Abstract
Hypomethylating agents (HMAs), azacitidine and decitabine, are standards of care in higher-risk myelodysplastic syndromes and in acute myeloid leukemia patients ineligible for intensive therapy. Over the last 10 years, research efforts have sought to better understand their mechanism of action, both at the molecular and cellular level. These efforts have yet to robustly identify biomarkers for these agents. The clinical activity of HMAs in myeloid neoplasms has been firmly established now but still remains of limited magnitude. Besides optimized use at different stages of the disease, most of the expected clinical progress with HMAs will come from the development of second-generation compounds orally available and/or with improved pharmacokinetics, and from the search, so far mostly empirical, of HMA-based synergistic drug combinations.
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Introduction
Azacitidine (5-azacitidine, AZA) and decitabine (5-aza-2′-deocytidicine, DAC) are two analogues of cytidine able, at low but clinically relevant concentrations, to inhibit DNA methyltransferases (DNMTs), resulting in transient and variable DNA hypomethylation. These so-called ‘hypomethylating agents’ (HMAs) are active in myeloid malignancies, including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). However, responses are heterogenous and rarely sustained. This review provides an update on the use of HMAs in myeloid malignancies. We will also provide an overview of the developing applications of HMAs, the efficacy of new HMAs currently in development and the utility of HMA-based combinations.
Mechanism of action of HMA
The metabolism of HMAs has recently been reviewed in detail [1], and is summarized in Fig. 1. After absorption, HMAs are instable in plasma owing to spontaneous hydrolysis and deamination by cytidine deaminase (CDA), explaining their relative short plasma half-life [2]. Following their cellular uptake, which is dependent on nucleoside transporters, they are successively phosphorylated by intracellular kinases. The active tri-phosphorylated metabolite of DAC (5-aza-dCTP) is directly incorporated into DNA during cell cycle. Regarding AZA, the majority of 5-aza-CTP is incorporated in RNA, whereas a minority is converted in 5-aza-dCTP by the ribonucleotide reductase and is incorporated in DNA during replication.
Molecular and cellular mechanisms of action of hypomethylating agents
5-aza-dCTP incorporated into DNA binds DNMT1 and leads to its degradation, promoting a progressive DNA hypomethylation after several rounds of replication. This has been postulated to lead to an activation of repressed tumor suppressor genes [3], inducing senescence and apoptosis. HMAs can also allow expression of tumor-associated antigens [4] that can trigger anti-tumoral immune response [5]. In solid tumors, HMAs promote the expression of endogenous retroviral elements leading to an interferon-dependent cell killing [6, 7]. Whether a similar phenomenon occurs in myeloid malignancies remains to be investigated. Finally, AZA have been shown to promote differentiation of leukemic cells in vivo in a tet2-mutated AML mouse model [8]. Besides these demethylating effects, DNA-incorporated 5-aza-dCTP can also induce DNA damage [9], although this effect is likely limited with current administration schemes of AZA and DAC, aiming at hypomethylation rather than cytotoxicity. Finally, incorporation of 5-aza-CTP in RNA may disrupt protein synthesis [10], a phenomenon that may also promote apoptosis. A novel mass spectrometry method has been developed to quantify the abundance of AZA incorporated in RNA, DNA and cytoplasm. This method showed that incorporation in DNA but not RNA seems to be the rate limiting step for response [11].
Approved use of HMA in hematological malignancies
In myelodysplastic syndromes
Since the AZA-001 study, AZA is the standard of care in higher-risk MDS not eligible for allogeneic stem cell transplant (ASCT). This randomized phase III trial showed that AZA was superior to best supportive care, low-dose aracytine or intensive chemotherapy, as it improved overall survival and delayed AML transformation in higher-risk MDS and low-blast count AML elderly patients [12]. Decitabine is also active in higher-risk MDS, but clinical trials failed to show an improvement in overall survival, possibly owing to trial design [13]. HMAs have been used as first-line therapy in lower-risk MDS [14, 15]. However, addition of AZA in patients who fail to respond to erythropoiesis-stimulating agents (ESA) has shown limited efficacy [16]. AZA and DAC are both licensed for MDS in the US, whereas only AZA is approved in int2/high-risk MDS in Europe.
