Have we already identified the entire repertoire of mutated genes?

We may have identified (most of) the major culprits [14]. First, there are hundreds of background mutations (i.e. that do not provide selective advantage) but only a limited number of driver mutations (i.e. that cause the disease) in each malignant disease. Second, many of the newly discovered mutated genes may affect the same pathways or networks as the major mutated genes. For example, deletions and mutations of NF1, which have been recently identified [17,44,45], or PTPN11[46] are thought to have the same effect as a RAS mutation; a mutation of the SHKBP1 gene [47] or a duplication of the SH3KBP1 gene [48], which both encode cytoplasmic regulators of the CBL pathway, may have the same effect as a CBL mutation [49,50]. Because EZH2, EED and SUZ12 proteins all belong to the same polycomb complex 2 (PRC2) the rare deletions or mutations of the EED[23,51] and SUZ12 genes [17,51] could have the same effect as EZH2 mutations. Third, several genes (e.g. ETV6[52] or RUNX1) can be structurally altered by mechanisms other than mutation, such as deletions and breakages. Fourth, some important regulatory genes could be affected not by structural alteration but through other mechanisms such as abnormal DNA methylation (e.g. CDKN2A/B[53], TRIM33[54], CTNNA1[55], SOCS1[56,57]), histone modifications, mRNA splicing, microRNA or long non-coding RNA (lncRNA) modulation, or product degradation. Fifth, when all known mutated genes are analyzed in a series of cases, the percentage of samples with at least one mutated candidate driver gene varies from 50% [58] to over 90% (in CMML; [33]; Gelsi-Boyer et al., submitted). Moreover, most samples studied by NGS were shown to harbor gene mutations [23,26]. Thus, we are soon approaching the days where all cases can be defined by combination of several alterations. The practical definition of leukemogenesis will then be based on a specific and limited repertoire of alterations, including translocations, mutations and copy number changes, affecting a defined set of driver genes.

However, some issues still need be addressed. First, many genes may be mutated or deleted with a very low frequency (i.e. under 1%); their involvement and recurrence may be hard to demonstrate. Second, because NGS studies of several malignancies have shown that hundreds of genes can be mutated in a single tumor, background mutations should be discarded and driver genes validated. Third, we still miss information in some diseases such as essential thrombocythemia (ET), in which JAK2 mutations are found in only half the cases, and TET2 mutations in less than 10%. We also lack knowledge about the targeted genes of some frequent genomic alterations such as the 20q11-q13 deletion (ASXL1 and DNMT3B, more centromeric, are not involved). Fortunately, this lack of information is bound to disappear. The example of refractory anemia with ring sideroblasts (RARS) is instructive; in three-quarters of RARS, mutations have been recently found in SF3B1, a gene encoding a subunit of a splicing factor (U2 snRNP) and histone acetyltransferase (STAGA) complexes [27,29,31].

Is there some specificity in gene alterations?

Gene fusions (e.g. BCR-ABL1, PML-RARA, FGFR1-associated fusions…), 5q deletion and JAK2 mutations are specific of some forms of myeloid diseases, although JAK2 mutations occur in three distinct subtypes of MPN. RUNX1 mutations are frequent in MDSs, CMML and AML but rare in MPNs. Among splicing factor genes, mutations in SF3B1 are highly specific of MDS with ring sideroblasts and SRSF2 mutations are most frequent in CMML [31]. In contrast, some mutated genes (e.g. ASXL1, DNMT3A, EZH2, TET2) occur in a wide range of myeloid diseases and with various frequencies. Future studies may identify mutations or combinations of mutations that drive a specific phenotype.

What are the functions of the mutated proteins ?

