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IntroductionNatural killer (NK) cells are CD56 ϩ CD3 Ϫ large granular lymphocytes that comprise a key cellular compartment of the innate immune system. NK cells have been shown to exert antitumor activity against the malignant plasma cell clone in multiple myeloma (MM). 1-4 However, through several established mechanisms, the NK-cell versus MM effect is attenuated as the disease inexorably progresses. 5-9 MM is increasing in incidence and remains incurable despite the advent of potent novel therapies such as lenalidomide and bortezomib. 10 In fact, both lenalidomide and bortezomib have been shown to confer anti-MM activity, in part, through recovery or enhancement of the NK-cell versus MM effect. 11,12 The NK-cell versus MM effect is subject to modulation through intracellular signal transduction cascades initiated by activating and inhibitory receptors at the NK-cell surface interacting with ligands expressed on MM tumor cells. Programmed death 1 (PD-1), a member of the B7 family of cosignaling molecules, and its associated ligands PD-L1 and PD-L2 have been shown to play a key role in down-regulating the T-cell immune response. 13 The constitutive or inducible expression of PD-1 has been characterized on several immune cell subsets, including T, B, and dendritic cells; however, to date, comparatively little is known regarding PD-1 expression on NK cells and whether or not the PD-1/PD-L1 axis is involved in the NK-cell versus MM effect. 14 CT-011 (CureTech, LTD; previously CT-AcTibody or BAT) is a novel immunoglobulin G1 (IgG1) humanized monoclonal antibody (mAb) that modulates the immune response through interaction with PD-1, with previously demonstrated antitumor efficacy in experimental models of both solid and liquid tumors. [15][16][17] Several human malignancies, including MM, express cognate ligands for PD-1 (eg, PD-L1) and play a key role in tumor immunoevasion. 18,19 In a phase 1 clinical trial of patients with advanced hematologic malignancies including MM, CT-011 was demonstrated to be safe and well tolerated with evidence of single-agent clinical beneficial responses in 33% of the patients. 20 Given the results of this phase 1 study and the potential complementary mechanisms of action between CT-011 and lenalidomide, we hypothesized these agents in combination may represent a promising novel therapy for MM.Lenalidomide (Revlimid; Celgene) exerts efficacy in part through enhancement of the NK-cell versus MM effect, 11 an effect likely mediated through T-cell production of interleukin-2 (IL-2) in response to this agent. 21 The numbers of both T cells and NK cells are increased in patients receiving lenalidomide therapy 22 ; however, NK-cell killing is also enhanced, including antibodydependent cellular cytotoxicity and natural cytotoxicity. 23,24 Moreover, these events correlate with clinical responses to lenalidomide therapy in patients with MM. 22 In this report, we show that the PD-1/PD-L1 signaling axis mediates NK-cell activation and cytotoxicity against MM. We show that freshly isolated NK cells...

Aberrant DNA hypermethylation contributes to myeloid leukemogenesis by silencing structurally normal genes involved in hematopoiesis. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by targeting protein-coding mRNAs. Recently, miRNAs have been shown to play a role as both targets and effectors in gene hypermethylation and silencing in malignant cells. In the current study, we showed that enforced expression of miR-29b in acute myeloid leukemia cells resulted in marked reduction of the expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B at both RNA and protein levels. This in turn led to decrease in global DNA methylation and reexpression of p15 INK4b and ESR1 via promoter DNA hypomethylation. Although down-regulation of DNMT3A and DNMT3B was the result of a direct interaction of miR-29b with the 3 untranslated regions of these genes, no predicted miR-29b interaction sites were found in the DNMT1 IntroductionDNA methylation consists of an enzymatic addition of a methyl group at the carbon 5 position of cytosine in the context of the sequence 5Ј-cytosine-guanosine (CpG) and is mediated by DNA methyltransferases (DNMTs). 1 The promoter regions of approximately 50% of human genes contain regions of DNA with a cytosine and guanine content greater than expected (so-called CpG islands) that, once hypermethylated, mediate gene transcriptional silencing. 2 Distinct roles in genomic methylation have been reported for DNMT isoforms. Whereas DNMT1 preferentially replicates already existing methylation patterns, DNMT3A and 3B are responsible for establishing de novo methylation. 2 Silencing of structurally normal tumor suppressor genes by aberrant DNA hypermethylation has been reported in hematologic malignancies, including subsets of acute myeloid leukemia (AML). 3,4 Although the mechanisms leading to aberrant DNA hypermethylation remain to be fully elucidated, increased levels of DNMT1 and DNMT3A and 3B have been observed in malignant myeloid blasts compared with normal bone marrow (BM) mononuclear cells (MNCs), suggesting that DNMT overexpression contributes to gene promoter hypermethylation and in turn to leukemogenesis. 4 Growing evidence supports a role for microRNAs (miRNAs) as both targets and effectors in aberrant mechanisms of DNA hypermethylation. 5,6 miRNAs are noncoding RNAs of 19 to 25 nucleotides in length that regulate gene expression by inducing translational inhibition or cleavage of their target mRNAs through base pairing at partially or fully complementary sites. 7 Several groups have shown that miRNAs are altered in human malignancies and can function as tumor suppressor genes or oncogenes through expression regulation of their target genes. 7 Similar to tumor suppressor genes, miRNAs with tumor suppressor activity are often located in deleted genomic areas or are silenced by mutations or promoter hypermethylation in malignant cells. 5,[8][9][10] Saito et al recently demonstrated that miR-127 is silenced by promoter DNA hypermethylation and down-regulated in human bladder ...

SUMMARY 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.