In chronic myelomonocytic leukemia
Small non-randomized prospective trials have reported activity of AZA [17] and DAC [18] in CMML. Because of the limited efficacy of conventional chemotherapy in CMML-2 and because the AZA-001 trial included some CMML-2 patients, AZA but not DAC was approved in Europe for CMML-2. Again, both drugs are approved in CMML by FDA. A prospective randomized phase III clinical trial comparing DAC (with or without hydroxyurea) versus hydroxyurea alone is currently ongoing to evaluate the efficacy of DAC in proliferative CMML (NCT02214407).
In acute myeloid leukemia
A subgroup analysis of the AZA-001 trial focusing on low-blast count AML [19], and a large randomized phase III trial in elderly AML patients with bone marrow blast counts > 30% showed that AZA improved survival compared to conventional care in older AML patients deemed ineligible for intensive chemotherapy [20]. The survival benefit was particularly apparent in high-risk subgroups with adverse cytogenetics or myelodysplasia-related changes [21]. DAC showed activity in older patients with poor/intermediate risk, but failed to improve survival when compared to best supportive care or low-dose AraC [22]. AZA and DAC are licensed for the treatment of low-blast count AML in US, whereas both are licensed in Europe for AML unfit for ASCT regardless of the blast count.
Biomarkers of response to HMAs
Only a subset of patients achieve response with HMAs alone. Moreover, responses are rarely sustained and are loosely correlated with prolonged survival [12, 18, 20]. Accumulative evidence suggests that HMAs mostly target secondary clones [23,24,25], whereas founder clones are spared and can acquire additional mutations, eventually leading to relapse [23,24,25,26,27]. Identification of robust biomarkers of response and survival with HMAs is thus warranted.
As gene mutations implicated in methylation are frequently mutated in myeloid malignancies, their impact on response to HMAs has been extensively investigated. Indeed, the presence of TET2 mutations has been associated with a higher likelihood of response in MDS [28,29,30,31] and CMML [32], especially in the absence of ASXL1 mutations. Mutations in other methylation regulators DNMT3A [30, 31, 33] and IDH1/2 [31, 34] may also predict response in AML and MDS. Whether TP53 mutations, which are classically associated with a poor prognosis, can be cleared by intensive DAC regimens remains controversial [18, 35,36,37,38]. This lack of robust association between mutational status and HMA benefit may be explained by the interaction between mutations, and by intra-tumoral heterogeneity. Indeed, in MDS and AML treated with AZA, lower clonal burden of secondary mutations in leukemic progenitors is associated with a higher response rate [23].
There is no correlation between baseline promoter methylation and response [39, 40], perhaps owing to intra-tumoral epigenetic heterogeneity [41]. However, an epigenetic classifier including analysis of distal enhancers has been developed in CMML, where it has been shown to predict response to DAC [42, 43]. Future studies will be required to determine the robust enhancer signatures predicting HMA activity. These will be facilitated by continuous technological progresses in assessing the methylome [44].
Chromatin organization may also influence HMA response [45]. Indeed, the RNA:m5C methyltransferase NSUN1 binds BRD4 and RNA-polymerase-II to form active chromatin structures that are insensitive to AZA. In samples from AZA-resistant MDS and AML, there is a significant increase in NSUN1/BRD4 recruitment to active chromatin that may be responsible for the AZA resistance. 5-Aza-dCTP is incorporated into DNA during the cell cycle. Recent data suggest patients resistant to AZA have more quiescent leukemic progenitors at treatment onset than responders [23, 25].
Finally, pharmacogenomics may explain in part the response heterogeneity. Indeed, somatic mutations and/or aberrant expression of genes encoding proteins implicated in HMAs uptake like hENT1 [46, 47], or in HMA metabolism like UCK and DCK kinases [47,48,49] have been reported in resistant MDS and AML. Increased activity of CDA, implicated in HMA degradation, is also associated with resistance to HMA [48]. Polymorphisms affecting CDA activity can predict response to other cytosin analogues [50, 51].
