Partial Tandem Duplication (PTD) of MLL Gene

Approximately 5–11% of CN-AML patients carry partial tandem duplications of the MLL gene (MLL-PTD). In MLL-PTD, duplication of genomic regions spanning exons 5–11 occurs where the duplicated segment is inserted into intron 4 of the gene. In contrast to MLL fusion protein, MLL-PTD retains all its functional domains. In the presence of MLL-PTD, the wild-type MLL allele is suppressed. This silencing is mediated through epigenetic mechanisms, wherein a combination of decitabine and depsipeptide, which inhibiting DNA methyltransferase and histone deacetylase, respectively, leads to transcriptional reactivation of the wild-type allele in MLL-PTD-positive blasts. Reactivation of wild-type MLL induces cell death in blasts. Clinically, MLL-PTD mutations associate with unfavorable outcomes (short-duration CR, high relapse rate and inferior EFS).6,16,18

MN1 Over-Expression


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In CN-AML, high expression of MN1 strongly occurs in un-mutated NPM1 cases and is an independent prognostic marker for poor response to the first course of induction therapy, higher relapse rate, shorter RFS and less OS. In treatment with ATRA, patients who express low levels of MN1 would have prolonged EFS and more OS, but patients with high-expression level of MN1 would exhibit higher relapse rate, shorter RFS and less OS. HPCs express MN1 in high levels while differentiated CD34+cells down-regulate it.15,8

TP53 Mutations

Tumor-suppressor p53 is a DNA-binding protein and the master transcription factor activated in response to diverse cellular stresses to induce cell cycle arrest and DNA repair or apoptosis. TP53 mutations involve single nucleotide changes which are prevailing in other types of cancer but not predominant in blood cancers. TP53 mutations occur in 75–78% of AML patients with complex karyotype, but rare in other AML subtypes. TP53 mutation is the most important marker of poor prognosis in both CK-AML and therapy-related AML. TP53 alterations frequently occur in old patients who display decreases in CR, EFS, RFS, and OS.3,16

CN-AML and cytogenetically abnormal AML patients have the chance to get more chromosomes in rearrangements in their leukemic karyotype. Late rearrangements or secondary aberrations are common in both ALL and AML, prevailingly follow a primary change and lead to substantial variability in patient outcome. These include del(7q) (−7), trisomy 8 (+8), del(16)/t(1;16)(q12-23;q12-24) and trisomy 21 (+21), which have been observed in both ALL and AML (Table 2)10

Mutations in Epigenetic Genes

Class 0/III mutations impairing epigenetic regulation in HPCs and defined in AML can be the earliest alteration occurring in an HSC. Importantly, mutations in the genes that encode DNA-methylation regulators (eg, DNMT3A, IDH1/2, TET2) are often acquired early and with a high recurrence rate with NPM1 and CEBPA alterations. Particularly, 73% of the NPM1-mutated AML carry mutations in DNA-methylation genes (DNMT3A, IDH1, IDH2, and TET2). Furthermore, there is evidence confirming that TET2 with FLT3 mutations and IDH1 with NPM1 mutations cooperate to induce AML. Moreover, the epigenetic changes are reversible by small molecules and therapeutics that reactivate epigenetically silenced genes and improve outcomes of the disease (Table 5).12,13

DNMT3A Mutations

DNMT3A mutations, one of the most important epigenetic-related alterations, are classified as the earliest and recurrent aberration in myeloid malignancies, occurring in 20–22% of adults with de novo AML (rare in children). Almost all CN-AML patients harbor at least a single point mutation in one of DNMT3A alleles and about 30–37% of them show DNMT3A loss-of-function mutations. In this regard, the CN-AML group is categorized into two subtypes, ie, with or without DNMT3A mutations with prognosis significance.12–14 Clinically, DNMT3A mutations frequently accompany intermediate-risk AML, where they may correlate with inferior outcomes.12,16 DNMT3A mutations have a negative impact on patient clinical outcome (a significant decrease in OS in comparison to those with wild-type DNMT3A). In particular, DNMT3A-mutation effects persist in HPCs and mature cells after remission (Table 5).3

At the molecular level, DNA methyltransferase 3A gene (DNMT3A) encodes DNMT3A that catalyzes the de novo addition of a methyl group to the cytosine residue of CpG dinucleotide. DNMT3A mutations have a severe impact on DNA-methylation patterns whereby give rise to global shifts in gene expression accompanied by increased self-renewal of hematopoietic cells at the blocking of normal differentiation.12 Mutations mainly disrupt the catalytic domain of the enzyme, which are associated with a loss-of-function of the enzyme activity.12–14 DNMT3A loss-of-function mutations result in hypomethylation of HSC specific genes commonly over-expressed in AML (eg Runx1, Erg, Myc, Smad3) which consequently impair HSC differentiation.1,3 In particular, DNMT3A function is required for HSC self-renewal and myeloid differentiation; its mutations have been detected in preleukemic HSCs and considered as an early event in AML. DNMT3A mutations lead to myeloid transformation in vivo and promote myeloid malignancies in impaired HSCs. Decitabine treatment caused an improved response in patients harboring mutated DNMT3A. Additionally, patients achieved a higher clinical remission rate and a superior OS when compared to those bearing the wild-type DNMT3A (75% vs 34% and 15.2 vs 11 months, respectively). High levels of miR-29b (which targets DNMT3A) would be a good marker for response to decitabine treatment. Importantly, patients with myeloid malignancies who are harboring DNMT3A, IDH1/IDH2 mutations show a favorable response to decitabine and azacitidine, specific DNMT inhibitors and hypomethylating agents (HMAs).12–14

IDH1/2 (Isocitrate Dehydrogenase) Mutations

The IDH1/2 genes encode tumor-suppressor proteins IDH1/2 whose mutations appear to induce DNA hypermethylation at specific sequences. About 15–20% of all AML subtypes and 25–30% of CN-AML subtype harbor IDH1/2 mutations. This frequency is similar in adults and children. IDH1/2 mutations prevailingly accompany NPM1 mutations but not FLT3-ITD.12,13 Epigenetic alterations occurring in IDH1/2 mutations intensify HPC proliferation and increase its pool. IDH1/2 impairment suppresses the histone demethylation process whereby associates with DNA hypermethylation, differentiation blocking and clonal expansion of HSCs.1,3 Biochemically, IDH1/2 are, respectively, cytosolic and mitochondria NADP-dependent dehydrogenases, decarboxylating isocitrate into αKG. The produced molecule is used by TET enzyme when catalyzing histone demethylation. Mutations give rise to the new catalytic activity by the enzyme whereby converts αKG to 2-HG. The oncometabolite 2-HG is a putative inhibitor of histone demethylase TET2. Blocking histone demethylation is accompanied by DNA hypermethylation phenotype (Table 5). All IDH1/2 mutations have been reported heterozygous and mutually exclusive to TET2 mutations, possibly due to overlapping molecular effects.12,13 IDH1/2 mutations are related to an adverse clinical outcome with poor prognosis, particularly when accompanying other mutations like as NMP1 and FLT3-ITD, however, compared to DNMT3A, IDH1/2 mutation has a better prognosis, DFS and superior OS. In the favorable risk groups, the coincidence of IDH1/2 mutations with other aberrations would lead to a lower rate of RFS and 5-year OS duration. Similar to DNMT3A mutations, IDH 1/2s show significantly higher remission rate and favorable response to anti-leukemia therapeutic decitabine and azacitidine. The IDH1 and IDH2 inhibitors are, respectively, AG-220 and AG-221, which reported to show a clinical response, with a significant reduction in 2-HG levels (~50–90%). It is reported that IDH 1/2 inhibitors are being evaluated in Phase III clinical trials and show a prominent impact on AML prognosis.13,16

The α-Ketoglutarate-Dependent Dioxygenase, Ten-Eleven Translocation (TET) Proteins

Approximately 10–20% of AMLs carry TET2 mutations (deletions, nonsense and missense mutations). Clinically, TET2 mutations associate with intermediated risk and short OS. Biologically, these loss-of-function mutations induce DNA hypermethylation, however, mutually exclusive with IDH1/2 mutations. TET2 mutations are prevalent in MDS and myeloproliferative disorders and are highly frequent in chronic myelomonocytic leukemia, wherein associated with monocytosis and poor outcomes.1,12 As an early event present in HSCs, TET2 inactivation induces pre-leukemic HSCs, clonal expansion and leukemogenesis. TET2 mutations may act in the same pathway as IDH1/IDH2 (Table 5). Biochemically, TET activation leads to DNA demethylation in enhancer regions of tumor-suppressor genes through the sequential conversion of 5-methylcytosines (5mC) to 5-hydroxymethylcytosine (5hmC), then 5-formylcytosine to 5-carboxylcytosine which finally accompanied by DNA glycosylase and base excision repair system. Specifically, TET2 mutations cause a reduction in 5hmC levels and an induction in DNA methylation mainly in enhancer regions of nearby tumor suppressors. TET2 inactivation correlates with hypermethylation of tumor suppressors in AML.1,3,6

Epigenetic Biomarkers in AML

The epigenetic regulators DNMT3A, IDH1/IDH2 and TET1/2 are epigenetic markers useful for risk stratification, therapy decision-making and clinical predicting of response to treatment. The epigenetic biomarkers can also include changes in DNA-methylation patterns or expression profile of non-coding RNAs (miRNAs). These epigenome alterations can be a result of class 0/III mutations which contribute to the cancer molecular pathogenesis. It is also said that epigenetic changes can be relatively reversible in response to combinations of epigenetic small molecular agents such as inhibitors of methyltransferases (cytarabine, azacitidine and decitabine), in combination with other available inhibitors including histone deacetylase (HDAC) inhibitors, which are now tested in clinical trials.12,13

Herein, purin and deoxynucleoside antimetabolites (eg thioguanine/mercaptopurine and cytarabine/azacytidine) are inhibitors of DNA methyltransferases (DNMTi) wherein their basic mechanism is quite similar. These compounds enter the cells and are converted into their respective nucleotide analogues and incorporated into the genomic DNA strands. Their incorporation into the DNA inhibits enzymes critical for DNA synthesis, repair, and methylation (acting as hypomethylating agents (HMAs)). Examples of HDAC inhibitors (HDACi) are vorinostat and valproic acid which numerously administrated in clinical trials in combination with sorafenib for treating patients with advanced/metastatic solid malignancies and refractory/relapsed AML. HDACi target directly the histone deacetylases. In clinical trials, rational combinations of HDACi and DNMTi result in the transcriptional activation of the corresponding genes including tumor-suppressor genes often silenced in cancers. Rational combinations and therapy duration of HDACi/DNMTi with DNA damage-inducing therapies (eg anthracyclines/TOP inhibitors) synergistically enhance the irradiation, growth inhibition and apoptotic effects of therapy. DNMT/HDAC inhibitors increase DNA accessibility by DNA damage-inducing therapies. Several cycles of therapeutics are required and the duration of inhibitors is emphasized for the manifestations of hematologic responses to improve survival in AML patients.25,26

Alteration in the Expression Profile of Non-Coding RNA (miRNAs)

Gene expression profiling (GEP) of non-coding RNAs (miRNAs) can discriminate different cytogenetic subtypes or act as prognostic factors. These small RNAs have been proposed as potential biomarkers of integrated panels for prognosis and risk stratification of disease. For example, a panel of 12 miRNAs is proposed which can divide CN-AML into poor and intermediate-risk categories independently of FLT-ITD.5,6 In particular, elevated expression of miR-17-92, miR196, miR-29, miR-125, miR-142, miR-146 and miR-155 is a characteristic of AML (Table 8), addressed as pathogenesis biomarkers. Generally, during normal myelopoiesis, there is a low expression of miRNA clusters whereas highly expressed in leukemic conditions. For instance, miR-125 family, encoded by three conserved clusters mapped to chromosomes 19/21/11, is over-expressed in leukemic HSCs. Over-expression of miR-21/10/196 is reported in NMP1 mutants wherein associated with blockage of normal HSC differentiation and down-regulation of PDCD4 (Programmed Cell Death 4).1,3 Moreover, MLL rearrangements are characterized by a high expression of miR-17-92 cluster, as well as, miR-196b which is located at 7p15, between HOXA9 and HOXA10 genes. Also, FLT3-ITD is associated with increased expression of miR-155/miR-181 family whereby leading to leukemic blast expansion. Moreover, miR-181a/b over-expression is a marker of unfavorable outcomes, in particular in CN-AML CEBPA+/FLT3-IDT+/NPM1-.11,16,18 Even more, expression of miRNAs changes during cytarabine therapy which correlates with outcomes and OS. For example, high expression of miR-191 and miR-199a can be associated with worse OS while down-regulation of miR29b (targeting DNMT3A & DNMT3B) is a marker of a better response to treatment.1,3

Changes in DNA-Methylation Patterns

Each cytogenetic or molecular subtype is supposed to have a distinct DNA-methylation profile. For example, cases with t(8;21), inv(16) or t(16;16), t(15;17) or t(v;11q23) exhibit unique DNA-methylation signatures whereby define AML subtypes. Methylome analysis has indicated that specific regions of the genome have lower methylation levels in AML compared to the normal, such as gene bodies and repetitive sequences. Hypomethylation of repetitive sequences with a high content of methylated CpGs (eg SINEs (short interspersed nuclear elements) and LINEs (long interspersed nuclear elements)) are associated with rearrangements.1,12 Additionally, in methylome analysis of CN-AML, the most pronounced DNA hypermethylation is detected in CpG islands representing tumor-suppressor promoters.3 Recurrent mutations (eg NPM1, CEBPA, RUNX1), can also be defined by distinct DNA-methylation patterns. For example, NPM1 mutations can be defined by four distinct DNA-methylation clusters. AML subtypes can be further classified according to the methylation profiles related to prognosis and clinical outcomes. CEBPA double mutations show two distinct DNA-methylation signatures whereby patients could be split into two distinct prognosis subtypes: one hypermethylated and one hypomethylated.12,13 Herein, initiation, progression and maintenance of the tumor phenotype associate with distinct DNA-methylation patterns.3

The underlying mechanism of aberrant DNA-methylation induction in these AML subsets is attributed to the fusion genes in recruiting DNMTs to their binding sites, or to a secondary epigenetic dysregulation including binding of PML-RARα to genomic regions of epigenetic modifiers such as DNMT3A and/or DNA-methylation disruption of AML1-ETO target genes. For instance, methylation of target genes involved in the genome stability were found to be changed in APL patients at diagnosis time, which predispose them to a hypomethylation phenotype seen in CBFB-MYH11 fusion gene, CEBPA promoter and in the regulatory regions of MN1.1,12 Methylation assays suggest that global DNA hypomethylation occurred in AML patients during treatment can be a marker associated with a successful response, an increased CR rate and an OS improvement. Methylome analyses indicate that hypomethylation of LINE-1 may also associate with low blast counts (<45%), better CR rate during the first cycle of azacitidine, and hematological improvement.

According to the DNA methylation-shifted loci observed in AML, samples can be defined into three categories: shifted loci unique to diagnosis, loci unique to relapse, lastly loci can be seen at both diagnosis and relapse. Accordingly, dividing AML samples into these categories do not correlate with age, white blood cell count, or the French–American–British (FAB) classification. Finally, DNA methylation-shifted loci are expected to be an independent classification for AML. Therefore, identification of mutations associated with DNA methylation and evaluation of changes occurred in methylation signatures would contribute to individual therapy of AML patients.3,12,13

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