N 6-Methyladenosine (m6A) is the most abundant RNA modification in mammal mRNAs and increasing evidence suggests the key roles of m6A in human tumorigenesis. However, whether m6A, especially its ‘reader’ YTHDF1, targets a gene involving in protein translation and thus affects overall protein production in cancer cells is largely unexplored. Here, using multi-omics analysis for ovarian cancer, we identified a novel mechanism involving EIF3C, a subunit of the protein translation initiation factor EIF3, as the direct target of the YTHDF1. YTHDF1 augments the translation of EIF3C in an m6A-dependent manner by binding to m6A-modified EIF3C mRNA and concomitantly promotes the overall translational output, thereby facilitating tumorigenesis and metastasis of ovarian cancer. YTHDF1 is frequently amplified in ovarian cancer and up-regulation of YTHDF1 is associated with the adverse prognosis of ovarian cancer patients. Furthermore, the protein but not the RNA abundance of EIF3C is increased in ovarian cancer and positively correlates with the protein expression of YTHDF1 in ovarian cancer patients, suggesting modification of EIF3C mRNA is more relevant to its role in cancer. Collectively, we identify the novel YTHDF1-EIF3C axis critical for ovarian cancer progression which can serve as a target to develop therapeutics for cancer treatment.

Clinical trials are testing oncolytic viruses (OVs) as therapies for cancer. We have shown that animals that have brain tumors and are treated with a herpes simplex virus (HSV)-derived OV live significantly longer when cyclophosphamide (CPA) is preadministered. Here, we explore the mechanisms behind this finding. In a syngeneic rat glioma model, intratumoral HSV administration is associated with rapid increase of natural killer cells, microglia͞ macrophages (CD68 ؉ and CD163 ؉ ), and IFN-␥. Pretreatment with CPA enhances HSV replication and oncolysis and reduces an HSVmediated increase in CD68 ؉ and CD163 ؉ cells and intratumoral IFN-␥. Molecular imaging shows CPA pretreatment to inhibit HSVinduced infiltration of tumor-associated phagocytic cells. Our results reveal molecular and cellular mechanisms that inhibit intratumoral spread of HSV and suggest a therapeutic path for improving the efficacy of virotherapy as a treatment for cancer.gene therapy ͉ innate immunity ͉ oncolytic virus ͉ brain tumor ͉ herpes simplex virus A dvances in virology and tumor biology have enabled development of oncolytic viruses (OVs), which replicate selectively in tumor cells (1-6). OV progeny propagate throughout tumors, lysing tumor cells but not normal cells. Phase I clinical trials have shown OV therapy to be safe (7-13) but with limited efficacy. A brisk host response to OV therapy has been seen. It includes intratumoral immune cells (7) and acute-phase reaction to intravascular virus (13). Innate immune responses may be a common side effect of OV therapy, similar to the radionecrosis of radiotherapy or myelosuppression of chemotherapy.The role of host immune responses in the efficacy or toxicity of OV therapy is poorly defined. Such responses are thought beneficial because oncolysis stimulates adaptive immunity, setting up an anticancer vaccination effect (14-16). However, initial innate responses to OVs may reduce efficient anticancer effects (17-21). For example, we have shown a herpes simplex virus (HSV)-based OV therapy to be more efficient when cyclophosphamide (CPA) is present (22-25), and this increased efficiency is credited to CPA's immunosuppressive action. However, the specific immune pathways of the observed effects have not been analyzed. ResultsPreadministering CPA Inhibits Clearance of Viral Particles and Increases HSV Replication Within Injected Tumors. We have reported that CPA allows increased replication of HSV in injected tumors by suppressing immune activity (25). To determine intratumoral persistence of HSV, we compared intratumoral viral-mediated LacZ (Fig. 1) and ICP4 (infected-cell protein 4) (Fig. 6, which is published as supporting information on the PNAS web site) gene expression in immunocompetent rats 6 and 72 h after treatment with HSV, with and without CPA pretreatment. Six hours after intratumoral HSV delivery, Ϸ50% of tumor cells showed viral-mediated gene expression, regardless of CPA ( Fig. 1 A and C). However, by 72 h, without CPA, Ͻ10% of tumor cells showed viral-mediated gene expression,...

The identification of distinct tissue-specific natural killer (NK) cell populations that apparently mature from local precursor populations has brought new insight into the diversity and developmental regulation of this important lymphoid subset. NK cells provide a necessary link between the early (innate) and late (adaptive) immune responses to infection. Gaining a better understanding of the processes that govern NK cell development should allow us to better harness NK cell functions in multiple clinical settings as well as to gain further insight into how these cells undergo malignant transformation. In this review, we summarize recent advances in understanding sites and cellular stages of NK cell development in humans and mice.

SUMMARY The biologic and clinical significance of KIT overexpression that associates with KIT gain-of- function mutations occurring in subsets of acute myeloid leukemia (AML) (i.e., core binding factor AML) is unknown. Here, we show that KIT mutations lead to MYC-dependent miR-29b repression and increased levels of the miR-29b target Sp1 in KIT-driven leukemia. Sp1 enhances its own expression by participating in a NFκB/HDAC complex that further represses miR-29b transcription. Upregulated Sp1 then binds NFκB and transactivates KIT. Therefore, activated KIT ultimately induces its own transcription. Our results provide evidence that the mechanisms of Sp1/NFκB/HDAC/miR-29b-dependent KIT overexpression contribute to leukemia growth and can be successfully targeted by pharmacological disruption of the Sp1/NFκB/HDAC complex or synthetic miR-29b treatment in KIT-driven AML.