New regimen for HMAs
Intensive therapy
Intensified schedules of HMAs have shown promising results in AML and MDS. A 10-day regimen of DAC in MDS and AML leads to high response rates in high-risk cytogenetic and TP53-mutated patients [35]. A recent study reported that an intensified schedule of AZA (75 mg/m2, d1–5 every 14 days) in high-risk MDS can also lead to high response rates [52]. Thus, intensified regimens could increase the efficacy of HMAs, probably at the expense of increased toxicity, prompting their randomized comparison with standard regimens.
Maintenance therapy
HMAs are active in relapsed or refractory myeloid malignancies, including after intensive treatment or ASCT [53]. Because of their ability to promote anti-tumor immune response, maintenance treatment with HMAs has been proposed to prevent relapse in patients who achieved CR. Pilot studies have shown the feasibility of the post-transplant maintenance with HMAs, despite significant toxicity leading to decreased dose of HMAs [53]. This led to the ongoing phase III randomized trial VZ-AML-PI-0129 that evaluates the efficacy of azacitidine maintenance (32 mg/m2, day 1–5) versus placebo after ASCT in AML and MDS (NCT00887068). The oral formulation of AZA, CC-486, has a good tolerance profile in post-transplant maintenance in AML and MDS [54] and may facilitate dose adaptation and adherence to treatment. CC-486 is currently evaluated as a maintenance therapy after intensive chemotherapy in previously untreated AML (QUAZAR AML-001, NCT01757535) and as a maintenance therapy after ASCT in AML and MDS patients (NCT01835587).
Following several negative or underpowered trials [55,56,57], the HOVON conducted a trial where high-risk MDS or AML patients older than 60 years, in CR/CRi after 2 courses of intensive chemotherapy were randomized to 12 cycles of attenuated dose AZA (50 mg/m2, day 1–5) or observation. This trial was the first to show a prolonged disease-free survival (DFS) with AZA. Although this did not translate in a meaningful OS benefit, probably owing to the limited number of patients accrued (n = 116), this study provides proof of concept for epigenetic therapy as a maintenance therapy in older AML [58].
Preemptive treatment
Another strategy to prevent relapse is to initiate a treatment as soon as there is evidence of incipient relapse, such as an increase in minimal residual disease (MRD). In the RELAZA-2 phase II trial, authors monitored AML and MDS patients achieving CR after intensive chemotherapy or ASCT and began AZA in MRD+ patients [59]. Despite the lack of a randomized control group, preemptive treatment with AZA might delay relapse in MRD+ patients compared to historical data, especially in patients with lower levels of MRD. These results should however be confirmed in a randomized trial.
Treatment of inflammatory manifestations associated to myeloid malignancies
Systemic autoimmune and inflammatory disorders (SAID) are present in up to 30% of MDS/CMML patients. HMA can control these manifestations, including in steroid-dependent or -resistant SAID. HMA activity on myeloid neoplasm and SAID was concordant in most cases [60], suggesting that this activity is caused by reversal of paracrine influence of leukemic cells on the immune system, rather than a direct action on the immune system. A phase II study is currently ongoing to evaluate the efficacy of AZA on steroid-dependent/resistant SAID in MDS and CMML patients (NCT02985190).
New hypomethylating agents
Oral azacitidine (CC-486)
CC-486 is an oral formulation of 5-azacitidine which has been developed to simplify AZA administration and dose adaptation, and eventually increase leukemic cells exposition to AZA [61]. CC-486 is active in AML, CMML and MDS [62], including low-risk MDS [63], with an acceptable safety profile. The phase III QUAZAR trial (NCT01566695) is currently comparing CC-486 to placebo in low-risk MDS patients with anemia or thrombocytopenia. Activity of CC-486 is also investigated as a maintenance therapy after intensive chemotherapy (NCT01757535) and after ASCT (NCT01835587) in AML and MDS patients.
