Effects of epigenetic therapy on natural killer cell function and development in hematologic malignancy.

Epigenetic therapy is an emerging field in the treatment of human cancer, including hematologic malignancies. This class of therapeutic agents approved by the US Food and Drug Administration for cancer treatment includes DNA hypomethylating agents, histone deacetylase inhibitors, IDH1/2 inhibitors, EZH2 inhibitors, and numerous preclinical targets/agents. Most studies measuring the biological effects of epigenetic therapy focus their attention on either their direct cytotoxic effects on malignant cells or their effects on modifying tumor cell antigen expression, exposing them to immune surveillance mechanisms. However, a growing body of evidence suggests that epigenetic therapy also has effects on the development and function of the immune system, including natural killer cells, which can alter their response to cancer cells. In this review, we summarize the body of literature studying the effects of different classes of epigenetic therapy on the development and/or function of natural killer cells.

Deletion of Glycogen Synthase Kinase 3 Beta Reprograms NK Cell Metabolism.

Loss of cytotoxicity and defective metabolism are linked to glycogen synthase kinase 3 beta (GSK3ß) overexpression in natural killer (NK) cells from patients with acute myeloid leukemia or from healthy donors after expansion ex vivo with IL-15. Drug inhibition of GSK3ß in these NK cells improves their maturation and cytotoxic activity, but the mechanisms of GSK3ß-mediated dysfunction have not been well studied. Here, we show that expansion of NK cells with feeder cells expressing membrane-bound IL-21 maintained normal GSK3ß levels, allowing us to study GSK3ß function using CRISPR gene editing. We deleted GSK3B and expanded paired-donor knockout and wild-type (WT) NK cells and then assessed transcriptional and functional alterations induced by loss of GSK3ß. Surprisingly, our data showed that deletion of GSK3B did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, GSK3B-KO cells demonstrated significant changes in expression of genes related to rRNA processing, cell proliferation, and metabolic function, suggesting possible metabolic reprogramming. Next, we found that key genes downregulated in GSK3B-KO NK cells were upregulated in GSK3ß-overexpressing NK cells from AML patients, confirming this correlation in a clinical setting. Lastly, we measured cellular energetics and observed that GSK3B-KO NK cells exhibited 150% higher spare respiratory capacity, a marker of metabolic fitness. These findings suggest a role for GSK3ß in regulating NK cell metabolism.

Single-cell RNA sequencing identifies a population of human liver-type ILC1s.

Group 1 innate lymphoid cells (ILCs) comprise a heterogeneous family of cytotoxic natural killer (NK) cells and ILC1s. We identify a population of "liver-type" ILC1s with transcriptional, phenotypic, and functional features distinct from those of conventional and liver-resident NK cells as well as from other previously described human ILC1 subsets. LT-ILC1s are CD49a+CD94+CD200R1+, express the transcription factor T-BET, and do not express the activating receptor NKp80 or the transcription factor EOMES. Similar to NK cells, liver-type ILC1s produce IFN-γ, TNF-α, and GM-CSF; however, liver-type ILC1s also produce IL-2 and lack perforin and granzyme-B. Liver-type ILC1s are expanded in cirrhotic liver tissues, and they can be produced from blood-derived ILC precursors in vitro in the presence of TGF-ß1 and liver sinusoidal endothelial cells. Cells with similar signature and function can also be found in tonsil and intestinal tissues. Collectively, our study identifies and classifies a population of human cross-tissue ILC1s.

A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells.