Leukemogenic alterations mainly affect five classes of proteins (Figure ​(Figure2):2): signaling pathway components, such as ABL, CBL, CBLB, FGFR1, FLT3, JAK2, KIT, LNK, MPL, PDGFRs, PTPN11, PTPRT [23,59] and RAS, transcription factors (TFs) such as CEBPA, ETV6 [58], GATA2 [30], IKZF1 [60], RARA and RUNX1, epigenetic regulators (ERs), such as ASXL1, BCORL1 [25], DAXX [23], DNMT3A, EZH2 [20,22], MLL, MYST3, NSD1 [30], PHF6 [61], SUZ12 [17,51], TET2 and UTX [28], tumor suppressors (TSG), such as CDKN2A, TP53, and WT1 and components of the spliceosome [27,29,31,38,39,41,42]. However, additional alterations occur in genes encoding proteins that it is too early to classified into these defined categories, such as DIS3, DDX41 [23], mitochondrial NAPDH dehydrogenase ND4 [62], or cohesin complex proteins [23,63].

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Figure 2

Schematic representation of five classes of leukemogenic genes. ERs (class III) can be subdivided into two subclasses (DNA methylation-associated and histone-associated).

In chronic stages, alterations in signaling molecules can be grouped in two major categories, a first one that is found in MPNs and affects oncogenic tyrosine kinases (ABL1, JAK2, FGFR1, PDGFRs) and the downstream JAK-STAT and/or PI3-kinase pathways, and a second one that is mutated in CMML and affect the RAS-MAP kinase pathway (RAS, PTPN11, NF1). CBL alterations occur in a wide variety of myeloid diseases [50].

TFs and ERs constitute the largest classes, which involve several categories of proteins (Figure ​(Figure3);3); because there are many ways to affect gene expression it is probable that not all of these categories are known yet. The existence of epigenetic alterations in myeloid malignancies has been known for long time [64,65]. For example, alterations of MLL, a histone methyltransferase (HMT), and MYST3, a histone acetyltransferase (HAT), have shown the importance of epigenetic deregulation in AMLs with translocation [45,64,65]. However, in chronic diseases and in AMLs with normal karyotype, the extent, causes, identities, exact roles and consequences of epigenetic alterations have long remained elusive. Molecular studies have recently shown that both DNA methylation and histone regulation are affected, and that epigenetic alterations may be due to genetic alterations, (i.e. mutations in genes encoding epigenetic regulators). The latter phenomenon has been observed in genome-wide analyses of many neoplasias [28,66,67]. However, not all epigenetic alterations may be due to an abnormal genetic background [1,53,54].

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Figure 3

Schematic representation of epigenetic regulation of a leukemogenic locus (LL) framed by histone H3. (a) Histone acetyltransferases (HAT; e.g. MYST3) and histone methyltransferases (HMT; e.g. MLL) can activate the locus. (b) Reciprocally, the locus is repressed by polycomb complex PRC2 (which comprised EED, EZH2 and SUZ12 proteins). ASXL1 would direct PRC2 to the locus. Loss-of-function mutations in PRC2 components or in ASXL1 remove PRC2 repression. DNMT3A is involved in the formation of 5-methylcytosines (5mC) from cytosines and interacts with HMTs as well as with PRC2 components. TET2 mediates hydroxylation of 5mC to 5hmC. To function, TET2 requires α-ketoglutarate (αKG), which is provided by IDH1/2 proteins. Aberrant methylation patterns are caused by mutation in TET2 or in IDH1/2, which produces 2-hydroxyglutarate instead of αKG.

