JAK2 and TET2 Mutation in Polycythemia Vera

The Ten-Eleven Translocation-2 (TET2) gene, located on chromosome 4q24, has been implicated in hematological malignancies. The TET2 gene shows mutations in variable myeloid malignancies with the involvement of 15% of myeloproliferative neoplasms (MPNs). The inactivation of the TET2 gene in both mice and humans has shown a high degree of deregulation of the hematopoiesis process leading to hematological malignancies. Polycythemia vera (PV), an MPN characterized by increased red blood cell mass, has been associated with the TET2 gene. Furthermore, TET2 genes have been found to facilitate Janus kinase-2 and signal transducer activator of transcription 5, as well as modulate the epigenetic composition of genomic DNA. However, little is known about the role of TET2 mutations in patients with PV. Several studies have been conducted to further assess the significant role of TET2 gene function in various disease processes and prognoses to enhance the management and care of these patients.


Introduction And Background
Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (MF), and chronic myeloid leukemia (CML) among several other disorders. CML is a Philadelphia chromosome-positive MPN, whereas others are negative for the Philadelphia chromosome. Several genes are implicated in myeloproliferative diseases including PV. In 2005, a recurrent point mutation in Janus kinase-2 (JAK2) exon 14 was identified in patients with MPNs. This mutation results in valine-to-phenylalanine substitution at position 617 in JAK homolog autoinhibitory domain. This substitution results in a gain-of-function of JAK2 which autonomously activates downstream signaling pathways, including Janus kinase/signal transducer and activator of transcription (JAK-STAT), phosphatidylinositol 3-kinase/protein kinase B, and extracellular signal-regulated kinases/microtubuleassociated protein kinase [1,2]. Mutations in JAK2 exon 12 and MPL515 genes have also been implicated in MPNs which alter the JAK-STAT signaling pathway. However, the contribution of these genes has not been clearly identified in MPN phenotype. Myeloproliferative leukemia (MPL) and JAK2, although strongly associated with MPN, do not necessarily specify clinicopathologic correlation. Growing evidence suggests the role of other genetic factors impacting the pathogenesis of MPNs. PV shows a higher expression of JAK2, but at the same time, JAK2 is not exclusive to PV in the MPN spectrum. Similar data have also been seen with the MPL mutant gene. This signifies the importance of the identification of more molecular alterations [1][2][3][4][5]. In recent studies, along with JAK2V617F, a coexisting Ten-Eleven Translocation-2 (TET2) gene, loss-offunction mutation has been found in the minimal loss of heterozygosity region of chromosome 4q24 in MPN patients [2]. TET2 has pleiotropic roles during hematopoiesis, including stem-cell self-renewal, lineage commitment, and terminal differentiation of monocytes [6]. TET2 is a known tumor-suppressor gene.
PV is a monoclonal proliferative disorder of multipotent myeloid progenitor cells, increasing the cell count of all three lineages [7]. The disorder has a prevalence of 0.68-2.6 per 100,000 individuals [8]. It is characterized by increased red blood cell (RBC) mass and is associated with an increased risk of thrombotic events, leukemic transformation, and MF. JAK2V617F mutations are found in greater than 95% of PV patients. However, in patients with PV who were negative for JAK2V617F, other abnormalities were found in JAK2 exon 12 which induced activation of the JAK-STAT pathway at a greater level than the JAK2V617F allele [3]. This has been the basis for the World Health Organization's definition of PV or other myeloproliferative disorders including ET which was revised in 2008. The diagnostic criteria include the detection of JAK2 mutation in exon 12 or 14. However, it is unclear how a single mutation in JAK2 can lead to different clinical phenotypes of MPN. JAK2 mutations do not explain the variable prognosis among patients with PV, which can perhaps be explained by the variable burden of this mutation in hematopoietic cells as well in genes other than JAK2. Furthermore, variations in the expression of alternative genes and epigenetic modification may account for some of these disparities. Several other genes are implicated in PV including TET2 genes [4]. TET2 genes facilitate JAK2 or STAT5 signal transduction as well as modulate the epigenetic composition of genomic DNA, including DNA and histone methylation and acetylation [5].
TET2 mutations often occur early during the development of human myeloid malignancies, including PV, ET, MF, myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). These mutations appear to target hematopoietic stem/progenitor cells [7,8]. However, studies have shown that TET2 gene mutations may also occur during later stages, which may explain the transformation of MPN to acute leukemia [6]. This implies that therapeutic targets may have to focus on hematopoietic stem cells or progenitor cells to eradicate these myeloproliferative conditions. Table 1 illustrates TET2 mutation prevalence in different myeloid malignancies [9], whereas Table 2 demonstrates the prevalence of TET2 mutation in MPNs [10].

Review
Overview of JAK2 and TET2 gene In 2009, the TET2 gene was described in myeloid malignancies along with its variants. The TET2 gene is located on chromosome 4q24. TET2 protein modulates DNA hydroxymethylation through the conversion of 5-methylcytosine to 5-hydroxymethylcytosine, promoting DNA demethylation. The functional domain of TET2 is located at the C-terminus; it consists of a cysteine-rich domain as well as a double-stranded β-helix fold domain. Significant functions of TET2 include the hematopoiesis role, stem-cell self-renewal promotion, monocyte differentiation on the terminal stage, and lineage commitment. In addition, the TET2 gene is highly expressed in hematopoietic progenitor cells [11]. TET2 gene shows mutations in variable myeloid malignancies with the involvement of 15% of the MPNs. The inactivation of the TET2 gene in both mice and humans has shown a high degree of deregulation of the hematopoiesis process leading to hematological malignancies [12]. Moreover, the TET2 gene has been described as a tumor-suppressor gene with its homozygous and heterozygous mutations leading to hematopoietic malignancies in humans. Amino acid substitutions, frameshifting, in-frame deletions, and generated stop codons are all possible TET2 gene mutations. However, there is no precise pattern of genotypes with the associated hematological malignancies such as MDS, CMML, and AML [11]. Jung-Sook et al. reported that all patients carrying the TET2 mutation also carried the JAK2V617F mutation. There was no relation between the occurrence of the TET2 mutation and age, JAK2V617F allele burden, frequency of organomegaly, fibrosis of the marrow, hematologic indices, as well as thrombotic or hemorrhagic complications in all MPN patients [13]. Most TET2 mutations result in the loss of enzymatic function. The most common types of mutations are nonsense and frameshift ones, which occur before the C-domain. However, missense mutations and in-frame deletions also occur within the C-domain [14]. Figure 1 demonstrates the loss of function of TET2 along with mutation of JAK2V617F, leading to MPN and AML [15].

Clinical implications of TET2 gene mutation and its association with polycythemia (in terms of diagnosis, treatment targets, and prognosis)
Patients with PV who are homozygous for JAK2V617F are prone to developing post-PV complications such as MF [16,17]. In addition, patients with JAK2V617F mutation who acquire BCR-ABL1 translocation are prone to developing CML. However, the role of TET2 in the progression or evolution of PV to MF, AML, or CML has not been established. Much controversy has been elicited regarding the prognosis of myeloid malignancies with TET2 mutations [18]. According to Tefferi et al., there were no clinically significant alterations in the prognosis or overall survival rate in patients diagnosed with MPNs based on findings from large cohort studies [19]. On the other hand, studies have demonstrated poorer outcomes in hematologic malignancies including CMML and AML associated with TET2 mutations with limited data on PV [20,21]. Moreover, TET2 mutations associated with malignancies have been shown to have a favorable response to hypomethylating agents in high-risk patients [11,22].
Uncertain prognosis in patients with TET2 mutations is largely due to studies lacking evidence of possible underlying and other associated mutations for the prognosis of TET2 mutations. In other words, there is a lack of evidence regarding whether TET2 mutations primarily dysregulate well-known pathways associated with hematopoietic transformation or constitute a novel, poorly discovered pathway toward malignancy pathogenesis [21]. To further elaborate, TET2 mutations may set the stage toward the pathogenesis of different hematologic malignancies and act jointly with other gene mutations at early stages. For example, TET2 mutations when combined with JAK2 and ASXL1 mutations give rise to PV and MF [23]. Overall, different combinations of gene mutations along with TET2 mutations markedly reflect on the prognosis. Of note, Swierczek et al. have suggested that TET2 mutations are not the PV initiating cascade, rather they occur after JAK2 mutations. It has also been suggested that combined JAK2 and TET2 mutations allow for dramatic proliferation over TET2-negative PV subclones, emphasizing the hypothesis that TET2 mutations increase the aggressive nature of JAK2 mutation-positive PV [24]. Interestingly, Ortmann et al. found that the order in which JAK2/TET2 mutations are acquired reflects on the clinical prognosis of patients diagnosed with PV. Patients with initial JAK2 mutation demonstrated a higher risk of thrombosis compared to a more indolent course in patients with initial PV-associated TET2 mutation [25].
With regard to the treatment, studies have shown that management with peginterferon alfa-2a can reduce JAK2V617F clones but not the TET2 mutant ones. As mentioned earlier, TET2 mutation leads to persistent clonal hematopoiesis [26]. Another study has shown that peginterferon alfa-2a-treated patients with both JAK2 and TET2 mutations had a less significant reduction in the burden of JAK2V617F compared to those with JAK2 but without TET2 mutations. The former group possessed a higher burden of JAK2V617F mutation at the beginning of the therapy. Furthermore, the same study revealed that patients without complete remission were more likely to have additional mutations apart from JAK2 [27].

Conclusions
Several studies suggest that TET2 mutations are an early event in the development of myeloid malignancies, yet their function in normal cells and pathologic conditions remains to be elucidated. It is unclear what triggers these mutations and at what point in time. JAK2 mutation is associated with most MPNs including PV. However, a single mutation in JAK2 does not explain the variable prognoses among patients with PV. Several other genes including mutations in TET2 have been implicated. TET2 genes facilitate JAK2 or STAT5 signal transduction and allow the accelerated proliferation of cells in patients with JAK2-positive PV. Further, studies have shown that patients with initial JAK2 mutation demonstrated a higher risk of thrombosis compared to a more indolent course in patients with initial PV-associated TET2 mutation. However, these are early days and much remains unknown about TET2 mutations. As newer studies continue to shed light on this subject, whether TET2 genes may be targeted as a part of disease management or prognostication remains to be seen. Targeted disruption of genes in animal models, as well as perturbation of TET2 levels in normal and malignant cell types in vitro and in vivo, may offer clues to the understanding of the function of TET2.

Conflicts of interest:
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