Epigenetic Changes in Neoplastic Mast Cells and Potential Impact in Mastocytosis

Systemic mastocytosis (SM) is a hematologic neoplasm with abnormal accumulation of mast cells in various organ systems such as the bone marrow, other visceral organs and skin. So far, only little is known about epigenetic changes contributing to the pathogenesis of SM. In the current article, we provide an overview of epigenetic changes that may occur and be relevant to mastocytosis, including mutations in genes involved in epigenetic processes, such as TET2, DNMT3A and ASXL1, and global and gene-specific methylation patterns in neoplastic cells. Moreover, we discuss methylation-specific pathways and other epigenetic events that may trigger disease progression in mast cell neoplasms. Finally, we discuss epigenetic targets and the effects of epigenetic drugs, such as demethylating agents and BET-targeting drugs, on growth and viability of neoplastic mast cells. The definitive impact of these targets and the efficacy of epigenetic therapies in advanced SM need to be explored in future preclinical studies and clinical trials.


Clinical Features and Classification of Mastocytosis
Systemic mastocytosis (SM) is a neoplastic disease of the bone marrow (BM) characterized by infiltration and abnormal accumulation of mast cells (MC) in the BM, liver, spleen, lymph nodes, skin and other organs. In a subset of patients, release of mediators from MC results in systemic symptoms, and sometimes even in anaphylaxis [1][2][3][4][5][6]. According to the World Health Organization (WHO) classification, mastocytosis can be divided into the following variants: cutaneous mastocytosis (CM), SM, and local MC tumors. SM is split into five categories with varying prognosis: the most prevalent form is indolent SM (ISM); less frequently detected variants of SM are smoldering SM (SSM), and advanced forms of SM; i.e., SM with an associated hematologic neoplasm (SM-AHN), aggressive SM (ASM) and MC leukemia (MCL) [4,7,8].
Clinical manifestations of mastocytosis are manifold. Several of the symptoms recorded are induced by MC-derived mediators. Other symptoms are caused by MC

Epigenetic Changes in Mastocytosis
Epigenetics refers to mechanisms connecting the genetic background with environmental signals in order to provide adaptations to various conditions and threats, like aging, lifestyle factors and also intrinsic pathologic processes [22]. For example, physiologic cell functions and genomic stability are regulated by maintenance of biochemical processes regulated by the epigenome [23]. Epigenetic alterations contribute to the regulation of gene product expression independent of the nucleotide sequence in DNA [24]. Both heritable as well as non-heritable changes can lead to epigenetic modifications. There are different levels of epigenetic regulation that include DNA methylation, posttranscriptional modifications by non-coding RNAs (ncRNAs), histone modifications and nucleosomes positioning, consequently affecting DNA and chromatin structure, respectively [25]. Somatic mutations in critical target genes regulating DNA methylation, chromatin shape and other nuclear processes may also act as epigenetic modifiers.

Histone Modifications in Neoplastic MC
A study by Martinelli et al. revealed that in advanced SM, specific histone modifications can be detected in neoplastic MC in SM, and the same was found in HMC-1 cells [17]. Several histone-modifying proteins were found to be expressed at lower levels in neoplastic MC compared to control cells. One of these proteins is the SET domain containing 2 histone lysine methyltransferase (SETD2) protein [17]. In fact, SETD2 showed reduced expression in patients with MCL. SETD2 is specific for lysine (K) -36 of histone H3, and methylation of this residue is associated with active chromatin. In the study by Martinelli et al., a decrease in H3K36Me3 methylation was correlated with SETD2 protein down-regulation in neoplastic MC in patients with MCL. Particularly, SETD2 loss of function may occur post-translationally, in the absence of mutations and other structural aberrations [26]. Loss of SETD2 function resulted in H3K36 trimethylation deficiency observed in human acute leukemia [27]. The interesting mechanism of epigenetic regulation is related with influence of tryptase on nuclei of MC. Tryptase possesses ability to cleave core histones at their N-terminal ends, leading to regulation of chromatin structure [28]. This part of histone is crucial for posttranslational modifications that have a wide epigenetic impact [29]. Tryptase clips nucleosomal histone 3 and histone 2B. Absence of tryptase leads to age-dependent accumulation of lysine 5-acetylated H2B which affects cell phenotype, up-regulation of markers of non-MC lineages and morphological alterations. The results prove that clipping of H2B by tryptase is a novel epigenetic mechanism and an example of intracellular action of tryptase [29]. Studies by Krajewski et al. showed that epigenetic regulation of MC activation during immune response may be caused by altered histone acetylation [30]. They showed that epigenetic properties of curcumin may suppress MC activation. Furthermore, trichostatin, a histone deacetylase inhibitor, may alter cytokine secretion (IL-4, IL-6, TNF-α, IL-13), and decrease expression of IL-33 by IgE-activated BM MC. In addition, continuous exposure with trichostatin led to MC apoptosis [28].