BACKGROUND: Mantle cell lymphoma (MCL) is a rare, highly heterogeneous type of B-cell non-Hodgkin's lymphoma. The sumoylation pathway is known to be upregulated in many cancers including lymphoid malignancies. However, little is known about its oncogenic role in MCL. METHODS: Levels of sumoylation enzymes and sumoylated proteins were quantified in MCL cell lines and primary MCL patient samples by scRNA sequencing and immunoblotting. The sumoylation enzyme SAE2 was genetically and pharmacologically targeted with shRNA and TAK-981 (subasumstat). The effects of SAE2 inhibition on MCL proliferation and cell cycle were evaluated using confocal microscopy, live-cell microscopy, and flow cytometry. Immunoprecipitation and orbitrap mass spectrometry were used to identify proteins targeted by sumoylation in MCL cells. RESULTS: MCL cells have significant upregulation of the sumoylation pathway at the level of the enzymes SAE1 and SAE2 which correlated with poor prognosis and induction of mitosis associated genes. Selective inhibition of SAE2 with TAK-981 results in significant MCL cell death in vitro and in vivo with mitotic dysregulation being an important mechanism of action. We uncovered a sumoylation program in mitotic MCL cells comprised of multiple pathways which could be directly targeted with TAK-981. Centromeric localization of topoisomerase 2A, a gene highly upregulated in SAE1 and SAE2 overexpressing MCL cells, was lost with TAK-981 treatment likely contributing to the mitotic dysregulation seen in MCL cells. CONCLUSIONS: This study not only validates SAE2 as a therapeutic target in MCL but also opens the door to further mechanistic work to uncover how to best use desumoylation therapy to treat MCL and other lymphoid malignancies.

ILC1s control leukemia stem cell fate and limit development of AML.

Type I innate lymphoid cells (ILC1s) are critical regulators of inflammation and immunity in mammalian tissues. However, their function in cancer is mostly undefined. Here, we show that a high density of ILC1s induces leukemia stem cell (LSC) apoptosis in mice. At a lower density, ILC1s prevent LSCs from differentiating into leukemia progenitors and promote their differentiation into non-leukemic cells, thus blocking the production of terminal myeloid blasts. All of these effects, which require ILC1s to produce interferon-γ after cell-cell contact with LSCs, converge to suppress leukemogenesis in vivo. Conversely, the antileukemia potential of ILC1s wanes when JAK-STAT or PI3K-AKT signaling is inhibited. The relevant antileukemic properties of ILC1s are also functional in healthy individuals and impaired in individuals with acute myeloid leukemia (AML). Collectively, these findings identify ILC1s as anticancer immune cells that might be suitable for AML immunotherapy and provide a potential strategy to treat AML and prevent relapse of the disease.

Adipocyte CD1d Gene Transfer Induces T Cell Expansion and Adipocyte Inflammation in CD1d Knockout Mice.

CD1d, a lipid Ag-presenting molecule for invariant NKT (iNKT) cells, is abundantly expressed on adipocytes and regulates adipose homeostasis through iNKT cells. CD1d gene expression was restored in visceral adipose tissue adipocytes of CD1d knockout (KO) mice to investigate the interactions between adipocytes and immune cells within adipose tissue. We developed an adipocyte-specific targeting recombinant adeno-associated viral vector, with minimal off-target transgene expression in the liver, to rescue CD1d gene expression in visceral adipose tissue adipocytes of CD1d KO mice, followed by assessment of immune cell alternations in adipose tissue and elucidation of the underlying mechanisms of alteration. We report that adeno-associated virus-mediated gene transfer of CD1d to adipocytes in CD1d KO mice fails to rescue iNKT cells but leads to massive and selective expansion of T cells within adipose tissue, particularly CD8+ T effector cells, that is associated with adipocyte NLRP3 inflammasome activation, dysregulation of adipocyte functional genes, and upregulation of apoptotic pathway proteins. An NLRP3 inhibitor has no effect on T cell phenotypes whereas depletion of CD8+ T cells significantly attenuates inflammasome activation and abolishes the dysregulation of adipocyte functional genes induced by adipocyte CD1d. In contrast, adipocyte overexpression of CD1d fails to induce T cell activation in wild-type mice or in invariant TCR α-chain Jα18 KO mice that have a normal lymphocyte repertoire except for iNKT cells. Our studies uncover an adipocyte CD1d → CD8+ T cell → adipocyte inflammasome cascade, in which CD8+ T cells function as a key mediator of adipocyte inflammation likely induced by an allogeneic response against the CD1d molecule.

Identification and Targeting of the Developmental Blockade in Extranodal Natural Killer/T-cell Lymphoma.

Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with poor clinical outcomes. Here we used phenotypic and molecular profiling, including epigenetic analyses, to investigate how ENKTL ontogeny relates to normal NK-cell development. We demonstrate that neoplastic NK cells are stably, but reversibly, arrested at earlier stages of NK-cell maturation. Genes downregulated in the most epigenetic immature tumors were associated with polycomb silencing along with genomic gain and overexpression of EZH2. ENKTL cells exhibited genome-wide DNA hypermethylation. Tumor-specific DNA methylation gains were associated with polycomb-marked regions, involving extensive gene silencing and loss of transcription factor binding. To investigate therapeutic targeting, we treated novel patient-derived xenograft (PDX) models of ENKTL with the DNA hypomethylating agent, 5-azacytidine. Treatment led to reexpression of NK-cell developmental genes, phenotypic NK-cell differentiation, and prolongation of survival. These studies lay the foundation for epigenetic-directed therapy in ENKTL. SIGNIFICANCE: Through epigenetic and transcriptomic analyses of ENKTL, a rare, aggressive malignancy, along with normal NK-cell developmental intermediates, we identified that extreme DNA hypermethylation targets genes required for NK-cell development. Disrupting this epigenetic blockade in novel PDX models led to ENKTL differentiation and improved survival. This article is highlighted in the In This Issue feature, p. 85.

Acute Myeloid Leukemia Alters Group 1 Innate Lymphoid Cell Differentiation from a Common Precursor.

NK cells are known to be developmentally blocked and functionally inhibited in patients with acute myeloid leukemia (AML), resulting in poor clinical outcomes. In this study, we demonstrate that whereas NK cells are inhibited, closely related type 1 innate lymphoid cells (ILC1s) are enriched in the bone marrow of leukemic mice and in patients with AML. Because NK cells and ILC1s share a common precursor (ILCP), we asked if AML acts on the ILCP to alter developmental potential. A combination of ex vivo and in vivo studies revealed that AML skewing of the ILCP toward ILC1s and away from NK cells represented a major mechanism of ILC1 generation. This process was driven by AML-mediated activation of the aryl hydrocarbon receptor (AHR), a key transcription factor in ILCs, as inhibition of AHR led to decreased numbers of ILC1s and increased NK cells in the presence of AML. These results demonstrate a mechanism of ILC developmental skewing in AML and support further preclinical study of AHR inhibition in restoring normal NK cell development and function in the setting of AML.

Functional expression of aryl hydrocarbon receptor as a potential novel therapeutic target in human multiple myeloma.

The etiology of multiple myeloma (MM) remains incompletely understood; however, epidemiologic studies have suggested a possible link between exposure to environmental aromatic hydrocarbons-which serve as exogenous ligands for the aryl hydrocarbon receptor (AHR), which has been implicated in cancer biology-and development of monoclonal gammopathy of undetermined significance (MGUS) and MM. Herein, we demonstrate the functional expression of AHR in MM cell lines and primary human MM samples. AHR is expressed in putative MM 'stem cells' and advanced clinical stages of MM, and functionally contributes to MM tumor cell phenotype and proliferation. Antagonism of AHR directly impairs MM cell viability and increases MM cell susceptibility to immune-mediated clearance. Furthermore, our findings indicate that AHR antagonism may represent an effective means to enhance the function of other drugs, such as anti-CD38 antibodies, in future clinical studies. Taken together, these data identify AHR as a novel target for MM therapy.

Differential Integrin Adhesome Expression Defines Human NK Cell Residency and Developmental Stage.

NK cells are innate immune cells that reside within tissue and circulate in peripheral blood. They interact with a variety of microenvironments, yet how NK cells engage with these varied microenvironments is not well documented. The adhesome represents a molecular network of defined and predicted integrin-mediated signaling interactions. In this study, we define the integrin adhesome expression profile of NK cells from human tonsil, peripheral blood, and those derived from human hematopoietic precursors through stromal cell coculture systems. We report that the site of cell isolation and NK cell developmental stage dictate differences in expression of adhesome associated genes and proteins. Furthermore, we define differences in cortical actin content associated with differential expression of actin regulating proteins, suggesting that differences in adhesome expression are associated with differences in cortical actin homeostasis. These data provide understanding of the diversity of human NK cell populations and how they engage with their microenvironment.

Unraveling the Role of Innate Lymphoid Cells in AcuteMyeloid Leukemia.

