Altered epigenetics at the center of NK-LGL leukemia

In this issue of Blood, Olson et al describe (epi)genetic changes in 58 patients with chronic natural killer large granular lymphocyte (NK-LGL) leukemia, uncovering recurrent loss-of function mutations in the TET2 gene. They highlight the associations of these lesions with altered global patterns of DNA methylation, with distinct cytopenias, and with treatment responses. This significantly adds to our understanding of the pathogenesis of and diagnostic approach to this rare disease.¹ 

NK-LGL leukemia, also referred to as chronic lymphoproliferative disorder of NK cells (CLPDNK), represents a neoplasm of clonal NK cells. Unlike T cells with their T-cell receptor genes, NK cells lack clonotypic structures. Therefore, NK-LGL leukemia is often hard to distinguish from reactive NK cell proliferations, and a restricted pattern of killer immunoglobulin-like receptors has been used as a less-than-ideal surrogate. On the basis of comparable cellular morphology, shared cytotoxic cell phenotypes, and similar clinical presentations, NK-LGL leukemia has been grouped together with T-cell LGL (T-LGL) leukemia.²  Although the true lymphoid vs myeloid nature of NK-LGL leukemia is uncertain,³  both T-LGL and NK-LGL leukemias share constitutive STAT signaling⁴  and recurrent gain-of-function mutations of STAT3 (∼30% in NK-LGL and ∼40% in T-LGL leukemia) as their established molecular hallmarks.^1,3,5 

Olson et al performed whole-genome sequencing of peripheral blood mononuclear cells (PBMCs) and saliva of 7 index patients with NK-LGL leukemia. Focusing on the most relevant candidate genes, they extended their approach to a targeted (re)sequencing strategy of those 7 and an additional 51 NK-LGL leukemia patients. They detected damaging mutations of the TET2 gene in 28% of patients. These mainly truncating variants predominantly resided within the catalytic domain of the molecule and were called at high variant allele frequencies. Besides the expected lesions in STAT3 (33% of patients¹ ), the authors also reported less common mutations in TNFAIP3 and PI3K pathway genes (10% and 5%, respectively). Comutations of TET2 and STAT3 were observed in 7 patients (12%).

The central finding of this work by Olson et al, namely the identification of defective TET2 variants in the NK cell compartment of nearly one-third of cases, was also independently reported recently in 34% of CLPD-NK patients by Pastoret et al.³  Both reports provide us with valuable means to determine the clonal nature of NK cell expansions (in at least a subset of them). Because both data sets report an incidence of TET2 and STAT3 comutations in 2%³  and 12%¹  of cases, detection of either of these clonal lesions would be expected in a cumulative proportion of at least 50% of NK-LGL leukemias.

Interestingly, the authors also showed methylations of TET2 promoter regions preferentially in those cases carrying TET2 gene mutations. Of those, TET2 promoter methylation was more frequent in samples with single TET2 mutations as compared with samples with compound TET2 mutations. It is, as the authors state, tempting to speculate that this reflects a mechanism of biallelic TET2 impairment, namely by monoallelic somatic mutation and transcriptional silencing of the wild-type allele.

The methylcytosine dioxygenase TET2 catalyzes the conversion of the modified DNA base methylcytosine to 5-hydroxy-methylcytosine, thus contributing to gene regulation by DNA demethylation. Olson et al investigated potential consequences of dysfunctional TET2 by assessing global DNA methylation in their samples. They identified distinct patterns of methylation between TET2-mutated and -unmutated NK-LGL leukemias and healthy donor–derived NK cells. In particular, TET2-mutated cases showed enhanced global methylation (eg, fivefold more differentially methylated regions than unmutated samples), fitting a defective demethylation pattern.

Olson et al also observed hypermethylation of 2 negative regulators of STAT3 (ie, PTPRD and PTPRN), which was significantly enriched in TET2-mutated samples. This is intriguing, because impaired negative regulation of the JAK/STAT pathway by methylation-induced silencing has been described in other cancers,⁶  suggesting alternative modes of constitutive STAT activation in LGL leukemias in addition to STAT3 mutations. This complements our current disease concept that is centered around cytokine-driven and genetically instructed STAT activation (see figure). These associative findings must be validated mechanistically in appropriate models.

TET2 mutations are frequently found in MDS and acute myeloid leukemia.^7,8  Fittingly, Pastoret et al³  also identified concurrent TET2 mutations in the myeloid compartment of NK-LGL leukemia, and the incidence of cooccurring hematologic malignancies was increased in TET2-mutated cases. Olson et al found these mutations to be limited to NK cells (CD94⁺); they were not observed in CD94⁻ PBMCs. Because ∼5% of LGL leukemia patients present with concurrent MDS, it is tempting to assume common precursor routes, especially because TET2 mutations are a known lesion in myeloid clonal hematopoiesis of indeterminate potential.⁹  By extension, it remains unresolved whether the cytopenias in LGL leukemias are merely a result of the cytotoxic activity of the clonal NK or T cells or to what extent the TET2 variant–burdened defective hematopoiesis contributes as well. Unexpectedly, Olson et al could not show a higher incidence of concomitant hematologic malignancies in their TET2-mutated cohort.

Importantly, the authors also examined the clinical correlations of these genetic lesions. They showed that TET2 mutations were exclusively associated with thrombocytopenia. STAT3 mutations were associated with anemia and neutropenia, particularly when TET2 lesions coexisted. The comutated subgroup also showed best clinical response rates to cyclophosphamide, whereas patients with single TET2 mutations were highly treatment resistant. Future work should evaluate if assessment of TET2 mutation status can be incorporated into the clinical decision-making process and if the presence of such mutations justifies specific treatment strategies.

Overall, this article by Olson et al contributes new important insights into the molecular landscape of NK-LGL leukemia. Although the recent work by Pastoret et al³  elaborated on the novel hallmark of TET2 variants to improve our diagnostic algorithms for and understanding of clonal origins in NK-LGL leukemia, Olson et al provide here the first insights into the functional relevance of defective TET2. Their finding of altered global and STAT regulator–associated methylation in NK-LGL leukemia, especially in TET2-mutated patients, implicates dysfunctional TET2 as a primer of reprogrammed gene expression and may provide rationales for approaches of epigenetic or gene-specific targeting. Fittingly, demethylating agents and histone deacetylase inhibition have shown promising results in the first in vitro studies.¹⁰