The recent reports of the interrelated functions of IDH1/2 and TET2 in DNA methylation represent a major breakthrough in our understanding of leukemogenesis [68]. It was initially hard to associate mutations of IDH1 and IDH2, two metabolic enzymes, with mutations in TET2, an unknown gene product, and as hard to suspect their role on DNA methylation. A very rapid series of elegant studies have shown i) that IDH1/2 and TET2 mutations are mutually exclusive in myeloid malignancies [68], ii) that mutated IDH1 and IDH2 produce 2-hydroxyglutarate instead of alpha-ketoglutarate (αKG) [69,70], iii) that TET2 encodes an αKG–dependent methyl cytosine dioxygenase whose mutation alters the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) [68,71] and iv) that both IDH1/2 and TET2 mutations impact on DNA methylation and are involved in the same biochemical pathway [72]. In addition, TET proteins can generate from 5-hmC 5-formylcytosine and 5-carboxylcytosine, but their roles are currently unknown [73]. The recent studies on TET proteins suggest a role in removing aberrant DNA methylation to ensure DNA methylation fidelity [74]. This has opened a new area of research since first, other factors involved in DNA demethylation may exist and second, several αKG–dependent enzymes, such as jumonji histone demethylases [75] are epigenetic regulators; therefore, some of these proteins could also be involved in malignancies. However, IDH1/2 and TET2 mutations, while mutually exclusive, are not equivalent because IDH1/2 mutations are more frequent in acute than in chronic myeloid diseases, whereas it is not the case for TET2 alterations, which are more evenly distributed between chronic and acute stages. Inactivation of TET2 increases self-renewal in hematopoietic stem cells and induces a disease resembling CMML in mouse models [76,77]. Mutated IDH1/2 enzymes may impact on self-renewal but with a different strength. The likely explanation is that IDH1/2 and TET2 have other, non-overlapping functions on the regulation of DNA methylation and histone marks. Also, an IDH-mutated product may depend on another, rate-limiting factor to exert a leukemogenic effect. DNMT3A is a de novo DNA methyltransferase involved in the formation of 5-mC and has complex interactions with polycomb and HMT proteins [78]. How DNMT3A mutations affect DNA methylation remains to be defined [24,30,79]; they probably do so in a different way from TET2 or IDH1/2 mutations since they may co-occur with either of them. A recent study showed that DNMT3A loss leads to upregulation of hematopoietic stem cell genes and downregulation of differentiation genes but is alone insufficient to induce a malignant disease in a mouse model [79].

Mutations in regulators of histone marks have become a major subject of research and the relationships between them are quickly unveiled. Central regulators of myelopoiesis and key players in leukemogenesis seem to be the polycomb regulatory complexes, especially PRC2, which, in addition to direct defects of its components (EED, EZH2, SUZ12), could be affected in its concerted action with several ERs, such as ASXL1, cohesins, DNMT3A, IDH1/2, MLL, TET2 and UTX. TET proteins could regulate pluripotency and self-renewal through interaction with PRC2 [74,80,81]. The cohesin complex is encoded by four genes (SMC1, SMC3, RAD21 and STAG2), which have been found mutated [23] and deleted [63]. A major interactor of cohesin complex is CTCF. PRC2 is recruited to specific loci through interaction of SUZ12 with CTCF [82]. Another main leukemogenic interactor of PRC2 components is ASXL1. A recent study showed that ASXL1 loss affects PRC2 complexes and H3K27me3 histone marks, and induces a strong hematopoietic phenotype consistent with an MDS in a conditional knock-out mouse model [83]. ASXL1 would direct PRC2 to leukemogenic loci such as HOXA genes. Thus, through direct alterations of its components or of proteins or lncRNAs [84] that recruit the complex, PRC2 has emerged as a key node in a network regulating hematopoietic stem cell self-renewal and proliferation and as a major factor in myeloid leukemogenesis. This is also true for T-cell leukemogenesis [85]. Correct functioning of polycomb repressive complex 1 (PRC1) seems also to be important for myeloid cells since the loss of BMI1 (a component of PRC1) in the mouse leads to a disease similar to PMF [86]. Structural alterations of the BMI1 gene occur but are rare in human myeloid diseases [87].

Whether other chromatin-associated complexes play a role in leukemogenesis should soon be revealed. ASXL1 could play a role in a cross-talk between major chromatin silencing systems, PRC1/PRC2, HP1α/CBX5 heterochromatin repressive complex and polycomb repressive deubiquitinase (PR-DUB) complex. Mutations in BCOR and BCORL1 suggest that the RAF/BCOR complex [84,88] might be involved in AML. The recent identification of a mutation in the DAXX gene in an AML case [23] further supports a wide participation of chromatin-regulatory complexes in leukemogenesis and cancer in general. DAXX and ATRX (which is mutated in X-linked α-thalassemia) are subunits of a chromatin remodeling complex and are both mutated in solid tumors [89,90].

The importance of the fifth class of mutated genes was more unexpected. Mutations in components of the spliceosome, which are mutually exclusive, lead to splicing defects including exon skipping, intron retention and use of incorrect splice site [31]. A recent study showed that a consequence of splicing gene mutations is accumulation of unspliced transcripts affecting a specific subset of mRNAs [41].