Over the past 50 years, few therapeutic advances have been made in treating acute myeloid leukemia (AML), an aggressive form of blood cancer, despite vast improvements in our ability to classify the disease. Emerging evidence suggests the immune system is important in controlling AML progression and in determining prognosis. Natural killer (NK) cells are important cytotoxic effector cells of the innate lymphoid cell (ILC) family that have been shown to have potent anti-leukemic functions. Recent studies are now revealing impairment or dysregulation of other ILCs in various types of cancers, including AML, which limits the effectiveness of NK cells in controlling cancer progression. NK cell development and function are inhibited in AML patients, which results in worse clinical outcomes; however, the specific roles of other ILC populations in AML are just now beginning to be unraveled. In this review, we summarize what is known about the role of ILC populations in AML.

Notch Regulates Innate Lymphoid Cell Plasticity during Human NK Cell Development.

Human NK cells develop in tonsils through discrete NK cell developmental intermediates (NKDIs), yet the mechanistic regulation of this process is unclear. We demonstrate that Notch activation in human tonsil-derived stage 3 (CD34-CD117+CD94-NKp80-) and 4A (CD34-CD117+/-CD94+NKp80-) NKDIs promoted non-NK innate lymphoid cell differentiation at the expense of NK cell differentiation. In contrast, stage 4B (CD34-CD117+/-CD94+NKp80+) NKDIs were NK cell lineage committed despite Notch activation. Interestingly, whereas NK cell functional maturation from stage 3 and 4A NKDIs was independent of Notch activation, the latter was required for high NKp80 expression and a stage 4B-like phenotype by the NKDI-derived NK cells. The Notch-dependent effects required simultaneous engagement with OP9 stromal cells and were also stage-specific, with NOTCH1 and NOTCH2 receptors regulating stage 3 NKDIs and NOTCH1 primarily regulating stage 4A NKDIs. These data establish stage-specific and stromal-dependent roles for Notch in regulating human NK cell developmental plasticity and maturation.

Activation of the Intracellular Pattern Recognition Receptor NOD2 Promotes Acute Myeloid Leukemia (AML) Cell Apoptosis and Provides a Survival Advantage in an Animal Model of AML.

TLRs, a family of membrane-bound pattern recognition receptors found on innate immune cells, have been well studied in the context of cancer therapy. Activation of these receptors has been shown to induce inflammatory anticancer events, including differentiation and apoptosis, across a wide variety of malignancies. In contrast, intracellular pattern recognition receptors such as NOD-like receptors have been minimally studied. NOD2 is a member of the NOD-like receptor family that initiates inflammatory signaling in response to the bacterial motif muramyl dipeptide. In this study, we examined the influence of NOD2 in human acute myeloid leukemia (AML) cells, demonstrating that IFN-γ treatment upregulated the expression of NOD2 signaling pathway members SLC15A3 and SLC15A4, downstream signaling kinase RIPK2, and the NOD2 receptor itself. This priming allowed for effective induction of caspase-1-dependent cell death upon treatment with muramyl tripeptide phosphatidylethanolamine (MTP-PE), a synthetic ligand for NOD2. Furthermore, the combination of MTP-PE and IFN-γ on AML blasts generated an inflammatory cytokine profile and activated NK cells. In a murine model of AML, dual treatment with MTP-PE and IFN-γ led to a significant increase in mature CD27- CD11b+ NK cells as well as a significant reduction in disease burden and extended survival. These results suggest that NOD2 activation, primed by IFN-γ, may provide a novel therapeutic option for AML.

MicroRNA regulation of natural killer cell development and function in leukemia.

MicroRNAs (miRNAs) are now recognized as important regulators of all cellular processes, including immune function and cancer survival. These evolutionary preserved, single-stranded, non-coding RNA molecules mediate important functional effects primarily through post-transcriptional regulation of protein expression. MiRNAs are known to mediate multiple oncogenic pathways in tumor cells, both tumor promoting and tumor suppressing. In addition to a direct tumor cell effect, miRNAs have also been shown to play a critical role in immune cell development, function and survival. Here we expand on previous reports to evaluate miRNA regulation in natural killer (NK) cells primarily in humans and focus on their influence on NK cell development and function in the setting of hematologic malignancies. In addition, we highlight the most recent miRNA discoveries in hematologic malignancies and discuss areas of future exploration relevant to the translational field of innate immunology and miRNA-based therapeutic intervention.

The IL-15-AKT-XBP1s signaling pathway contributes to effector functions and survival in human NK cells.

Interleukin 15 (IL-15) is one of the most important cytokines that regulate the biology of natural killer (NK) cells1. Here we identified a signaling pathway-involving the serine-threonine kinase AKT and the transcription factor XBP1s, which regulates unfolded protein response genes2,3-that was activated in response to IL-15 in human NK cells. IL-15 induced the phosphorylation of AKT, which led to the deubiquitination, increased stability and nuclear accumulation of XBP1s protein. XBP1s bound to and recruited the transcription factor T-BET to the gene encoding granzyme B, leading to increased transcription. XBP1s positively regulated the cytolytic activity of NK cells against leukemia cells and was also required for IL-15-mediated NK cell survival through an anti-apoptotic mechanism. Thus, the newly identified IL-15-AKT-XBP1s signaling pathway contributes to enhanced effector functions and survival of human NK cells.

CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway.

According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.

Human AML activates the aryl hydrocarbon receptor pathway to impair NK cell development and function.

Acute myeloid leukemia (AML) can evade the mouse and human innate immune system by suppressing natural killer (NK) cell development and NK cell function. This is driven in part by the overexpression of microRNA (miR)-29b in the NK cells of AML patients, but how this occurs is unknown. In the current study, we demonstrate that the transcription factor aryl hydrocarbon receptor (AHR) directly regulates miR-29b expression. We show that human AML blasts activate the AHR pathway and induce miR-29b expression in NK cells, thereby impairing NK cell maturation and NK cell function, which can be reversed by treating NK cells with an AHR antagonist. Finally, we show that inhibition of constitutive AHR activation in AML blasts lowers their threshold for apoptosis and decreases their resistance to NK cell cytotoxicity. Together, these results identify the AHR pathway as a molecular mechanism by which AML impairs NK cell development and function. The results lay the groundwork in establishing AHR antagonists as potential therapeutic agents for clinical development in the treatment of AML.

The Broad Spectrum of Human Natural Killer Cell Diversity.

Natural killer (NK) cells provide protection against infectious pathogens and cancer. For decades it has been appreciated that two major NK cell subsets (CD56bright and CD56dim) exist in humans and have distinct anatomical localization patterns, phenotypes, and functions in immunity. In light of this traditional NK cell dichotomy, it is now clear that the spectrum of human NK cell diversity is much broader than originally appreciated as a result of variegated surface receptor, intracellular signaling molecule, and transcription factor expression; tissue-specific imprinting; and foreign antigen exposure. The recent discoveries of tissue-resident NK cell developmental intermediates, non-NK innate lymphoid cells, and the capacity for NK cells to adapt and differentiate into long-lived memory cells has added further complexity to this field. Here we review our current understanding of the breadth and generation of human NK cell diversity.

MicroRNA-29b mediates altered innate immune development in acute leukemia.

Natural killer (NK) cells can have potent antileukemic activity following haplo-mismatched, T cell-depleted stem cell transplantations for the treatment of acute myeloid leukemia (AML), but they are not successful in eradicating de novo AML. Here, we have used a mouse model of de novo AML to elucidate the mechanisms by which AML evades NK cell surveillance. NK cells in leukemic mice displayed a marked reduction in the cytolytic granules perforin and granzyme B. Further, as AML progressed, we noted the selective loss of an immature subset of NK cells in leukemic mice and in AML patients. This absence was not due to elimination by cell death or selective reduction in proliferation, but rather to the result of a block in NK cell differentiation. Indeed, NK cells from leukemic mice and humans with AML showed lower levels of TBET and EOMES, transcription factors that are critical for terminal NK cell differentiation. Further, the microRNA miR-29b, a regulator of T-bet and EOMES, was elevated in leukemic NK cells. Finally, deletion of miR-29b in NK cells reversed the depletion of this NK cell subset in leukemic mice. These results indicate that leukemic evasion of NK cell surveillance occurs through miR-mediated dysregulation of lymphocyte development, representing an additional mechanism of immune escape in cancer.