The Genomic and Epigenomic Landscapes of AML Luca Mazzarella,a Laura Riva,b Lucilla Luzi,a,c Chiara Ronchini,b and Pier Giuseppe Peliccia,d A progressively better understanding of the genetic and epigenetic abnormalities underlying acute myeloid leukemia has changed clinical practice and affected the outcome of thousands of patients. Over the past decades, approaches focused on cloning, sequencing, and functional characterization of one or a few genes were the preferred (and the only possible) modality of investigation. The advent of disruptive new sequencing technologies brought about an unprecedented acceleration in our learning curve. Our view of the abnormalities required to generate and sustain leukemia is evolving from a piecemeal account based on individual lines of research into a comprehensive view of how all the important components (eg, transcriptional program, chromatin modifications, DNA sequence, alterations in noncoding genome) interact, in each patient and each leukemic cell. In this article, we provide an overall look at this complicated landscape and highlight outstanding issues for future research. Semin Hematol 51:259–272. C 2014 Elsevier Inc. All rights reserved. he idea that underlying genetic abnormalities A complex picture has emerged, with some AML- might dictate clinical decisions, now a common distinctive features. concept in oncology, was pioneered for acute The average number of mutations in coding sequences T 4 1–3 myeloid leukemia (AML) 25 years ago. Treatment of is very low in AML, compared with the majority of solid AML was indeed revolutionized by the recognition of tumors (13 mutations per patient in AML vs 52 in microscopically visible chromosomal abnormalities, as breast cancers, 290 in bladder cancers, and 500 in exemplified by the case of acute promyelocytic leukemia smoking-associated lung adenocarcinomas). A functional – and the associated t(15;17) PML-RARa translocation.4 7 logic governs the type of mutations, which recurrently About one half of all AML cases are cytogenetically affect specific pathways controlling lineage identity, cell normal,8 and it took a long time to appreciate the role of survival, and proliferation, as well as genomic and smaller-scale genomic abnormalities (single nucleotide epigenomic stability. This logic is parsimonious in that variations or small insertions/deletions), such as those each pathway suffers the minimum number of hits that – involving NPM1, FLT3, or RAS.9 11 The advent of are sufficient to affect its outcome: mutations of genes high-throughput sequencing has radically changed the within each pathway are usually mutually exclusive. By field and finally allowed an assessment of the panoply of estimating the mutant allele fraction within leukemia cell genetic aberrations in AML, providing a preliminary view populations, it has become evident that each leukemia of the numbers and types of mutations, their distribution exhibits an oligoclonal anatomy, which evolves over time within leukemic and preleukemic populations, and their under the effect of a selective environment, as in the case – evolution during the natural history of the disease.12 17 of systemic treatments. Furthermore, mutations arise in a clearly ordered temporal fashion, with some being invariably present in the founding clones (eg, mutations aDepartment of Experimental Oncology, European Institute of Oncol- of NPM1) and others occurring at later stages, often in ogy, Milan, Italy. FLT3 b subclones (eg, mutations of ). All known major Center for Genomic Science of IIT@SEMM, Fondazione Istituto players appeared, together with new ones, such as Italiano di Tecnologia (IIT), Milan, Italy. fi cIFOM, FIRC Institute of Molecular Oncology Foundation, Milan, epigenetic modi ers (DNA methyltransferase [DNMT] 12 Italy. 3, ten-eleven translocation [TET]), metabolic enzymes dDipartimento di Scienze della Salute, Università degli Studi di (isocitrate dehydrogenase 1 and 2 [IDH1/2]), and Milano, Milan, Italy. components of the cohesin complex.18,19 Finally, dereg- Conflicts of interest: none. Address correspondence to: Luca Mazzarella MD PhD or Pier ulation of epigenetic factors, caused directly by gene Giuseppe Pelicci MD PhD, Department of Experimental Oncology, mutations or indirectly by alterations of transcriptional European Institute of Oncology, Via Adamello 16, 20139 Milan, networks, has emerged as major driving forces of the Italy. E-mail: [email protected], [email protected] 0037-1963/$ - see front matter disease. The details of this picture are evolving at & 2014 Elsevier Inc. All rights reserved. stunning pace. We provide here an overview of the http://dx.doi.org/10.1053/j.seminhematol.2014.08.007 current state-of-the-art. Seminars in Hematology, Vol 51, No 4, October 