methods Aurora kinase gene expression profiles were assessed in 101 samples from patients with acute myeloid leukemia. Subsequently, aurora kinase inhibitors were investigated for their in vitro effects on cell viability, histone H3 phosphorylation, cell cycle and morphology in acute myeloid leukemia cell lines and primary acute myeloid leukemia samples. Results The aurora kinase inhibitors AZD1152-HQPA and ZM447439 induced growth arrest and the accumulation of hyperploid cells in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. Furthermore, both agents inhibited histone H3 phosphorylation and this preceded perturbations in cell cycle and the induction of apoptosis. Single cell cloning assays were performed on diploid and polyploid cells to investigate their colony-forming capacities. Although the polyploid cells showed a reduced capacity for colony formation when compared with their diploid counterparts, they were consistently able to form colonies. Conclusions AZD1152-HQPA- and ZM447439 are effective apoptosis-inducing agents in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. However, their propensity to induce polyploidy does not inevitably result in apoptosis.

Transcriptional dysregulation mediated by RUNX1-RUNX1T1 in normal human progenitor cells and in acute myeloid leukaemia.

The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and γ-catenin). Further, we show that overexpression of CD200 and γ-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and γ-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.

RARalpha-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

Leukemia-associated chimeric oncoproteins often act as transcriptional repressors, targeting promoters of master genes involved in hematopoiesis. We show that CRABPI (encoding cellular retinoic acid binding protein I) is a target of PLZF, which is fused to RARα by the t(11;17)(q23;q21) translocation associated with retinoic acid (RA)-resistant acute promyelocytic leukemia (APL). PLZF represses the CRABPI locus through propagation of chromatin condensation from a remote intronic binding element culminating in silencing of the promoter. Although the canonical, PLZF-RARα oncoprotein has no impact on PLZF-mediated repression, the reciprocal translocation product RARα-PLZF binds to this remote binding site, recruiting p300, inducing promoter hypomethylation and CRABPI gene up-regulation. In line with these observations, RA-resistant murine PLZF/RARα+RARα/PLZF APL blasts express much higher levels of CRABPI than standard RA-sensitive PML/RARα APL. RARα-PLZF confers RA resistance to a retinoid-sensitive acute myeloid leukemia (AML) cell line in a CRABPI-dependent fashion. This study supports an active role for PLZF and RARα-PLZF in leukemogenesis, identifies up-regulation of CRABPI as a mechanism contributing to retinoid resistance, and reveals the ability of the reciprocal fusion gene products to mediate distinct epigenetic effects contributing to the leukemic phenotype.

Gene expression profiling in MDS and AML: potential and future avenues.

Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet, and even within some cytogenetically well-defined subclasses there is considerable clinical heterogeneity. Recent advances in genomics technologies such as gene expression profiling (GEP) provide powerful tools to further characterize myeloid malignancies at the molecular level, with the goal to refine the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML with regard to diagnosis, prediction of clinical outcome, discovery of novel subclasses and identification of novel therapeutic targets and novel drugs. As many challenges remain ahead, we discuss the pitfalls of this technology and its potential including future integrative studies with other genomics technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients.