1. Introduction
Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system, which is caused by malignant transformation of hematopoietic stem- or progenitor cells [ 1 , 2 , 3 ]. AML may occur “de novo”, secondary to antecedent hematological disorders or after chemo- and/or radiotherapy for a primary disease [ 1 , 2 , 4 , 5 ]. AML is the most common form of acute leukemia in adults with an incidence of approximately 2–4/100,000 per year ( http://seer.cancer.gov/ ) [ 1 , 6 ]. Although everyone can be affected by this disorder, it is mainly a disease of the elderly with a median age at diagnosis of 72 years ( http://seer.cancer.gov/ ). With current high-intensity treatment approaches, approximately 35%–40% of younger AML patients can be cured, however, in older patients >60 years of age, long lasting remissions can be achieved in up to 15% of cases only [ 1 , 2 , 7 ]. Unfortunately, outcomes are even worse in very old patients, or in those with additional comorbidities excluding them from high-dose approaches. Although the introduction of low-intensity therapeutic regimens (LITR), in particular the hypomethylating agents (HMA) azacitidine and decitabine, has resulted in both increased response rates and extended survival within this cohort, the vast majority of these patients ultimately succumbs to therapy-resistant, progressive disease [ 1 , 8 , 9 ]. Prognosis of AML patients is influenced by patient-associated factors on the one hand and by disease-related factors on the other hand. Particularly, research on disease-related factors has made significant progress in the last years, as a variety of molecular markers with prognostic relevance have been identified via the introduction of next generation sequencing techniques. This has led to the development of robust risk stratification models that have helped to accurately predict the clinical course of a given patient and to plan the therapeutic approach accordingly [ 1 , 7 , 10 ]. Unfortunately, despite all this progress in diagnosis and risk evaluation, the major problem in AML is still the development of chemorefractory disease. Although a large proportion of patients achieves complete remission (CR) after cytotoxic therapy, a significant subset presents with primary refractory disease. Even in patients achieving CR, a substantial subgroup ultimately develops chemoresistant relapse [ 1 , 6 , 7 , 11 , 12 ], which is caused by the fact that cytotoxic therapy failed to eradicate a therapy-resistant (sub)clone within these patients [ 13 ]. In any case, the vast majority of these patients still succumbs to this complication, making chemoresistance one of the primary targets of AML research with a tremendous potential to increase survival if overcome.
Non-coding RNAs (ncRNAs) are RNA transcripts of variable length, which are basically not transcribed and translated into proteins. Quite frequently, they are produced from introns, intergenic stretches or from antisense transcripts of protein coding genes [ 14 , 15 , 16 , 17 ]. Given the fact that only a minority of RNA transcripts within mammalian cells is truly transcribed into protein-coding mRNAs, and that the function of ncRNAs has been unknown for a long time, they have been considered as genomic debris initially. However, it is now evident, that they play a central role in many cellular functions, primarily by regulation of gene expression levels. This is orchestrated in a variety of ways, including inhibition of transcription and translation, as well as by epigenetic modification and destabilization of protein coding genes [ 14 ]. Although a broad range of ncRNAs has been discovered, the probably best studied examples, especially in cancer, are micro-RNAs (miRNAs) and long non-coding RNAs (lncRNAs). While miRNAs are usually only 19–24 nucleotides long, lncRNAs display a length of more than 200 nucleotides. Aberrant expression of miRNAs and lncRNAs has been described in all types of cancers, and their functional involvement in pathogenesis of these malignant disorders is continuously being unraveled [ 15 , 16 ]. In AML, aberrant expression of ncRNAs has also been described and shown to be a pivotal process in malignant transformation of hematopoietic stem and/or progenitor cells [ 18 ]. Aberrant ncRNA expression thereby often acts as part of complex networks which additionally includes other genetic alterations in oncogenes and/or tumor-suppressors, as well as altered activation of transcription factors and signaling cascades. Quite frequently, ncRNAs thereby influence the activation status of these networks by targeting one or more of their members in a complex manner. On the other hand, expression of ncRNAs is frequently under the control of other players within these networks as well, thereby forming complex circuits which finally orchestrate malignant transformation. Among others, these networks include prominent players in leukemogenesis, such as fms-like tyrosine kinase 3 ( FLT3 ), nucleophosmin ( NPM1 ), CCAAT/Enhancer Binding Protein Alpha ( CEBPA ), tumor protein P53 ( TP53 ) or the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS-MAPK/ERK) signaling cascade [ 19 , 20 , 21 , 22 , 23 ]. Lately, the role of ncRNA in mediating sensitivity and resistance to cytotoxic agents, respectively, has been discovered, which makes them attractive targets of novel anti-cancer therapies. Here, we review the role of miRNAs and lncRNAs in therapeutic resistance of AML and discuss potential therapeutic options arising thereof.
2. miRNAs and lncRNAs in Sensitivity and Resistance to High-Intensity Therapeutic Regimens
In patients eligible to high-intensity therapeutic regimens, induction chemotherapy consisting of continuous infusion cytarabine over seven days, usually combined with three days of an anthracycline (in most cases daunorubicin) is administered. This so-called “3 + 7” scheme has been largely unchanged for 30 years now and results in CR rates in 60% to 85% in patients younger than 60 years and 40% to 60% in patients older than 60 years [ 1 , 7 ]. In the case CR is achieved, consolidation therapy has to be employed. Currently, postremission consolidation comprises two to four cycles of intermediate-dose cytarabine or allogeneic hematopoietic stem cell transplantation (HSCT) [ 1 , 7 ]. Whether a patient receives cytarabine consolidation or HSCT mainly depends on the risk profile of the respective AML. In this respect, a risk stratification model has been proposed by the European LeukemiaNet and has evolved as widely accepted standard since [ 7 ]. This model is based on cytogenetic and molecular information, subdividing AML patients into four groups (favorable, intermediate-I, intermediate-II and adverse). While cytarabine consolidation is the treatment of choice for patients within the favorable group who achieve CR after induction therapy, allogeneic HSCT has to be considered for all other cases [ 1 , 7 , 10 , 24 ]. In the case chemoresistant disease or relapse after CR occurs, a variety of re-induction schemes have been tested. In the case the patient is still eligible for high-intensity approaches, they all aim to induce AML remission with subsequent allogeneic first (or second) HSCT [ 1 ]. Unfortunately, however, outcomes within these patients are dismal, which highlights the need for novel therapeutic approaches with the potential to overcome therapeutic resistance.
2.1. The Role of miRNAs
A wide range of studies dealing with miRNA expression and therapeutic resistance to intensive AML treatment have been published and allow classification of miRNAs into two major groups ( Table 1 ). Group I comprises miRNAs, in which increased expression mediates sensitivity to chemotherapy, or, on the contrary, in which decreased expression mediates resistance. Group II comprises miRNAs, in which increased expression relays therapeutic resistance, whereas decreased expression mediates sensitivity.
One of the first insights into group I were revealed via a landmark paper by Marcucci and coworkers, who studied diagnostic leukemia specimens from AML patients treated within Cancer and Leukemia Group B (CALGB) trials [ 25 ]. Their analyses comprised a training group of 64 and a validation group of 55 adult patients <60 years with a normal karyotype and high-risk molecular features. By employing expression arrays with 305 miRNA probes, they were able to identify that increased expression of the miR-181 family was associated with a favorable clinical outcome ( Figure 1 ). These results were later corroborated in another CALGB analysis, studying the expression of miR-181a in 309 diagnostic cytogenetically normal (CN) AML specimens [ 26 ]. Again, miR-181a proved to be a favorable prognostic marker and in addition to the previous study, the authors were also able to demonstrate, that the favorable prognostic value of miR-181a did not only apply to younger patients with molecular high-risk features, but also to an older cohort comprising patients >60 years of age. Most importantly, however, as all patients were treated on similar high-dose induction protocols and as high miR-181a expression was associated with achievement of CR in uni- and multivariate analyses, these data suggested a potential impact of this miRNA on mechanisms of sensitivity to AML high-intensity chemotherapeutic regimens. In this respect, the authors tried to identify potential target genes by performing mRNA arrays and correlating the results to the expression of miR-181a. In total, 1174 probe sets showed a statistically significant correlation, which was inversely in 1002 of them, thereby making them potential targets of miR-181a. Although functional experiments were missing, this list included interesting candidates, such as the homeobox ( HOX ) A and B clusters, whose overexpression had been linked to myeloid leukemogenesis and adverse prognosis in AML previously [ 57 ]. This correlation, as well as its prognostic value could later be confirmed in another study of 454 AML patients, this time comprising a cohort of cytogenetically abnormal (CA) patients [ 27 ]. Functional insight into the role of miR-181a in therapeutic resistance came from two studies that analyzed the resistance of AML cell lines to cytarabine and daunorubicin treatment, respectively [ 49 , 50 ]. Both studies demonstrated that resistant subclones were characterized by decreased miR-181a expression levels and that re-expression, as achieved by miR-181a mimics, was able to restore chemosensitivity. Interestingly, the BCL2 genes were identified as direct miR-181a targets in both studies and the miR-181a induced chemosensitivity was shown to be mediated via this miR-181a/ BCL2 axis. Finally, an interesting therapeutic approach to overcome the therapeutic resistance induced by decreased miR-181a was presented by Hickey and coworkers [ 58 ]. They observed that high expression of miR-181a correlated with N-terminal mutations in CEBPA . These mutations account for approximately 90% of CEBPA mutations in AML and enable translation of a truncated CEBPA -p30 isoform. The authors could show that this isoform directly targets the miR-181a promoter and thereby causes its upregulation. Most importantly, however, lenalidomide, which is currently used for the treatment of multiple myeloma and myelodysplastic syndromes, induced the expression of CEBPA -p30 and miR-181a, thereby inhibiting leukemogenic effects in vitro and in vivo. The authors thereby provided a possible explanation for the favorable prognostic impact of mutated CEBPA on the one hand, and a possible therapeutic intervention to overcome therapeutic resistance via a miR-181a axis on the other hand.
Another interesting example constitutes the family of let-7 miRNAs. This miRNA family was first discovered in nematodes and describes one of the first human miRNAs described at all [ 59 ]. Downregulation of let-7f has been described in a doxorubicin-resistant subclone of K562 leukemia cells [ 28 ]. In a subsequent series of experiments, the authors performed artificial re-expression of this miRNA and measured doxorubicin IC 50 values, defined as the doxorubicin concentration required to inhibit cell growth by 50%. Importantly, re-expression of let-7f in the doxorubicin-resistant subclone resulted in a significant decrease of IC 50 values, thereby indicating a role of let-7f downregulation in development of anthracycline resistance. Indeed, when 60 patients with de novo and 50 with refractory AML were analyzed, let-7f expression levels turned out to be decreased in specimens from the refractory cohort. Chen and coworkers demonstrated that increased levels of let-7a cause enhanced sensitivity to cytarabine by downregulation of the BCL proteins and subsequent induction of apoptosis [ 51 ]. Importantly, however, the value of aberrant let-7a expression as prognostic marker still remains a matter of debate. Current data are conflicting in this respect [ 60 , 61 ], which might also be due to the fact that distinct let-7a family members have been reported [ 62 ]. The same problem seems to apply for other miRNAs as well, among others including miR-128, miR-331 and miR-27a, respectively [ 25 , 52 , 53 , 63 ]. All of these have been studied in functional in vitro and/or in vivo approaches and their overexpression has been shown to enhance sensitivity to cytotoxic agents used in high-intensity treatment approaches used in AML. However, association of these miRNA expression patterns with response rates in clinical datasets, which would further corroborate these functional observations, is either missing or contradictory. Larger, prospective and uniformly treated patient cohorts will definitely be needed to unambiguously clarify these issues and to delineate the role of these miRNAs in the development of therapeutic resistance. Whereas association with therapeutic resistance has been backed by functional in vitro and/or in vivo approaches in the miRNAs described so far, a group of potential candidate miRNAs exists, in which clinical data demonstrated that their increased expression levels are associated with a favorable prognosis but where functional data supporting these clinical correlations are still missing. Among others, these comprise miR-9* (miR-9-3p), miR-96, miR135a and miR-409 [ 29 , 30 , 31 ]. An interesting example from this group is the miR-10 family, which has been linked to mutations in NPM1 previously [ 22 , 64 ]. In subsequent follow-up studies, miR-10 expression and NPM1 mutations indeed correlated with higher CR rates in uni- and multivariate models, thereby making it a likely candidate for modulating therapeutic resistance [ 32 ]. However, when the authors employed functional analyses, including cytarabine treatment after miR-10 modulation, they did not see any impact of miR-10 overexpression. These data suggest that—although miR-10 and mutated NPM1 seem to be linked—the favorable prognostic effects seem to be mediated primarily via NPM1 and that miR-10 seems to be a bystander of these events. Furthermore, they clearly highlight the importance of functional experiments, in which the effects of miRNA modulation are tested after treatment with the respective cytotoxic agents. Relying solely on clinical correlations, i.e., the achievement of CR, might result in misinterpretation of the results.
While this part of the review focused on miRNAs categorized as members of group I, a large fraction has to be assigned to group II, which means that increased expression mediates therapeutic resistance, or, conversely, decreased expression relays sensitivity. One such example is miR-155. This miRNA has been shown to be upregulated in AML specimens carrying an internal tandem duplication within the FLT3 gene ( FLT3 -ITD) [ 22 , 44 , 64 , 65 ]. Indeed, a causal relation could be shown, as the expression of miR-155 could be induced by the FLT3 -ITD downstream targets nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 5 STAT5. The role of miR-155 in the pathogenesis of AML has also been demonstrated in functional approaches. It has been shown to directly target the PU.1 transcription factor, thereby promoting proliferation of myeloid cells on the one hand and inhibiting their apoptosis on the other hand [ 23 ]. miR-155 is also of interest from a therapeutic view, as its expression can be inhibited by Silvestrol, a compound isolated from the Indonesian plant Aglaia foveolata [ 66 ] and by the synthetic Nedd8-activating enzyme inhibitor MLN4924 [ 67 ]. Indeed, these approaches are able to induce anti-leukemogenic effects both in vitro and in vivo [ 66 , 67 ]. The prognostic value of miR-155, and particularly its association with therapeutic sensitivity, was first shown in a study comprising 363 leukemic specimens of newly diagnosed AML patients with a normal karyotype. Increased expression correlated with decreased CR rates and a shorter overall survival (OS) in both uni- and multivariate analyses within this cohort [ 34 ]. As all patients were treated with high-dose induction treatments, these data suggested a potential role of increased miR-155 expression in the development of therapeutic resistance. Again, high miR-155 expression correlated with FLT3 -ITD and the expression of genes involved in NF-κB activation, which again highlights the existence of the FLT3 -ITD/NF-κB/miR-155 axis described above. In a subsequent study comprising 138 AML patient specimens, high miR-155 expression was included in a prognostic scoring system, including information on the expression of specific miRNAs [ 35 ]. Again, this model proved to be of prognostic relevance as the subgroup with high miR-155 levels correlated with a shortened OS. The link to therapeutic resistance was further strengthened by data showing that a doxorubicin resistant subclone of K562 leukemia cells demonstrated significantly increased expression levels of miR-155 [ 68 ]. However, functional studies exploring the mechanisms of miR-155 induced chemoresistance in more detail are still missing. This is of particular relevance, as conflicting results in this area have been reported. In discordance with the results presented above, one study observed resistance to cytarabine in AML cells not by miR-155a overexpression but by employing its knockdown by the means of miRNA inhibitors [ 69 ]. Another miRNA, in which increased expression plays a role is miR-125b. This miRNA has been shown to exhibit increased expression in a variety of leukemias, including AML [ 70 ]. Furthermore, enforced expression of miR-125b has been shown to cause leukemia in murine transplant models [ 70 ]. Zhang and colleagues first demonstrated its involvement in therapeutic resistance, when studying 169 primary AML specimens (including 114 samples with acute promyelocytic leukemia [APL] as well as specimens obtained at diagnosis and following therapy) [ 36 ]. They observed that miR-125b expression was upregulated in diagnostic AML samples when compared to healthy controls. Importantly, however, while it was downregulated in post-therapeutic specimens in patients achieving a CR, it increased again in those showing relapsed disease. To gain more insight into this phenomenon, the authors performed cellular cytotoxicity assays in AML and APL cell lines with and without transfection of miR-125b and assessed IC 50 values for treatment with doxorubicin and all trans retinoic acid (ATRA), respectively. Indeed, overexpression of miR-125b caused a significant increase in IC 50 of both drugs, thereby confirming its role in development of therapeutic resistance. These data were subsequently corroborated in a cohort of 46 acute leukemia patients [ 37 ]. Again, high miR-125b expression was observed and associated with decreased event-free survival (EFS). In a series of AML cell lines, the authors demonstrated again that overexpression of miR-125b contributes to daunorubicin resistance, and that these effects are mediated via downregulation of PUMA and GRK2 , which in turn resulted in decreased apoptosis. Another interesting example of increased miRNA expression conferring resistance is miR-126. This miRNA was originally identified as overexpressed in core binding factor (CBF) AML [ 71 ]. The authors could show that increased expression of miR-126 is associated with hypomethylation of its promoter region and causes leukemogenic effects in vitro. Leeuw and colleagues could subsequently show, that miR-126 expression was significantly increased in hematopoietic (HSC) and leukemic stem cells (LSC), when compared to more mature leukemic progenitors [ 38 ]. When analyzing 92 non-CBF AML patient specimens, high miR-126 expression proved to be an adverse prognostic marker, which was associated with poorer EFS, relapse-free survival (RFS) and OS. In functional experiments, miR-126 knockdown decreased the growth of AML cells by inducing apoptosis. Similar results were observed in a cohort of 126 AML patients treated with high-dose induction chemotherapy within the CALGB protocols [ 39 ]. High expression of miR-126 correlated with decreased CR rates, as well as with shortened EFS and OS in older patients >60 years. As previously demonstrated, the authors also showed that increased expression of miR-126 correlated with hypomethylation of its promoter as well, and that its expression was particularly increased within the LSC compartment. Most interestingly, the authors present a nanoparticle based miR-126 inhibitor, which enabled the pronounced downregulation of this miRNA, both in vitro and in vivo. Indeed, this approach caused strong anti-leukemic effects in murine leukemia transplant models. A real link to the development of resistance to chemotherapy was established by Shibayama and coworkers, who modified miR-126 expression in AML cell lines and subsequently assessed their sensitivity to cytarabine and idarubicin, respectively, in MTT assays [ 55 ]. While miR-126 overexpression had no effect on the sensitivity to idarubicin, it caused resistance to cytarabine, as evidenced by significantly increased IC 50 values. More detail in the mechanisms behind these phenomena could be provided very recently by Lechman and colleagues [ 40 ]. In a landmark study, they could prove that, indeed, the LSC compartment exhibits the highest levels of miR-126 expression and could confirm the adverse prognostic impact of high miR-126 levels in AML patients. In a series of in vitro and in vivo approaches, they could demonstrate that miR-126 preserves LSC in a quiescent state by targeting the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/MTOR) pathway, which in turn mediates resistance of LSC to daunorubicin. Despite all these progress, again caution has to be employed, when the value of miR-126 as therapeutic target is revisited. As already shown above for miR-155, a certain two-faceted role of miR-126 seems to exist, as a recent publication observed the same leukemogenic effects, both after overexpression and knockdown of miR-126 [ 72 ]. These results again highlight the fact that the regulation and effects of changes in miRNA expression are part of a complex network depending on a variety of co-factors, most of them hitherto unknown. Furthermore, they demonstrate the importance of thorough preclinical evaluation of miR-targeting drugs, before the step into clinical trials is dared. Beside these well studied miRNAs, again a group of candidates exists, in which functional relevance of their increased expression in the development of therapeutic resistance has been shown in vitro and/or in vivo, however, where clinical data corroborating these results are still missing or preliminary. Among others, these include miR-20a and miR-32 [ 33 , 54 , 56 ]. Finally, as already described above, a group of miRNAs in which increased expression could be linked to adverse outcome (or in which decreased expression was linked to favorable prognosis) in clinical association studies, but where functional experiments are missing, has been described. Among others, this group comprises miR-210, miR-3151, miR-196b, miR-644, miR-199a and miR-191 [ 31 , 41 , 42 , 43 , 44 ]. Further functional and or clinical association studies, respectively, will be needed for these two miRNA groups to unambiguously clarify their role in the development of therapeutic resistance.
2.2. The Role of lncRNAs
While a lot of data are available on the role of miRNAs in development of resistance to high intensity therapeutic regimens in AML, data about the role of lncRNAs within this field are scarce until now ( Table 1 ). A first lead into this direction came from Garzon and colleagues in 2014, who studied the association of lncRNAs with prognosis in a cohort of 148 CN-AML patients treated within the CALGB trials by microarray expression profiling [ 73 ]. By selecting lncRNAs associated with EFS, the authors were able to establish a lncRNA-based scoring system, which included the expression status of 48 lncRNAs and which was able to accurately predict CR, disease-free survival (DFS) and OS rates. The reliability of this score could subsequently be validated in a second cohort of 71 AML patients, where lncRNA expression was studied by an independent technique (RNA sequencing). Again, the lncRNA score correlated with CR, DFS and OS in uni- and multivariate analyses. Although no functional analyses were included, the fact that lncRNA expression profiles correlated with CR rates in patients treated with similar high-dose approaches suggested a potential involvement of lncRNAs in the development of therapeutic resistance for the first time.
A lncRNA of potential interest might be Hox transcript antisense intergenic RNA (HOTAIR), which displays a length of 2.2 kb and which is expressed from the HOXC locus on chromosome 12 [ 74 , 75 ]. Aberrant expression of HOTAIR has been shown in a variety of solid tumors and its involvement in malignant transformation could be proven in functional in vitro and in vivo approaches [ 74 ]. Recently, increased expression of HOTAIR was observed in AML as well. Xing and colleagues studied the expression of HOTAIR in 136 diagnostic AML specimens and compared the results to normal controls. They observed a significant upregulation of HOTAIR in AML and could prove the functional involvement of HOTAIR in AML cell growth, apoptosis and colony formation by means of knockdown experiments. Finally, when looking on clinical parameters, high expression of HOTAIR correlated with worse DFS and OS [ 45 ]. Similar results were observed in two additional studies [ 46 , 47 ]. Again, high expression of HOTAIR was associated with an adverse clinical course and proved to be involved in malignant transformation of hematopoietic cells. Although a definitive link to resistance to therapeutic high-intensity AML treatment regimens is still missing, HOTAIR induced resistance to anthracyclines in functional in vitro models of solid tumors previously [ 76 ]. It will be of interest to see whether these mechanisms apply to AML as well. Another lncRNA showing at least a clinical indication to therapeutic resistance is Hox antisense intergenic RNA myeloid 1 (HOTAIRM1). In an analysis of 241 AML patient specimens, increased levels of HOTAIRM1 were observed and correlated with shorter OS, shorter leukemia-free survival and a higher cumulative incidence of relapse in uni- and multivariate analyses [ 48 ]. Again, functional experiments further proving that therapeutic resistance is indeed mediated via HOTAIRM1 will be needed to further clarify the role of HOTAIRM1 in this respect.
3. miRNAs and lncRNAs in Sensitivity and Resistance to Low-Intensity Therapeutic Regimens
Although a subset of patients can be cured with high-dose chemotherapy and/or allogeneic hematopoietic stem cell transplantation, a large subset of AML patients are ineligible to these approaches, mainly because of older age and/or comorbidities [ 1 , 2 , 6 ]. While best supportive care was the only treatment option in these cases for a long time, the introduction of LITR therapies was able to significantly increase both OS and quality of life in affected patients [ 1 , 9 , 77 ]. The first LITR to be introduced was low-dose cytarabine (LDAC). At a dose of only 20 mg twice daily for 10 days every four weeks, it demonstrated increased survival as compared to best supportive care only [ 78 ]. While LDAC served as the mainstay of AML LITR for a long time, the recent introduction of HMA treatments remarked a further significant development in this area [ 1 , 8 ], with the cytidine analogues azacitidine (5-azacytidine) and decitabine (5-aza-2′-deoxycytidine) representing the currently best studied substances within this category. Two main anti-tumorigenic effects of these drugs have been described [ 8 ]: (a) Both drugs act as antimetabolites and are incorporated into DNA (decitabine) or DNA and RNA (azacitidine), respectively. Consequently, DNA damage response is induced; (b) Both drugs cause hypomethylation of DNA by inhibiting DNA methyltransferases, which is thought to restore the expression of silenced tumor suppressor genes. Both drugs have been tested in multicenter, randomized phase III trials, where they have shown efficacy when compared to LDAC, BSC and/or intensive chemotherapy [ 8 ]. Consequently, these drugs evolved as mainstay of AML treatment in older patients who are ineligible to high-intensity chemotherapy, thereby causing a significant amelioration of survival within this subgroup. However, despite all this progress in the development of LITR approaches, a significant proportion of patients exhibits refractory disease. Furthermore, even in patients showing partial or complete remission of AML under LITR therapy, development of therapeutic resistance is observed in the vast majority of patients, thereby causing recurrence or progression of AML [ 1 , 8 ]. Hence, research on strategies overcoming the mechanisms behind this therapeutic resistance is desperately needed to further improve survival within these patients.
3.1. The Role of miRNAs
Aberrant expression of miRNAs has been associated with differential sensitivity to HMA treatment in AML ( Table 2 ). The probably best studied example is miR-29b ( Figure 2 ). This miRNA has been shown to be downregulated in AML via activation of a KIT-MYC axis [ 79 ] and has been demonstrated to be involved in the regulation of DNA methylation by directly targeting DNA methyltransferases 3A ( DNMT3A ) and 3B ( DNMT3B ) [ 80 ]. Indeed, artificial overexpression of miR-29b caused inhibition of proliferation of AML cells in vitro and of leukemogenesis in vivo [ 81 ]. In a phase II clinical trial of decitabine in older AML patients, Blum and coworkers studied miR-29b expression in diagnostic specimens and observed that increased levels of miR-29b correlated with superior clinical response to decitabine treatment [ 82 ]. Likewise, increased expression of miR-29b did also correlate with decreased expression levels of DNMT3A . To further study this phenomenon, the same group performed overexpression of miR-29b using a transferrin-conjugated nanoparticle delivery system for synthetic miR-29b in AML cell lines and primary patient specimens followed by treatment with decitabine [ 83 ]. Interestingly, overexpression of miR-29b increased the sensitivity to decitabine thereby increasing the anti-leukemic properties of this drug in in vitro and in vivo assays. They further observed that miR-29b expression in AML is repressed via histone deacetylases (HDACs) and can be restored by the application of HDAC inhibitors [ 84 ]. Indeed, sequential treatment with HDAC inhibitors and decitabine resulted in stronger anti-leukemic activities in vitro and in vivo as compared to either drug alone.
Another member of the miR-29 family studied in this respect is miR-29c. Butrym and colleagues studied miR-29c in AML patients treated with azacitidine and observed increased expression of this miRNA in AML specimens when compared to a healthy control group [ 85 ]. Unexpectedly, lower expression of miR-29c correlated with good clinical response to azacitidine, which is in sharp contrast to the findings observed in miR-29b. It will be of interest to delineate whether these discrepancies are due to differences between azacitidine and decitabine, or whether they are due to differences between these miR-29 subforms. Beside the miR-29 family, miR-331 could be associated with chemosensitivity in LITR treatment of AML. Butrym and colleagues studied its expression in 95 bone marrow specimens of newly diagnosed AML patients, who were treated either with intensive ( n = 56), or non-intensive therapies ( n = 27) or best supportive care ( n = 12) [ 63 ]. Among the non-intensively treated patients, LDAC and azacitidine were used. Interestingly, increased expression of miR-331 correlated to lower CR rates and a greater risk of death. Unfortunately, functional data proving that miR-331 indeed plays a causal role in chemoresistance to azacitidine are still missing, therefore, these experiments will be important for future research within this area.
Another interesting group of miRNAs for HMA treatment encompasses miRNAs that are suppressed in AML by hypermethylation occurring within their promoter regions. Many of these miRNAs fulfill a tumor-suppressor function under normal circumstances, as they directly target and thus inhibit a broad range of oncogenes. Consequently, their suppression in AML results in increased oncogenic activity, thereby aggravating the leukemogenic potential. In light of the fact that treatment with the hypomethylating agents azacitidine and decitabine might restore the expression of these miRNAs, analysis of their pre-treatment expression levels might provide important information on patients profiting most from HMA therapy. One such example is miR-193a, which has been shown to be silenced in AML by methylation [ 86 ]. Additionally, it has been demonstrated to target the KIT oncogene. Treatment of AML cells characterized by activating KIT mutations and/or KIT overexpression with azacitidine caused the re-expression of this miRNA, which in turn downregulated the KIT oncogene and inhibited cellular growth [ 86 ]. miR-663 has also been shown to be hypermethylated in both AML and chronic myeloid leukemia (CML) [ 87 , 88 ]. Again, this induced oncogenes, such as HRAS , and leukemogenic transformation [ 87 ]. Importantly, treatment with hypomethylating agents was able to reverse this miR-663 silencing [ 88 ]. Other examples are miR-125a and miR-370. Both have been shown to be methylated and suppressed in AML, which in turn resulted in activation of the ErbB pathway in the case of miR-125a [ 89 ] and the transcription factor FoxM1 in the case of miR-370 [ 90 ]. Importantly, however, treatment with hypomethylating agents was able to reverse these effects. miR-124-1 is a tumor suppressor miRNA downregulated in various hematologic malignancies including AML [ 91 ]. Again, treatment with decitabine was able to restore its expression and caused the downregulation of miR-124-1 target genes, including CDK6 [ 91 ]. Evidently, clinical studies demonstrating that AML patients with decreased expression of these miRNAs truly exhibit a particular benefit from HMA treatment are still missing. Answering these questions within prospective clinical trials will be of interest and is currently addressed, as miRNA expression profiling is increasingly introduced into clinical trials, also including those with LITR treatment.
Another possibility of chemoresistance mediated by hypermethylation of miRNAs has recently been observed in CML. miR-217, miR-23a and miR-203 are miRNAs that frequently show a hypermethylated promoter region and decreased expression within this malignancy [ 94 , 95 , 96 ]. Importantly, this mechanism has been shown to confer resistance to tyrosine kinase inhibitors, the current gold-standard in CML treatment [ 95 ]. Simultaneous treatment with hypomethylating agents restored the expression of these miRNAs [ 92 , 95 ], which in turn re-sensitized the cells to TKI treatment [ 95 ]. Consequently, the hypothesis that resistance to standard induction therapy of AML might be caused by the epigenetic inactivation of miRNAs as well and therefore might be overcome by simultaneous administration of HMA is appealing. These hopes were further nurtured by both in vitro and in vivo data, where synergistic effects between cytarabine and azacitidine could be shown, and particularly, where cytarabine-resistant cell lines could be re-sensitized by simultaneous administration of azacitidine [ 97 , 98 ]. Additionally, data from a clinical phase 1 study analyzing a combination of decitabine with standard induction chemotherapy suggested its use to be safe and feasible [ 99 ]. Unfortunately, however, the authors of a large and randomized phase II trial, who studied the combination of azacitidine and standard induction chemotherapy in older AML patients, failed to observe a beneficial effect of this regimen [ 100 ]. It will be interesting to see, whether these results can be ameliorated by altered dosing schedules or by the use of newer hypomethylating agents, such as guadecitabine [ 101 ]. Another obstacle in interpretation of the CML data mentioned above, is that AML constitutes a distinct genetic disease. Whereas miR-203 shows downregulation in approximately 10% of AML patient samples as well [ 92 ], this does not apply for miR-23a. In previous work of our own and other groups, including both in vitro and in vivo approaches, miR-23a was demonstrated to act rather as oncogene within this disease [ 21 , 102 ]. From a mechanistic point of view, increased expression of miR-23a caused a downregulation of the tumor- and metastasis suppressor RKIP thereby increasing the proliferation of leukemic cells. Taken together, these results clearly demonstrate that different hematologic neoplasms are characterized by distinct clonal architectures and that, although the concept of hypermethylated miRNAs conferring resistance to therapy might apply for all of them, the potential culprit miRNAs have to be identified in unbiased approaches specifically addressing each malignancy separately.
3.2. The Role of lncRNAs
Much less is known about the role of long non-coding RNAs within the process of sensitivity to LITR, in particular HMA treatment ( Table 2 ). Treppendahl and colleagues studied the 106-nucleotide lncRNA vault RNA2-1 (vtRNA2-1; previously mislabeled as miR-886) in AML [ 93 ]. They observed its frequent methylation in AML patient specimens, which correlated to decreased expression of this lncRNA and worse clinical outcome. Importantly, treatment with azacitidine was able to restore vtRNA2-1 expression and to down-regulate the phosphorylated RNA-dependent protein kinase (pPKR), a known vtRNA2-1 target that has been shown to promote cell survival in AML. As for miRNAs, prospective clinical trials specifically addressing this question will be needed to truly prove that patients with methylation of vtRNA2-1 demonstrate a special profit from HMA treatment.
4. Conclusions and Outlook
ncRNAs are increasingly recognized as central players in development of therapeutic resistance to cytotoxic agents administered for the treatment of human malignancies. In this review, we discuss miRNAs and lncRNAs involved in therapeutic resistance and sensitivity to intensive and non-intensive treatment regimens currently used in AML. Firstly, this is of relevance as prognostic risk stratification in AML has improved dramatically within recent years by inclusion of cytogenetic and molecular markers [ 1 , 2 , 7 , 103 ]. This has led to treatment algorithms, where the molecular profile of individual patients is able to predict their response to treatment, and consequently, where intensity and duration of therapy is routinely planned according to this genetic information [ 1 ]. It seems a likely option that these risk stratification schemes could be further improved by the inclusion of selected ncRNA expression profiles. Additionally, however, it is a tempting vision, that therapeutic sensitivity to conventional chemotherapy can be restored or further increased by the artificial modulation of ncRNA expression(s) in potential future therapeutic approaches. The likeliness of such a scenario is further supported by the fact that therapeutic targeting of miRNAs has become a feasible option and that the first miRNA therapeutics are already available [ 104 ]. Although this sounds promising, it has to be mentioned that beside the specific disease-modifying effects, these substances seem to come along with unique side effect profiles as well. These can also be severe, as in the case of the miR-34 mimic MRX34, where a phase 1 trial was recently halted following multiple immune-related severe adverse events ( http://investor.mirnarx.com/releases.cfm ; 20 September 2016). Therefore, although ncRNA therapeutics are promising new drugs with the potential to overcome therapeutic resistance in AML, their establishment as therapeutic agents that can be safely used in the clinical routine setting, is still at its beginning. To achieve this goal, ongoing research will be needed in identification of the most promising ncRNA targets on the one hand and design of the best ncRNA therapeutics with appropriate efficacy and safety profiles on the other hand. Finally, it will also be important to identify the ideal cohort of patients that truly benefit from a specific ncRNA-targeting therapeutic agent.
Author Contributions
Armin Zebisch designed the review, performed an intensive literature review and wrote the manuscript; Stefan Hatzl, Martin Pichler, Albert Wölfler and Heinz Sill made substantial contributions to the conception, performed an intensive literature review and critically revised the manuscript. All authors proofread and approved the final manuscript.
Conflicts of Interest
Armin Zebisch reports receiving honoraria from Celgene. Heinz Sill reports receiving research support and honoraria from Celgene. Albert Wölfler reports receiving research support from Celgene and honoraria from Celgene and Janssen-Cilag.
References
1. Dohner H. Weisdorf D.J. Bloomfield C.D. Acute Myeloid Leukemia N. Engl. J. Med. 2015 373 1136 1152 26376137 10.1056/NEJMra1406184
2. Sill H. Olipitz W. Zebisch A. Schulz E. Wolfler A. Therapy-Related Myeloid Neoplasms: Pathobiology and Clinical Characteristics Br. J. Pharmacol. 2011 162 792 805 10.1111/j.1476-5381.2010.01100.x 21039422 PMC3042191
3. Zebisch A. Czernilofsky A.P. Keri G. Smigelskaite J. Sill H. Troppmair J. Signaling through RAS-RAF-MEK-ERK: From Basics to Bedside Curr. Med. Chem. 2007 14 601 623 10.2174/092986707780059670 17346150
4. Schulz E. Valentin A. Ulz P. Beham-Schmid C. Lind K. Rupp V. Lackner H. Wolfler A. Zebisch A. Olipitz W. Germline Mutations in the DNA Damage Response Genes BRCA1 , BRCA2 , BARD1 and TP53 in Patients with Therapy Related Myeloid Neoplasms J. Med. Genet. 2012 49 422 428 10.1136/jmedgenet-2011-100674 22652532
5. Zebisch A. Haller M. Hiden K. Goebel T. Hoefler G. Troppmair J. Sill H. Loss of RAF Kinase Inhibitor Protein is a Somatic Event in the Pathogenesis of Therapy-Related Acute Myeloid Leukemias with C-RAF Germline Mutations Leukemia 2009 23 1049 1053 10.1038/leu.2009.68 19357705
6. Juliusson G. Lazarevic V. Horstedt A.S. Hagberg O. Hoglund M. Swedish Acute Leukemia Registry Group Acute Myeloid Leukemia in the Real World: Why population-based registries are needed Blood 2012 119 3890 3899 10.1182/blood-2011-12-379008 22383796 PMC3358248
7. Dohner H. Estey E.H. Amadori S. Appelbaum F.R. Buchner T. Burnett A.K. Dombret H. Fenaux P. Grimwade D. Larson R.A. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet Blood 2010 115 453 474 10.1182/blood-2009-07-235358 19880497
8. Diesch J. Zwick A. Garz A.K. Palau A. Buschbeck M. Gotze K.S. A Clinical-Molecular Update on Azanucleoside-Based Therapy for the Treatment of Hematologic Cancers Clin. Epigenet. 2016 8 1 11 10.1186/s13148-016-0237-y 27330573 PMC4915187
9. Bainschab A. Quehenberger F. Greinix H.T. Krause R. Wolfler A. Sill H. Zebisch A. Infections in Patients with Acute Myeloid Leukemia Treated with Low-Intensity Therapeutic Regimens: Risk Factors and Efficacy of Antibiotic Prophylaxis Leuk. Res. 2016 42 47 51 10.1016/j.leukres.2016.01.014 26866663
10. Estey E.H. How to Manage High-Risk Acute Myeloid Leukemia Leukemia 2012 26 861 869 10.1038/leu.2011.317 22116555
11. Zebisch A. Lal R. Muller M. Lind K. Kashofer K. Girschikofsky M. Fuchs D. Wolfler A. Geigl J.B. Sill H. Acute Myeloid Leukemia with TP53 Germline Mutations Blood 2016 128 2270 2272 10.1182/blood-2016-08-732610 27621308 PMC5095760
12. Juliusson G. Antunovic P. Derolf A. Lehmann S. Mollgard L. Stockelberg D. Tidefelt U. Wahlin A. Hoglund M. Age and Acute Myeloid Leukemia: Real World Data on Decision to Treat and Outcomes from the Swedish Acute Leukemia Registry Blood 2009 113 4179 4187 10.1182/blood-2008-07-172007 19008455
13. Ding L. Ley T.J. Larson D.E. Miller C.A. Koboldt D.C. Welch J.S. Ritchey J.K. Young M.A. Lamprecht T. McLellan M.D. Clonal Evolution in Relapsed Acute Myeloid Leukaemia Revealed by Whole-Genome Sequencing Nature 2012 481 506 510 10.1038/nature10738 22237025 PMC3267864
14. Ling H. Fabbri M. Calin G.A. MicroRNAs and Other Non-Coding RNAs as Targets for Anticancer Drug Development Nat. Rev. Drug Discov. 2013 12 847 865 10.1038/nrd4140 24172333 PMC4548803
15. Ling H. Vincent K. Pichler M. Fodde R. Berindan-Neagoe I. Slack F.J. Calin G.A. Junk DNA and the Long Non-Coding RNA Twist in Cancer Genetics Oncogene 2015 34 5003 5011 10.1038/onc.2014.456 25619839 PMC4552604
16. Pichler M. Calin G.A. MicroRNAs in Cancer: From Developmental Genes in Worms to their Clinical Application in Patients Br. J. Cancer 2015 113 569 573 10.1038/bjc.2015.253 26158421 PMC4647691
17. Calin G.A. Dumitru C.D. Shimizu M. Bichi R. Zupo S. Noch E. Aldler H. Rattan S. Keating M. Rai K. Frequent Deletions and Down-Regulation of Micro-RNA Genes miR15 and miR16 at 13q14 in Chronic Lymphocytic Leukemia Proc. Natl. Acad. Sci. USA 2002 99 15524 15529 10.1073/pnas.242606799 12434020 PMC137750
18. Schotte D. Pieters R. Den Boer M.L. MicroRNAs in Acute Leukemia: From Biological Players to Clinical Contributors Leukemia 2012 26 1 12 10.1038/leu.2011.151 21701489
19. Chiu Y.C. Tsai M.H. Chou W.C. Liu Y.C. Kuo Y.Y. Hou H.A. Lu T.P. Lai L.C. Chen Y. Tien H.F. Prognostic Significance of NPM1 Mutation-Modulated microRNA-mRNA Regulation in Acute Myeloid Leukemia Leukemia 2016 30 274 284 10.1038/leu.2015.253 26376228
20. Hermeking H. MicroRNAs in the p53 Network: Micromanagement of Tumour Suppression Nat. Rev. Cancer. 2012 12 613 626 10.1038/nrc3318 22898542
21. Hatzl S. Geiger O. Kuepper M.K. Caraffini V. Seime T. Furlan T. Nussbaumer E. Wieser R. Pichler M. Scheideler M. Increased Expression of miR-23a Mediates a Loss of Expression in the RAF Kinase Inhibitor Protein RKIP Cancer Res. 2016 76 3644 3654 10.1158/0008-5472.CAN-15-3049 27197200 PMC5024755
22. Jongen-Lavrencic M. Sun S.M. Dijkstra M.K. Valk P.J. Lowenberg B. MicroRNA Expression Profiling in Relation to the Genetic Heterogeneity of Acute Myeloid Leukemia Blood 2008 111 5078 5085 10.1182/blood-2008-01-133355 18337557
23. Gerloff D. Grundler R. Wurm A.A. Brauer-Hartmann D. Katzerke C. Hartmann J.U. Madan V. Muller-Tidow C. Duyster J. Tenen D.G. NF-κB/STAT5/miR-155 Network Targets PU.1 in FLT3-ITD-Driven Acute Myeloid Leukemia Leukemia 2015 29 535 547 10.1038/leu.2014.231 25092144 PMC4490787
24. Cornelissen J.J. Gratwohl A. Schlenk R.F. Sierra J. Bornhauser M. Juliusson G. Racil Z. Rowe J.M. Russell N. Mohty M. The European LeukemiaNet AML Working Party Consensus Statement on Allogeneic HSCT for Patients with AML in Remission: An Integrated-Risk Adapted Approach Nat. Rev. Clin. Oncol. 2012 9 579 590 10.1038/nrclinonc.2012.150 22949046
25. Marcucci G. Radmacher M.D. Maharry K. Mrozek K. Ruppert A.S. Paschka P. Vukosavljevic T. Whitman S.P. Baldus C.D. Langer C. MicroRNA Expression in Cytogenetically Normal Acute Myeloid Leukemia N. Engl. J. Med. 2008 358 1919 1928 10.1056/NEJMoa074256 18450603
26. Schwind S. Maharry K. Radmacher M.D. Mrozek K. Holland K.B. Margeson D. Whitman S.P. Hickey C. Becker H. Metzeler K.H. Prognostic Significance of Expression of a Single microRNA, miR-181a, in Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study J. Clin. Oncol. 2010 28 5257 5264 10.1200/JCO.2010.29.2953 21079133 PMC3018359
27. Li Z. Huang H. Li Y. Jiang X. Chen P. Arnovitz S. Radmacher M.D. Maharry K. Elkahloun A. Yang X. Up-Regulation of a HOXA-PBX3 Homeobox-Gene Signature Following Down-Regulation of miR-181 is Associated with Adverse Prognosis in Patients with Cytogenetically Abnormal AML Blood 2012 119 2314 2324 10.1182/blood-2011-10-386235 22251480 PMC3311258
28. Dai C.W. Bai Q.W. Zhang G.S. Cao Y.X. Shen J.K. Pei M.F. Yin C.C. MicroRNA Let-7f is Down-Regulated in Patients with Refractory Acute Myeloid Leukemia and is Involved in Chemotherapy Resistance of Adriamycin-Resistant Leukemic Cells Leuk. Lymphoma 2014 55 1645 1648 10.3109/10428194.2013.847936 24067109
29. Nowek K. Sun S.M. Dijkstra M.K. Bullinger L. Dohner H. Erkeland S.J. Lowenberg B. Jongen-Lavrencic M. Expression of a Passenger miR-9* Predicts Favorable Outcome in Adults with Acute Myeloid Leukemia Less than 60 Years of Age Leukemia 2016 30 303 309 10.1038/leu.2015.282 26464168
30. Zhao J. Lu Q. Zhu J. Fu J. Chen Y.X. Prognostic Value of miR-96 in Patients with Acute Myeloid Leukemia Diagn. Pathol. 2014 9 1 6 10.1186/1746-1596-9-76 24678958 PMC3975266
31. Diaz-Beya M. Brunet S. Nomdedeu J. Tejero R. Diaz T. Pratcorona M. Tormo M. Ribera J.M. Escoda L. Duarte R. MicroRNA Expression at Diagnosis Adds Relevant Prognostic Information to Molecular Categorization in Patients with Intermediate-Risk Cytogenetic Acute Myeloid Leukemia Leukemia 2014 28 804 812 10.1038/leu.2013.281 24072101
32. Havelange V. Ranganathan P. Geyer S. Nicolet D. Huang X. Yu X. Volinia S. Kornblau S.M. Andreeff M. Croce C.M. Implications of the miR-10 Family in Chemotherapy Response of NPM1-Mutated AML Blood 2014 123 2412 2415 10.1182/blood-2013-10-532374 24596420 PMC3983615
33. Xiang L. Li M. Liu Y. Cen J. Chen Z. Zhen X. Xie X. Cao X. Gu W. The Clinical Characteristics and Prognostic Significance of MN1 Gene and MN1-Associated microRNA Expression in Adult Patients with De Novo Acute Myeloid Leukemia Ann. Hematol. 2013 92 1063 1069 10.1007/s00277-013-1729-x 23515710
34. Marcucci G. Maharry K.S. Metzeler K.H. Volinia S. Wu Y.Z. Mrozek K. Nicolet D. Kohlschmidt J. Whitman S.P. Mendler J.H. Clinical Role of microRNAs in Cytogenetically Normal Acute Myeloid Leukemia: MiR-155 Upregulation Independently Identifies High-Risk Patients J. Clin. Oncol. 2013 31 2086 2093 10.1200/JCO.2012.45.6228 23650424 PMC3731981
35. Chuang M.K. Chiu Y.C. Chou W.C. Hou H.A. Chuang E.Y. Tien H.F. A 3-microRNA Scoring System for Prognostication in De Novo Acute Myeloid Leukemia Patients Leukemia 2015 29 1051 1059 10.1038/leu.2014.333 25428263
36. Zhang H. Luo X.Q. Feng D.D. Zhang X.J. Wu J. Zheng Y.S. Chen X. Xu L. Chen Y.Q. Upregulation of microRNA-125b Contributes to Leukemogenesis and Increases Drug Resistance in Pediatric Acute Promyelocytic Leukemia Mol. Cancer. 2011 10 1 13 10.1186/1476-4598-10-108 21880154 PMC3189170
37. Zhou L. Bai H. Wang C. Wei D. Qin Y. Xu X. MicroRNA125b Promotes Leukemia Cell Resistance to Daunorubicin by Inhibiting Apoptosis Mol. Med. Rep. 2014 9 1909 1916 24604579 10.3892/mmr.2014.2011
38. De Leeuw D.C. Denkers F. Olthof M.C. Rutten A.P. Pouwels W. Schuurhuis G.J. Ossenkoppele G.J. Smit L. Attenuation of microRNA-126 Expression that Drives CD34 + 38 − stem/progenitor Cells in Acute Myeloid Leukemia Leads to Tumor Eradication Cancer Res. 2014 74 2094 2105 10.1158/0008-5472.CAN-13-1733 24477595
39. Dorrance A.M. Neviani P. Ferenchak G.J. Huang X. Nicolet D. Maharry K.S. Ozer H.G. Hoellarbauer P. Khalife J. Hill E.B. Targeting Leukemia Stem Cells in Vivo with antagomiR-126 Nanoparticles in Acute Myeloid Leukemia Leukemia 2015 29 2143 2153 10.1038/leu.2015.139 26055302 PMC4633325
40. Lechman E.R. Gentner B. Ng S.W. Schoof E.M. van Galen P. Kennedy J.A. Nucera S. Ciceri F. Kaufmann K.B. Takayama N. MiR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells Cancer Cell 2016 29 214 228 10.1016/j.ccell.2015.12.011 26832662 PMC4749543
41. Tang X. Chen L. Yan X. Li Y. Xiong Y. Zhou X. Overexpression of miR-210 is Associated with Poor Prognosis of Acute Myeloid Leukemia Med. Sci. Monit. 2015 21 3427 3433 10.12659/MSM.894812 26549593 PMC4644019
42. Eisfeld A.K. Marcucci G. Maharry K. Schwind S. Radmacher M.D. Nicolet D. Becker H. Mrozek K. Whitman S.P. Metzeler K.H. MiR-3151 Interplays with its Host Gene BAALC and Independently Affects Outcome of Patients with Cytogenetically Normal Acute Myeloid Leukemia Blood 2012 120 249 258 10.1182/blood-2012-02-408492 22529287 PMC3398762
43. Diaz-Beya M. Brunet S. Nomdedeu J. Cordeiro A. Tormo M. Escoda L. Ribera J.M. Arnan M. Heras I. Gallardo D. The Expression Level of BAALC-Associated microRNA miR-3151 is an Independent Prognostic Factor in Younger Patients with Cytogenetic Intermediate-Risk Acute Myeloid Leukemia Blood Cancer. J. 2015 5 e352 10.1038/bcj.2015.76 26430723 PMC4635188
44. Garzon R. Volinia S. Liu C.G. Fernandez-Cymering C. Palumbo T. Pichiorri F. Fabbri M. Coombes K. Alder H. Nakamura T. MicroRNA Signatures Associated with Cytogenetics and Prognosis in Acute Myeloid Leukemia Blood 2008 111 3183 3189 10.1182/blood-2007-07-098749 18187662 PMC2265455
45. Xing C.Y. Hu X.Q. Xie F.Y. Yu Z.J. Li H.Y. Zhou B. Wu J.B. Tang L.Y. Gao S.M. Long Non-Coding RNA HOTAIR Modulates c-KIT Expression through Sponging miR-193a in Acute Myeloid Leukemia FEBS Lett. 2015 589 1981 1987 10.1016/j.febslet.2015.04.061 25979172
46. Wu S. Zheng C. Chen S. Cai X. Shi Y. Lin B. Chen Y. Overexpression of Long Non-Coding RNA HOTAIR Predicts a Poor Prognosis in Patients with Acute Myeloid Leukemia Oncol. Lett. 2015 10 2410 2414 10.3892/ol.2015.3552 26622861 PMC4580046
47. Hao S. Shao Z. HOTAIR is Upregulated in Acute Myeloid Leukemia and that Indicates a Poor Prognosis Int. J. Clin. Exp. Pathol. 2015 8 7223 7228 26261618 PMC4525952
48. Diaz-Beya M. Brunet S. Nomdedeu J. Pratcorona M. Cordeiro A. Gallardo D. Escoda L. Tormo M. Heras I. Ribera J.M. The lincRNA HOTAIRM1 , Located in the HOXA Genomic Region, is Expressed in Acute Myeloid Leukemia, Impacts Prognosis in Patients in the Intermediate-Risk Cytogenetic Category, and is Associated with a Distinctive microRNA Signature Oncotarget 2015 6 31613 31627 26436590 10.18632/oncotarget.5148 PMC4741628
49. Li H. Hui L. Xu W. MiR-181a Sensitizes a Multidrug-Resistant Leukemia Cell Line K562/A02 to Daunorubicin by Targeting BCL-2 Acta Biochim. Biophys. Sin. 2012 44 269 277 10.1093/abbs/gmr128 22285729
50. Bai H. Cao Z. Deng C. Zhou L. Wang C. MiR-181a Sensitizes Resistant Leukaemia HL-60/Ara-C Cells to Ara-C by Inducing Apoptosis J. Cancer Res. Clin. Oncol. 2012 138 595 602 10.1007/s00432-011-1137-3 22209977 PMC11824326
51. Chen Y. Jacamo R. Konopleva M. Garzon R. Croce C. Andreeff M. CXCR4 Downregulation of Let-7a Drives Chemoresistance in Acute Myeloid Leukemia J. Clin. Investig. 2013 123 2395 2407 10.1172/JCI66553 23676502 PMC3668829
52. Seca H. Lima R.T. Almeida G.M. Sobrinho-Simoes M. Bergantim R. Guimaraes J.E. Vasconcelos M.H. Effect of miR-128 in DNA Damage of HL-60 Acute Myeloid Leukemia Cells Curr. Pharm. Biotechnol. 2014 15 492 502 10.2174/1389201015666140519122524 24846063
53. Feng D.D. Zhang H. Zhang P. Zheng Y.S. Zhang X.J. Han B.W. Luo X.Q. Xu L. Zhou H. Qu L.H. Down-Regulated miR-331–5p and miR-27a are Associated with Chemotherapy Resistance and Relapse in Leukaemia J. Cell. Mol. Med. 2011 15 2164 2175 10.1111/j.1582-4934.2010.01213.x 21070600 PMC4394226
54. Gocek E. Wang X. Liu X. Liu C.G. Studzinski G.P. MicroRNA-32 Upregulation by 1,25-Dihydroxyvitamin D3 in Human Myeloid Leukemia Cells Leads to Bim Targeting and Inhibition of AraC-Induced Apoptosis Cancer Res. 2011 71 6230 6239 10.1158/0008-5472.CAN-11-1717 21816906 PMC5572464
55. Shibayama Y. Kondo T. Ohya H. Fujisawa S. Teshima T. Iseki K. Upregulation of microRNA-126–5p is Associated with Drug Resistance to Cytarabine and Poor Prognosis in AML Patients Oncol. Rep. 2015 33 2176 2182 10.3892/or.2015.3839 25759982 PMC4391586
56. Weng H. Huang H. Dong B. Zhao P. Zhou H. Qu L. Inhibition of miR-17 and miR-20a by Oridonin Triggers Apoptosis and Reverses Chemoresistance by Derepressing BIM-S Cancer Res. 2014 74 4409 4419 10.1158/0008-5472.CAN-13-1748 24872388
57. Alharbi R.A. Pettengell R. Pandha H.S. Morgan R. The Role of HOX Genes in Normal Hematopoiesis and Acute Leukemia Leukemia 2013 27 1000 1008 10.1038/leu.2012.356 23212154
58. Hickey C.J. Schwind S. Radomska H.S. Dorrance A.M. Santhanam R. Mishra A. Wu Y.Z. Alachkar H. Maharry K. Nicolet D. Lenalidomide-Mediated Enhanced Translation of C/EBPα-p30 Protein Up-Regulates Expression of the Antileukemic microRNA-181a in Acute Myeloid Leukemia Blood 2013 121 159 169 10.1182/blood-2012-05-428573 23100311 PMC3538328
59. Reinhart B.J. Slack F.J. Basson M. Pasquinelli A.E. Bettinger J.C. Rougvie A.E. Horvitz H.R. Ruvkun G. The 21-Nucleotide Let-7 RNA Regulates Developmental Timing in Caenorhabditis Elegans Nature 2000 403 901 906 10706289 10.1038/35002607
60. Li Y. Lin J. Yang J. Qian J. Qian W. Yao D.M. Deng Z.Q. Liu Q. Chen X.X. Xie D. Overexpressed Let-7a-3 is Associated with Poor Outcome in Acute Myeloid Leukemia Leuk. Res. 2013 37 1642 1647 10.1016/j.leukres.2013.09.022 24138945
61. Jinlong S. Lin F. Yonghui L. Li Y. Weidong W. Identification of Let-7a-2-3p or/and miR-188-5p as Prognostic Biomarkers in Cytogenetically Normal Acute Myeloid Leukemia PLoS ONE 2015 10 e0118099 10.1371/journal.pone.0118099 25646775 PMC4315415
62. Lee H. Han S. Kwon C.S. Lee D. Biogenesis and Regulation of the Let-7 miRNAs and their Functional Implications Protein Cell 2016 7 100 113 10.1007/s13238-015-0212-y 26399619 PMC4742387
63. Butrym A. Rybka J. Baczynska D. Tukiendorf A. Kuliczkowski K. Mazur G. Expression of microRNA-331 can be used as a Predictor for Response to Therapy and Survival in Acute Myeloid Leukemia Patients Biomark. Med. 2015 9 453 460 10.2217/bmm.14.112 25620533
64. Garzon R. Garofalo M. Martelli M.P. Briesewitz R. Wang L. Fernandez-Cymering C. Volinia S. Liu C.G. Schnittger S. Haferlach T. Distinctive microRNA Signature of Acute Myeloid Leukemia Bearing Cytoplasmic Mutated Nucleophosmin Proc. Natl. Acad. Sci. USA 2008 105 3945 3950 10.1073/pnas.0800135105 18308931 PMC2268779
65. Cammarata G. Augugliaro L. Salemi D. Agueli C. La Rosa M. Dagnino L. Civiletto G. Messana F. Marfia A. Bica M.G. Differential Expression of Specific microRNA and their Targets in Acute Myeloid Leukemia Am. J. Hematol. 2010 85 331 339 10.1002/ajh.21667 20425795
66. Alachkar H. Santhanam R. Harb J.G. Lucas D.M. Oaks J.J. Hickey C.J. Pan L. Kinghorn A.D. Caligiuri M.A. Perrotti D. Silvestrol Exhibits Significant in Vivo and in Vitro Antileukemic Activities and Inhibits FLT3 and miR-155 Expressions in Acute Myeloid Leukemia J. Hematol. Oncol. 2013 6 421 422 10.1186/1756-8722-6-21 23497456 PMC3623627
67. Khalife J. Radomska H.S. Santhanam R. Huang X. Neviani P. Saultz J. Wang H. Wu Y.Z. Alachkar H. Anghelina M. Pharmacological Targeting of miR-155 Via the NEDD8-Activating Enzyme Inhibitor MLN4924 (Pevonedistat) in FLT3-ITD Acute Myeloid Leukemia Leukemia 2015 29 1981 1992 10.1038/leu.2015.106 25971362 PMC4868182
68. Cao Y.X. Dai C.W. Zhang G.S. Screening for Drug Resistance Related microRNAs in K562 and K562/A02 Cell Lines Zhonghua Xue Ye Xue Za Zhi 2010 31 361 365 21122348
69. Palma C.A. Al Sheikha D. Lim T.K. Bryant A. Vu T.T. Jayaswal V. Ma D.D. MicroRNA-155 as an Inducer of Apoptosis and Cell Differentiation in Acute Myeloid Leukaemia Mol. Cancer 2014 13 1 15 10.1186/1476-4598-13-79 24708856 PMC4021368
70. Shaham L. Binder V. Gefen N. Borkhardt A. Izraeli S. MiR-125 in Normal and Malignant Hematopoiesis Leukemia 2012 26 2011 2018 10.1038/leu.2012.90 22456625
71. Li Z. Lu J. Sun M. Mi S. Zhang H. Luo R.T. Chen P. Wang Y. Yan M. Qian Z. Distinct microRNA Expression Profiles in Acute Myeloid Leukemia with Common Translocations Proc. Natl. Acad. Sci. USA 2008 105 15535 15540 10.1073/pnas.0808266105 18832181 PMC2563085
72. Li Z. Chen P. Su R. Li Y. Hu C. Wang Y. Arnovitz S. He M. Gurbuxani S. Zuo Z. Overexpression and Knockout of miR-126 both Promote Leukemogenesis Blood 2015 126 2005 2015 10.1182/blood-2015-04-639062 26361793 PMC4616234
73. Garzon R. Volinia S. Papaioannou D. Nicolet D. Kohlschmidt J. Yan P.S. Mrozek K. Bucci D. Carroll A.J. Baer M.R. Expression and Prognostic Impact of lncRNAs in Acute Myeloid Leukemia Proc. Natl. Acad. Sci. USA 2014 111 18679 18684 10.1073/pnas.1422050112 25512507 PMC4284555
74. Bhan A. Mandal S.S. LncRNA HOTAIR: A Master Regulator of Chromatin Dynamics and Cancer Biochim. Biophys. Acta 2015 1856 151 164 10.1016/j.bbcan.2015.07.001 26208723 PMC4544839
75. Rinn J.L. Kertesz M. Wang J.K. Squazzo S.L. Xu X. Brugmann S.A. Goodnough L.H. Helms J.A. Farnham P.J. Segal E. Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs Cell 2007 129 1311 1323 10.1016/j.cell.2007.05.022 17604720 PMC2084369
76. Shang C. Guo Y. Zhang H. Xue Y.X. Long Noncoding RNA HOTAIR is a Prognostic Biomarker and Inhibits Chemosensitivity to Doxorubicin in Bladder Transitional Cell Carcinoma Cancer Chemother. Pharmacol. 2016 77 507 513 10.1007/s00280-016-2964-3 26781446
77. Montalban-Bravo G. Garcia-Manero G. Novel Drugs for Older Patients with Acute Myeloid Leukemia Leukemia 2015 29 760 769 10.1038/leu.2014.244 25142817
78. Burnett A.K. Milligan D. Prentice A.G. Goldstone A.H. McMullin M.F. Hills R.K. Wheatley K. A Comparison of Low-Dose Cytarabine and Hydroxyurea with or without all-Trans Retinoic Acid for Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome in Patients Not Considered Fit for Intensive Treatment Cancer 2007 109 1114 1124 10.1002/cncr.22496 17315155
79. Liu S. Wu L.C. Pang J. Santhanam R. Schwind S. Wu Y.Z. Hickey C.J. Yu J. Becker H. Maharry K. Sp1/NFκB/HDAC/miR-29b Regulatory Network in KIT-Driven Myeloid Leukemia Cancer Cell 2010 17 333 347 10.1016/j.ccr.2010.03.008 20385359 PMC2917066
80. Garzon R. Liu S. Fabbri M. Liu Z. Heaphy C.E. Callegari E. Schwind S. Pang J. Yu J. Muthusamy N. MicroRNA-29b Induces Global DNA Hypomethylation and Tumor Suppressor Gene Reexpression in Acute Myeloid Leukemia by Targeting Directly DNMT3A and 3B and Indirectly DNMT1 Blood 2009 113 6411 6418 10.1182/blood-2008-07-170589 19211935 PMC2710934
81. Garzon R. Heaphy C.E. Havelange V. Fabbri M. Volinia S. Tsao T. Zanesi N. Kornblau S.M. Marcucci G. Calin G.A. MicroRNA 29b Functions in Acute Myeloid Leukemia Blood 2009 114 5331 5341 10.1182/blood-2009-03-211938 19850741 PMC2796138
82. Blum W. Garzon R. Klisovic R.B. Schwind S. Walker A. Geyer S. Liu S. Havelange V. Becker H. Schaaf L. Clinical Response and miR-29b Predictive Significance in Older AML Patients Treated with a 10-Day Schedule of Decitabine Proc. Natl. Acad. Sci. USA 2010 107 7473 7478 10.1073/pnas.1002650107 20368434 PMC2867720
83. Huang X. Schwind S. Yu B. Santhanam R. Wang H. Hoellerbauer P. Mims A. Klisovic R. Walker A.R. Chan K.K. Targeted Delivery of microRNA-29b by Transferrin-Conjugated Anionic Lipopolyplex Nanoparticles: A Novel Therapeutic Strategy in Acute Myeloid Leukemia Clin. Cancer Res. 2013 19 2355 2367 10.1158/1078-0432.CCR-12-3191 23493348 PMC3644023
84. Mims A. Walker A.R. Huang X. Sun J. Wang H. Santhanam R. Dorrance A.M. Walker C. Hoellerbauer P. Tarighat S.S. Increased Anti-Leukemic Activity of Decitabine via AR-42-Induced Upregulation of miR-29b: A Novel Epigenetic-Targeting Approach in Acute Myeloid Leukemia Leukemia 2013 27 871 878 10.1038/leu.2012.342 23178755 PMC3764604
85. Butrym A. Rybka J. Baczynska D. Poreba R. Kuliczkowski K. Mazur G. Clinical Response to Azacitidine Therapy Depends on microRNA-29c (miR-29c) Expression in Older Acute Myeloid Leukemia (AML) Patients Oncotarget 2016 7 30250 30257 26862847 10.18632/oncotarget.7172 PMC5058678
86. Gao X.N. Lin J. Li Y.H. Gao L. Wang X.R. Wang W. Kang H.Y. Yan G.T. Wang L.L. Yu L. MicroRNA-193a Represses C-Kit Expression and Functions as a Methylation-Silenced Tumor Suppressor in Acute Myeloid Leukemia Oncogene 2011 30 3416 3428 10.1038/onc.2011.62 21399664
87. Yang Y. Wang L.L. Wang H.X. Guo Z.K. Gao X.F. Cen J. Li Y.H. Dou L.P. Yu L. The Epigenetically-Regulated miR-663 Targets H-Ras in K-562 Cells FEBS J. 2013 280 5109 5117 10.1111/febs.12485 23953123
88. Yan-Fang T. Jian N. Jun L. Na W. Pei-Fang X. Wen-Li Z. Dong W. Li P. Jian W. Xing F. The Promoter of miR-663 is Hypermethylated in Chinese Pediatric Acute Myeloid Leukemia (AML) BMC Med. Genet. 2013 14 1 9 10.1186/1471-2350-14-74 23870168 PMC3726388
89. Ufkin M.L. Peterson S. Yang X. Driscoll H. Duarte C. Sathyanarayana P. miR-125a Regulates Cell Cycle, Proliferation, and Apoptosis by Targeting the ErbB Pathway in Acute Myeloid Leukemia Leuk. Res. 2014 38 402 410 10.1016/j.leukres.2013.12.021 24484870 PMC4117580
90. Zhang X. Zeng J. Zhou M. Li B. Zhang Y. Huang T. Wang L. Jia J. Chen C. The Tumor Suppressive Role of miRNA-370 by Targeting FoxM1 in Acute Myeloid Leukemia Mol. Cancer. 2012 11 2271 2278 10.1186/1476-4598-11-56 22900969 PMC3533721
91. Wong K.Y. So C.C. Loong F. Chung L.P. Lam W.W. Liang R. Li G.K. Jin D.Y. Chim C.S. Epigenetic Inactivation of the miR-124–1 in Haematological Malignancies PLoS ONE 2011 6 e19027 10.1371/journal.pone.0019027 21544199 PMC3081325
92. Chim C.S. Wong K.Y. Leung C.Y. Chung L.P. Hui P.K. Chan S.Y. Yu L. Epigenetic Inactivation of the Hsa-miR-203 in Haematological Malignancies J. Cell. Mol. Med. 2011 15 2760 2767 10.1111/j.1582-4934.2011.01274.x 21323860 PMC4373446
93. Treppendahl M.B. Qiu X. Sogaard A. Yang X. Nandrup-Bus C. Hother C. Andersen M.K. Kjeldsen L. Mollgard L. Hellstrom-Lindberg E. Allelic Methylation Levels of the Noncoding VTRNA2–1 Located on Chromosome 5q31.1 Predict Outcome in AML Blood 2012 119 206 216 10.1182/blood-2011-06-362541 22058117 PMC3251229
94. Xishan Z. Xianjun L. Ziying L. Guangxin C. Gang L. The Malignancy Suppression Role of miR-23a by Targeting the BCR/ABL Oncogene in Chromic Myeloid Leukemia Cancer Gene Ther. 2014 21 397 404 10.1038/cgt.2014.44 25213664
95. Nishioka C. Ikezoe T. Yang J. Nobumoto A. Tsuda M. Yokoyama A. Downregulation of miR-217 Correlates with Resistance of Ph(+) Leukemia Cells to ABL Tyrosine Kinase Inhibitors Cancer Sci. 2014 105 297 307 10.1111/cas.12339 24350829 PMC4317938
96. Bueno M.J. Perez de Castro I. Gomez de Cedron M. Santos J. Calin G.A. Cigudosa J.C. Croce C.M. Fernandez-Piqueras J. Malumbres M. Genetic and Epigenetic Silencing of microRNA-203 Enhances ABL1 and BCR-ABL1 Oncogene Expression Cancer Cell 2008 13 496 506 10.1016/j.ccr.2008.04.018 18538733
97. Neil G.L. Berger A.E. Bhuyan B.K. DeSante D.C. Combination Chemotherapy of L1210 Leukemia with 1-Beta-D-Arabinofuranosylcytosine and 5-Azacytidine Cancer Res. 1976 36 1114 1120 56229
98. Kong X.B. Tong W.P. Chou T.C. Induction of Deoxycytidine Kinase by 5-Azacytidine in an HL-60 Cell Line Resistant to Arabinosylcytosine Mol. Pharmacol. 1991 39 250 257 1705001
99. Scandura J.M. Roboz G.J. Moh M. Morawa E. Brenet F. Bose J.R. Villegas L. Gergis U.S. Mayer S.A. Ippoliti C.M. Phase 1 Study of Epigenetic Priming with Decitabine Prior to Standard Induction Chemotherapy for Patients with AML Blood 2011 118 1472 1480 10.1182/blood-2010-11-320093 21613261 PMC3156041
100. Muller-Tidow C. Tschanter P. Rollig C. Thiede C. Koschmieder A. Stelljes M. Koschmieder S. Dugas M. Gerss J. Butterfass-Bahloul T. Azacitidine in Combination with Intensive Induction Chemotherapy in Older Patients with Acute Myeloid Leukemia: The AML-AZA Trial of the Study Alliance Leukemia Leukemia 2016 30 555 561 10.1038/leu.2015.306 26522083
101. Issa J.P. Roboz G. Rizzieri D. Jabbour E. Stock W. O’Connell C. Yee K. Tibes R. Griffiths E.A. Walsh K. Safety and Tolerability of Guadecitabine (SGI-110) in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukaemia: A Multicentre, Randomised, Dose-Escalation Phase 1 Study Lancet Oncol. 2015 16 1099 1110 10.1016/S1470-2045(15)00038-8 26296954 PMC5557041
102. Zhang Y.C. Ye H. Zeng Z. Chin Y.E. Huang Y.N. Fu G.H. The NF-κB p65/miR-23a-27a-24 Cluster is a Target for Leukemia Treatment Oncotarget 2015 6 33554 33567 26378023 10.18632/oncotarget.5591 PMC4741785
103. Zebisch A. Wolfler A. Fried I. Wolf O. Lind K. Bodner C. Haller M. Drasche A. Pirkebner D. Matallanas D. Frequent Loss of RAF Kinase Inhibitor Protein Expression in Acute Myeloid Leukemia Leukemia 2012 26 1842 1849 10.1038/leu.2012.61 22388727
104. Christopher A.F. Kaur R.P. Kaur G. Kaur A. Gupta V. Bansal P. MicroRNA Therapeutics: Discovering Novel Targets and Developing Specific Therapy Perspect. Clin. Res. 2016 7 68 74 27141472 10.4103/2229-3485.179431 PMC4840794