2014, pp 259–272 259 260 L. Mazzarella et al 31–33 MECHANISMS OF GENOMIC ALTERATIONS IN not require either oxidative damage or DNA replication. BLOOD CELLS Because CpG density is 10 times higher in coding than in noncoding DNA, the exome is more prone to Genomic alterations are the net result of DNA damage replication-independent mutations, suggesting that spon- and subsequent DNA repair, coupled with the cell's taneous deamination of methylated cytosines could be inability to eliminate itself (apoptosis). Accumulation of particularly relevant to cancer development.32,34 Notably, DNA damage is inevitable because DNA, unlike all other overrepresentation of C4T substitutions is usually con- macromolecules, cannot be substituted in its entirety served in leukemia.35 Overrepresentation of A4T trans- during the life span of a cell. Established sources of versions in the TpA dinucleotide, through unknown exogenous DNA damage for the development of AML underlying molecular mechanisms, have also been include ionizing radiation, smoke, benzene, and chemo- reported,29 which may be in line with hematopoietic- therapeutic treatment with alkylating drugs.20 However, specific mutational patterns associated with aging identi- clear exposure to any of these agents can only rarely be fied in experimental animals.33 identified. Thus, a significant amount of DNA-damaging A further mechanism leading to genomic instability in agents are thought to derive from inside the cells, as stem cells is their reliance on replication-independent byproducts of intracellular metabolic pathways. Central to strand-break repair mechanisms, such as nonhomologous the damaging effect of both exogenous and endogenous end-joining.23 This mechanism is inherently more error agents is the formation of reactive oxygen species, which prone than homologous recombination because it does not cause alterations to both bases and the sugar backbone of rely on strand complementarity to repair the DNA breaks. DNA, resulting in nucleotide mismatching or strand HSCs have evolved a specific molecular circuitry of breaks. An additional- and thus far underappreciated- resilience to DNA damage characterized by lack of p53- potential source of DNA damage comes from endogenously triggered apoptosis response,36 which is fundamental for formed aldehydes, as recently reported.21 The number of the maintenance of the HSC pool. damaging insults suffered by each DNA molecule every day Thus, HSCs accumulate mutations over time, at a is estimated on the order of thousands.22 consistent rate, with aging being the most relevant risk DNA damage is continuously generated and needs to factor for AML.37 be continuously corrected by a wide array of repair mechanisms. The bone marrow mainly relies on compo- METHODS TO MEASURE MUTATION nents of the homologous recombination and interstrand 22,23 ACCUMULATION IN BLOOD CELLS crosslink repair systems. Hereditary deficiencies in such components lead to syndromes characterized by bone Measuring the actual rate of mutation accumulation in marrow failure and leukemia, such as Fanconi anemia, the hematopoietic tissue, and in the HSCs in particular, is Bloom syndrome, and ataxia-telangiectasia.24 Treatment critical to understanding the effects of metabolism and with topoisomerase inhibitors or alkylating agents also external environments on leukemogenesis, and might entail a major risk of accumulating genetic alterations for provide clues to prevent or delay leukemia initiation. Is AML.25 the rate of spontaneous mutation in HSCs sufficient, in To be “fixed” into the genome, most DNA alterations the long run, to initiate leukemogenesis, or, alternatively, “require” a round of DNA replication; therefore, the are exogenous stimuli needed to reach a critical threshold accumulation of mutations increases with each cell's repli- of mutation accumulation? In the latter case, which type of cative history. The hierarchical organization of cell prolifer- environment favors mutation accumulation? Because ation in any given tissue, however, reduces its mutation mutations are rare events and HSCs are rare cells, burden. Most tissue cells divide and amplify rapidly but are estimating their true mutation rate has proven challenging periodically eliminated and replaced, thus impeding, in the and has relied mostly on the use of indirect methods. The long run, the accumulation of mutations. In the bone classical strategy has consisted of quantifying cells that marrow, only a small subset of stem cells persists through- spontaneously lose, through a single genetic hit, a meas- out a life span, and these are maintained in a quiescent urable biological property that is assumed to be irrelevant replicative state, thus favoring genomic integrity of the for the fitness of the