Epigenetic Modifications of KIT
It has turned out recently that chromatin epigenetic silencing might be one of the key regulators of mutated KIT inactivation [31]. However, the specific mechanism of action requires further investigation. Lyberg at al. in 2017 showed that epigenetic treatment with the histone deacetylase inhibitor (HDACi) suberoyl anilide hydroxamid acid (SAHA), is especially effective in KIT D816V-mutated BM MC and HMC-1 cells [32]. Among the applied HDACi, panobinostat, romidepsin and valproic acid, SAHA treatment rapidly increased total acetylated histones, followed by a decrease in phosphorylated KIT in the KIT-mutated human MC lines HMC-1 and ROSA (KIT D816V). Using ChIP qPCR resulted in a decreased active chromatin mark H3K18ac/H3 only in the KIT region but not in control genes upstream and downstream of KIT (PDGFRα and KDR). MC exposed to SAHA showed downregulation of KIT mRNA expression followed by decreased KIT protein expression, and apoptosis. Interestingly, primary MC isolated from SM patients, including one with MCL, were highly sensitive to SAHA-mediated cell death, while normal BM cells were resistant [32].

Methylation Status of Apoptosis-Associated and Tumor Suppressor Genes in HMC-1 Cells
Ghanim et al. explored the methylation status of 24 classical apoptosis-associated genes and 24 classical tumor suppressor genes (TSG) in HMC-1 cells and in control (normal BM) cells [31]. Their data showed that several of them were hypermethylated in the neoplastic MC line. Seven apoptosis-associated genes (CIDEB, GADD45A, HRK, TNFRSF25, BIK, BID and TNFRSF21) and six TSG (NEUROG1, CDH1, GSTP1, CDH13, TP73 and WIF1) were found to be methylated in HMC-1 cells but not in normal BM cells, suggesting that these genes are aberrantly hypermethylated in these neoplastic MC.