Guadecitabine
Guadecitabine is a dinucleotide of DAC and deoxyguanosine. It was developed to be resistant to CDA, leading to a longer exposure of leukemic cells to the active metabolite of DAC [64]. It is administered sub-cutaneously. Guadecitabine is well tolerated and active in untreated [65] and relapsed/refractory [66] MDS and AML, and the schedule of 5 days at 60 mg/m2 has the best safety profile. Two phase III trials have evaluated guadecitabine versus treatment of choice in untreated AML patients unfit for intensive chemotherapy (ASTRAL-1 trial, NCT02348489), and in relapsed/refractory AML (ASTRAL-2, NCT02920008). The sponsors of ASTRAL-1 have announced that the trial had failed to meet its co-primary endpoints of superior CR rate and prolonged overall survival, but the detailed scientific report of this trial has not been made available yet (https://www.otsuka.co.jp/en/company/newsreleases/2018/20180731_1.html). Guadecitabine is also compared to conventional care in MDS and CMML previously treated with HMA in a phase III study (NCT02907359).
Astx727
An oral formulation of DAC is currently in development. ASTX727 is an association of DAC with the CDA inhibitor cedazuridine (E7727). Pharmacokinetic analysis in MDS and CMML revealed similar DAC exposition between a standard 5-day IV DAC course and 5 days of oral ASTX727 [67]. The treatment was well tolerated, and response rate was 61%.
Combinations based on HMAs
Combination with histone deacetylase (HDAC) inhibitors
In myeloid malignancies, deacetylation of histone tails can participate in silencing tumor suppressor genes. HDAC inhibitors (HDACi) in monotherapy are modestly active in high-risk MDS and AML, and in vitro evidence supported the synergy between HMAs and HDACi [68]. However, randomized trials have repeatedly failed to demonstrate a benefit for the combination of HMA with HDACi including entinostat [69], valproic acid [70, 71], vorinostat [72, 73] or pracinostat [74]. The combinations were potentially hampered by increased toxicity, and one study even suggested an antagonism between AZA and entinostat, perhaps owing to the reduction of proliferation by HDACi [75].
Combination with lenalidomide
Lenalidomide is active in del(5q) MDS, where it induces synthetic lethality by promoting degradation of CK1α, which is encoded by a gene located in the deleted regions of these diseases [76]. Lenalidomide also promotes erythropoiesis in non-del(5q) MDS [77]. The rationale of its association with azacitidine in multiple trials with various regimens was largely empirical, and multiple randomized studies in non-del(5q) higher-risk MDS, CMML and AML failed to demonstrate a benefit for the combination [71, 73].
Combination with sapacitabine
Sapacitabine, an oral cytarabine analogue, is active in monotherapy in AML [78]. However, the large randomized phase III trial SEAMLESS comparing DAC to alternating cycles of DAC and sapacitabine in older untreated AML failed to demonstrate a survival advantage in the experimental group [79].
Combination with checkpoint inhibitors
Gene mutations and epigenetic abnormalities give rise to expression of tumor-associated antigens that can be recognized by the immune system. However, checkpoint proteins overexpressed by tumor and T cells from AML [80] and MDS [81] cells can lead to exhaustion of immune cells and thus evasion from anti-leukemia immunity. Inhibitors of the CLTA-4 and PD-1 checkpoints are efficient in solid tumors. Studies so far reported modest activity in myeloid malignancies [82]. Their combination with HMAs in these diseases is attractive as HMAs can enhance anti-tumor immune response by inducing expression of endogenous retroviral elements [6, 7] and tumor-associated antigens [4], as well as induce overexpression of checkpoint molecules by T cells at the same time [81]. Several clinical trials evaluating azacitidine with anti-PD1 nivolumab (NCT02397720, NCT02397720), anti-PDL1 durvalumab (NCT02775903), anti-PDL1 atezolizumab (NCT02508870) and the combination nivolumab + anti-CTLA-4 ipilimumab (NCT02397720) in myeloid malignancies are currently ongoing. The non-randomized phase II trial evaluating AZA+ nivolumab in relapsed/refractory AML reported moderate response rates, and manageable immune-related adverse events [83]. Non-responders were shown to express higher rates of CTLA-4 on T cells. Anti-CTLA-4 ipilimumab is active in association to AZA in AML [84] and a phase II trial is currently evaluating the combination AZA+ nivolumab with or without ipilimumab in those diseases (NCT02397720).
Combination with venetoclax
BCL-2 is an anti-apoptotic protein frequently overexpressed in myeloid malignancies, and is a potential vulnerability of leukemic stem cells (LSCs) [85, 86]. Venetoclax is a specific inhibitor of BCL-2 which is active in AML, with modest single-agent activity in relapsed AML [87]. Ex vivo studies have highlighted a synergy between venetoclax and HMA [88], justifying the evaluation of the combination. The phase Ib trial evaluating the combination VEN + HMA in untreated AML of the elderly reported an acceptable tolerance with probably more myelosuppression than with HMAs alone, but high response rates (composite CR rate 73%) [89]. In this study, 400 mg of venetoclax seemed to have the best safety profile while providing deep and durable responses in this poor-risk population, although the follow-up duration of this cohort is still limited. HMAs + VEN combination has also been reported to provide a response when used as a salvage therapy in relapsed/refractory AML and MDS patients [90]. Detailed biological studies have shown that the combination of HMAs with VEN uniquely altered the metabolic activity of LSCs, possibly explaining the high rate of flow cytometry-based MRD negativity in AML patients treated upfront with the combination [86]. Thus, the combination of HMAs and VEN has the potential to challenge single-agent HMA therapy in the near future, although this will require additional studies and prolonged follow up.
Combination with IDH inhibitors
IDH1 and IDH2 mutations are frequent in myeloid malignancies, and lead to a gain of function with production of the oncometabolite 2-hydroxyglutarate (2-HG). 2-HG inhibits α-ketoglutarate-dependent enzymes such as TET2, leading to a dysregulation of methylation. Selective inhibitors of IDH2 mutations enasidenib (AG-221 [91]) and of IDH1 mutations ivosidenib (AG-120 [92]) have shown efficacy in monotherapy in relapsed/refractory AML. Preliminary results from a phase 1b/2 trial (NCT02677922) in untreated AML report efficiency of the association in older patients with newly diagnosed AML [93]. Evaluation of the ivosidenib + AZA combination is ongoing in the randomized phase 3 AGILE trial (NCT03173248).
Combination with pevonidestat
Pevonedistat (PEV) is an inhibitor of NEDD8-activating enzyme modestly active as a single agent in relapsed or refractory MDS and AML [94]. A high-throughput ex vivo screen in AML cells showed a synergy between PEV and HMAs [95]. A phase 1b study in untreated elderly AML unfit for intensive chemotherapy that evaluated PEV in association with AZA [96] reported an acceptable safety profile with an overall response rate of 50%. The randomized phase 3 clinical trial PANTHER is currently comparing AZA + PEV to AZA alone in untreated MDS, CMML and low-blast count AML (NCT03268954).
Conclusion
HMAs have been used in myeloid malignancies for more than a decade. Progress in single cell epigenomics should help in improving our understanding of HMAs’ mechanisms of action. This will eventually help us to identify robust biomarkers to predict which patients will benefit from the HMA treatment. The spectrum of HMA indications is currently widening, especially as a maintenance or preemptive treatment. Second-generation HMAs are being evaluated in myeloid malignancies. Preliminary results show they may not be superior to AZA or DAC, but oral formulation is at least more convenient and will certainly optimize compliance and dose adaptation. Combinations may finally prove superior to single agents. Whether they should be sought empirically or through a rational pre-clinical screen remains uncertain. Nevertheless, HMAs have the potential to remain an important part of the armamentarium against myeloid neoplasms in the coming decade.
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MD declares no competing financial interests. RI has received research funding from Janssen, Novartis and Oncoethix (now Merck), honoraria from Sanofi, BMS and Celgene and consulting fees from Novartis, Otsuka Pharma, Jazz Pharmaceuticals, Karyopharm, StemLine Therapeutics and Abbvie.
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Duchmann, M., Itzykson, R. Clinical update on hypomethylating agents. Int J Hematol 110, 161–169 (2019). https://doi.org/10.1007/s12185-019-02651-9
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DOI: https://doi.org/10.1007/s12185-019-02651-9