Somatic Mutations in Genes Regulating Epigenetic Mechanisms
Clinical presentations of the disease vary widely from indolent to advanced forms, and to the exceedingly rare MCL. KIT D816V mutation solely does not explain heterogeneity of SM with different disease phenotypes. Thus, extensive mutational analyses of patients may provide a better explanation of mastocytosis pathogenesis. Interestingly, several somatic mutations have been observed in epigenetically relevant genes.
The overall survival (OS) was significantly shorter in patients with additional aberrations as compared to those with KIT D816V alone [34]. Occurrence of 3 or more non-KIT mutations led to reduced OS (n = 4, 7 months) compared with fewer than 3 mutations (48 months) [17]. Interestingly, TET2, DNMT3A and ASXL1 mutations may affect prognosis, as demonstrated by worse OS in mutated patients. For example, mutation in epigenetic regulators, namely TET2 and/or ASXL1, was associated with reduced OS (11 months) compared with the absence of these mutations (84 months) [17]. In addition, significant differences in OS between patients with KIT-mutated SM and patients with combined TET2/DNMT3A/ASXL1 mutations independent of KIT and sole TET2 mutations (p < 0.001) have been observed [36]. To add, in SM-AHN patients ASXL1, but not TET2, mutation was associated with OS, suggesting different epigenetic changes in heterogeneous SM prognosis [37].
Importantly, mutations in TET2, DNMT3A, and ASXL1, EZH2 and IDH1/IDH2 have been observed more frequently in advanced SM, e.g., ASM, SM-AHN and MCL than in ISM patients ( Table 2). Mutations in epigenetic driver targets in four groups of SM patients have been observed with the following relative frequencies: SM-AHN > ASM > ISM. In the whole group of SM patients, the most frequent mutations have been observed in TET2 (29%), ASXL1 (17%), and DNMT3A (6%). The only additional mutations observed in ISM patients are age-related somatic mutations that have also been associated with epigenetic changes, e.g., mutations in TET2 (7%) and DNMT3A (5%) [33]. Thus, mutations in three genes: TET2, DNMT3A, and ASXL1, may be associated with epigenetic features in SM. [36]. However, it remains unknown whether these mutations indeed lead to abnormal methylation patterns or other critical epigenetic changes in clonal cells and, if so, whether these changes indeed contribute to disease evolution and/or the aggressiveness of SM.
Mutations in the discussed epigenetic modulators frequently occur in other hematological diseases. Recurrent TET2, DNMT3A, IDH1/2 mutations have been observed in patients with myeloproliferative neoplasms and other hematological malignancies, such as acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), or myelodysplastic syndromes (MDS) [38,39]. Thus, two questions can be raised: are these point mutations prognostic in SM and do these mutations have an impact on the responsiveness of neoplastic MC to various targeted drugs.
Alterations of the numbers, location and function of MC are frequently observed in patients with SM [41]. Mutations in TET2 and other critical target genes are often detected in primary neoplastic cells in patients with advanced SM, suggesting a possible role of the gene products in abnormal MC accumulation and function in these neoplasms (see Table 2). For example, loss of Tet2 associated with Kit D816V mutation has been related to a transformation of the MC disease into a more aggressive phenotype in mice [42].
Recently, Montagner et al. found that TET2 might be a crucial regulator of MC proliferation and differentiation, and also of cytokine production [42]. MC lacking TET2 revealed disrupted gene expression, profound down-regulation of many genomic regionsenhancers, and reduced global 5-hmC deposition, delay in differentiation, defects in cytokine production and a marked increase in proliferation. Interestingly, while cell differentiation under TET2 absence has been found to be regulated with compensatory mechanisms mediated by TET1 and TET3 family members, MC proliferation was only dependent on TET2 without a compensatory effect by other TET family members [43].

DNMT3
Three DNMT enzymes have been described. DNMT1 is responsible for maintenance of DNA methylation, which recognizes hemimethylated CpG sites and replicates DNA methylation pattern in newly synthetized nucleotides in the cell. DNMT3A and DNMT3B are responsible for de novo DNA methylation [25].
DNA methylation and specifically DNA methyltransferase enzyme DNMT3A, involved in de novo methylation, modulates pathogenesis of various hematological diseases and is involved in regulating the function of immune cells. The role of DNA methylation and DNMT3A in MC regulation requires further investigations.
Moreover, it has been observed that DNMT3A is essential for hematopoietic stem cells (HSC) differentiation. HSC lacking Dnmt3a presented a level of global 5-mC levels comparable with control cells [44] and loss of Dnmt3a immortalized these cells in vivo [45]. Therefore, Dnmt3a seems to be critical in the epigenetic silencing of regulatory genes, thereby enabling efficient differentiation of HSC. Interestingly, in murine MC, overall levels of genomic 5-mC were not significantly different in both Dnmt3aknockout and wildtype cells [46].

5-mC, 5-hmC Level
The only study on global DNA methylation indices, like 5-hmC and 5-mC levels in patients with SM, has revealed that the correlation between SM diagnosis and significantly low 5-hmC levels may represent a systemic effect of SM. Overall reduced 5-hmC levels in all patients with SM have been reported. In SM, reduced level of 5-hmC was associated with the effect of KIT D816V mutated oncogene. Neither the loss of 5-hmC correlated with overall MC burden in those patients, nor was it due to inactivating mutations of TET2 or reduced TET2 expression [47]

MicroRNA
Non-coding RNA transcripts (ncRNAs), constituting almost 98% of human genome, have been demonstrated to play a significant role in various pathological processes. It is regarded that microRNAs (miRNAs) possess capacity to act as putative negative regulators of gene expression at transcriptional, posttranscriptional and epigenetic levels of the target genes [48].
miRNAs are recognized to be critical regulators of immune responses in various allergic diseases and are involved in cell differentiation, proliferation, survival and IgEmediated allergic response [49]. In addition, specific miRNAs might be important for mouse MC differentiation. The analysis of expression of 181 mature miRNAs in selected highly purified hematopoietic murine cell types at immature, mature, and effector stages has revealed specific differences between the related cell types [50].
Additionally, in murine MC, miR-221-222 (miR-221 and its paralogue miR-222) has occurred to be significantly up-regulated upon cell activation. Overexpression of miR-221-222 resulted in altered cell morphology and cell cycle regulation without altering viability. Overall, this miRNA was involved in cell cycle regulation in MC in response to acute activation stimuli [51].
Currently, there is excessive evidence that exosome might provide cell-to-cell communication. In 2007, Valadi et al. showed that exosomes derived from a mouse and a human MC line (MC/9 and HMC-1, respectively), and primary bone marrow-derived mouse MC, contained RNA, both microRNA and mRNA. MC-derived RNAs can be further delivered to another cell, and can be functional in this new location. The authors have thus proposed that this RNA could be called "exosomal shuttle RNA" [52].
Characteristics of detailed RNA content have been determined in human MC line, HMC-1 using the microarray assay and miRCURY TM RNA array analysis. Note that 116 mi-croRNAs were found in exosomes and 134 were identified in donor MC, the most abundant microRNAs in the exosomes, compared to the MC were: hsa-miR-451, hsa-miR-503, miR-Plus_27560, miRPlus_2843, miRPlus_27564, hsa-miR-583, miRPlus_1795, miRPlus_17890, hsa-miR-663 and hsa-miR-30b. Predictions according to canonical pathways and networks showed involvement of MC-derived exosomal microRNA in cellular development, cellular movement, hematological system development and function as well as in cell cycle regulation [53].
However, it remains unknown whether all these miR species play a role in human (neoplastic) MC and it also remains unknown whether such miR molecules contribute to the pathogenesis of SM.

MITF and MicroRNA
A protein encoded by MITF is a transcription factor that contains both basic helix-loophelix and leucine zipper structural features. The encoded protein regulates melanocyte development and is responsible for pigment cell-specific transcription of the melanogenesis enzyme genes [53]. The transcription factor, microphthalmia-associated transcription factor (MITF) is critical for MC function and it is closely related to the regulation of KIT expression and activity. MITF may also be required for the neoplastic phenotype of MC in the SM context. It has been observed that MITF-deficient MC have down-regulated KIT expression, suggesting that KIT may be a target of MITF [54,55]. MITF has also been found to be highly expressed in neoplastic cells in BM biopsies in 9 of 10 patients with SM carrying KIT D816V. A study of Lee et al. indicated that MC with activating KIT mutation require MITF expression for MC proliferation [56].
Interestingly, it has been observed that miRNA regulation can link these two factors essential for MC function. Lee at al. have revealed that miR-539 and miR-381 are repressed by KIT D816V signaling through conserved miRNA binding sites in the MITF 3'-untranslated region. Normally, expression of these miRNAs is involved in MITF suppression and inhibition of colony-forming capacity of mastocytosis cell lines. Importantly, dysregulation of this pathway may contribute to abnormal MC proliferation and thus to the pathogenesis of aggressive MC diseases, both in humans and in the murine system [56].
Studies on the regulation of miRNA with other epigenetic key players are still at an initial stage. For example, findings from acute myeloid leukemia cells have indicated strong impact of miR-29 family members on three target DNMT izoenzymes: DNMT1, DNMT3A and DNMT3B resulting in global DNA hypomethylation as well as reexpression of tumor suppressor genes [57].
In the study on murine MC, although primary screening has not revealed modification of knockout Dnmt3a MC by any microRNA, it has been observed that miR-223 was dysregulated in cells lacking Dnmt3a [46]. Additionally, degranulation of MC followed by down-regulation of miR-223 has been observed in IgE-activated cells [58], suggesting the importance of miR-223 in epigenetic regulation of MC regardless of a MC immune response or differentiation.

Effects of Epigenetic Drugs on Growth and Viability of Neoplastic MC
The classical epigenetic drugs are the hypomethylating agents, such as azacytidine and decitabine. Both drugs are highly active in patients with advanced MDS, CMML and AML [59,60]. So far, however, only a few studies have examined the potential effects of these epigenetic drugs in the SM context or in SM-AHN.
A study of Ghanim et al. examined the antineoplastic effects of demethylating agents on human neoplastic MC. They found that the death regulator FAS is hypermethylated in neoplastic MC, but not in normal BM cells, and that azacytidine and decitabine can induce demethylation and thereby can promote expression of FAS mRNA, p21 mRNA, Noxa mRNA, and Bim mRNA in neoplastic MC [61].
Another potential epigenetic target in neoplastic MC is the epigenetic reader BRD4 which regulates expression of MYC in normal and neoplastic cells, including AML cells [62]. Wedeh et al. have shown that neoplastic MC in SM, including advanced SM, display BRD4 and MYC. The MC lines HMC-1 and ROSA also express BRD4 and MYC at the mRNA and protein level. Moreover, the BRD4-targeting drug JQ1 was found to induce dosedependent growth inhibition and apoptosis in HMC-1 cells and ROSA cells and to suppress proliferation of primary neoplastic MC obtained from patients with ASM or MCL [63].
More recently, SETD2 histone methyltransferase was identified as a potential target in advanced SM. Indeed, Martinelli et al. have reported that very low or absent SETD2 protein expression was especially found in cases with advanced SM, and proteasomal degradation was found to have a major role in the observed lack this protein. Inhibitors of the proteasome, such as bortezomib, can rescue (the epigenetically decreased) expression of SETD2 and H3K36 trimethylation in neoplastic MC, resulting in a marked accumulation of ubiquitinated SETD2. Bortezomib was also found to counteract viability in neoplastic MC and to cooperate with midostaurin in inducing apoptosis in HMC-1 cells and neoplastic MC from patients with advanced SM [26].
The use of epigenetic drugs may pave the way for the development of improved treatment approaches in advanced SM. However, more preclinical studies and clinical trials need to be performed to confirm the impact of epigenetic targets and the efficacy of epigenetic therapies in patients with MC neoplasms.

Concluding Remarks
Mastocytosis is a group of heterogeneous diseases with a broad range of molecular patterns and markers and a variable clinical course. Several disease-specific processes may be triggered by the epigenome in these patients. However, only little is known about the role of epigenetic modifications in neoplastic cells in SM.
The current paper provides a summary of recent findings regarding epigenetic changes in neoplastic cells in various types of mastocytosis. In general, data published recently suggest that epigenetic changes may indeed trigger the pathogenesis of SM through several different mechanisms, including aberrant methylation of promoters of genes critically involved in DNA/RNA processing, apoptosis, and activation of MC, and modulation of KIT expression by epigenetic regulators. Moreover, genes whose products influence epigenetic processes including TET2, DNMT3A or ASXL1 may be altered (mutated) in mastocytosis. Such mutations are frequently observed in advanced SM and these mutations may also affect clinical behavior of SM and thus prognosis. In addition, TET2 and DNMT3A may play a role in MC responsiveness to acute and chronic activation. These observations suggest a potential influence of epigenetic processes on the pathogenesis of MC-related disorders.   Ten-Eleven-Translocation 2 U2AF1 U2 auxiliary factor 1 5-caC 5-carboxylcytosine 5-fC 5-formylcytosine 5-hmC 5-hydroxymethylcytosine 5-mC 5-methylcytosine