XPO1 blockade with KPT-330 promotes apoptosis in cutaneous T-cell lymphoma by activating the p53-p21 and p27 pathways.

Dysregulated nuclear-cytoplasmic trafficking has been shown to play a role in oncogenesis in several types of solid tumors and hematological malignancies. Exportin 1 (XPO1) is responsible for the nuclear export of several proteins and RNA species, mainly tumor suppressors. KPT-330, a small molecule inhibitor of XPO1, is approved for treating relapsed multiple myeloma and diffuse large B-cell lymphoma. Cutaneous T-cell lymphoma (CTCL) is an extranodal non-Hodgkin lymphoma with an adverse prognosis and limited treatment options in advanced stages. The effect of therapeutically targeting XPO1 with KPT-330 in CTCL has not been established. We report that XPO1 expression is upregulated in CTCL cells. KPT-330 reduces cell proliferation, induces G1 cell cycle arrest and apoptosis. RNA-sequencing was used to explore the underlying mechanisms. Genes associated with the cell cycle and the p53 pathway were significantly enriched with KPT-330 treatment. KPT-330 suppressed XPO1 expression, upregulated p53, p21WAF1/Cip1, and p27Kip1 and their nuclear localization, and downregulated anti-apoptotic protein (Survivin). The in vivo efficacy of KPT-330 was investigated using a bioluminescent xenograft mouse model of CTCL. KPT-330 blocked tumor growth and prolonged survival (p < 0.0002) compared to controls. These findings support investigating the use of KPT-330 and next-generation XPO1 inhibitors in CTCL.

The role of interleukin-15 in the development and treatment of hematological malignancies.

Cytokines are a vital component of the immune system that controls the activation and growth of blood cells. However, chronic overexpression of cytokines can trigger cellular events leading to malignant transformation. The cytokine interleukin-15 (IL-15) is of particular interest, which has been shown to contribute to the development and progression of various hematological malignancies. This review will provide an overview of the impact of the immunopathogenic function of IL-15 by studying its role in cell survival, proliferation, inflammation, and treatment resistance. We will also review therapeutic approaches for inhibiting IL-15 in blood cancers.

Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.

Cytokines in the Pathogenesis of Large Granular Lymphocytic Leukemia.

Large granular lymphocytic leukemia (LGLL) is a lymphoproliferative disorder of older adults characterized by the clonal expansion of cytotoxic T/natural killer cells due to constitutive pro-survival signaling. In recent years, it has become clear that cytokines and their receptors are aberrantly expressed in LGLL cells. The exact initiation process of LGLL is unknown, although several cytokine-driven mechanisms have emerged. Elevated levels of several cytokines, including interleukin-15 (IL-15) and platelet-derived growth factor (PDGF), have been described in LGLL patients. Evidence from humans and animal models has shown that cytokines may also contribute to the co-occurrence of a wide range of autoimmune diseases seen in patients with LGLL. The goal of this review is to provide a comprehensive analysis of the link between cytokines and pro-survival signaling in LGLL and to discuss the various strategies and research approaches that are being utilized to study this link. This review will also highlight the importance of cytokine-targeted therapeutics in the treatment of LGLL.

Defining the AHR-regulated transcriptome in NK cells reveals gene expression programs relevant to development and function.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular processes in cancer and immunity, including innate immune cell development and effector function. However, the transcriptional repertoire through which AHR mediates these effects remains largely unexplored. To elucidate the transcriptional elements directly regulated by AHR in natural killer (NK) cells, we performed RNA and chromatin immunoprecipitation sequencing on NK cells exposed to AHR agonist or antagonist. We show that mature peripheral blood NK cells lack AHR, but its expression is induced by Stat3 during interleukin-21-driven activation and proliferation, coincident with increased NCAM1 (CD56) expression resulting in a CD56bright phenotype. Compared with control conditions, NK cells expanded in the presence of the AHR antagonist, StemRegenin-1, were unaffected in proliferation or cytotoxicity, had no increase in NCAM1 transcription, and maintained the CD56dim phenotype. However, it showed altered expression of 1004 genes including those strongly associated with signaling pathways. In contrast, NK cells expanded in the presence of the AHR agonist, kynurenine, showed decreased cytotoxicity and altered expression of 97 genes including those strongly associated with oxidative stress and cellular metabolism. By overlaying these differentially expressed genes with AHR chromatin binding, we identified 160 genes directly regulated by AHR, including hallmark AHR targets AHRR and CYP1B1 and known regulators of phenotype, development, metabolism, and function such as NCAM1, KIT, NQO1, and TXN. In summary, we define the AHR transcriptome in NK cells, propose a model of AHR and Stat3 coregulation, and identify potential pathways that may be targeted to overcome AHR-mediated immune suppression.

Genetic and epigenetic modification of human primary NK cells for enhanced antitumor activity.

Cancer immunotherapy using genetically modified immune cells such as those expressing chimeric antigen receptors has shown dramatic outcomes in patients with refractory and relapsed malignancies. Natural killer (NK) cells as a member of the innate immune system, possessing both anticancer (cytotoxic) and proinflammatory (cytokine) responses to cancers and rare off-target toxicities have great potential for a wide range of cancer therapeutic settings. Therefore, improving NK cell antitumor activity through genetic modification is of high interest in the field of cancer immunotherapy. However, gene manipulation in primary NK cells has been challenging because of broad resistance to many genetic modification methods that work well in T cells. Here we review recent successful approaches for genetic and epigenetic modification of NK cells including epigenetic remodeling, transposons, mRNA-mediated gene delivery, lentiviruses, and CRISPR gene targeting.

Efficient and Robust NK-Cell Transduction With Baboon Envelope Pseudotyped Lentivector.

NK-cell resistance to transduction is a major technical hurdle for developing NK-cell immunotherapy. By using Baboon envelope pseudotyped lentiviral vectors (BaEV-LVs) encoding eGFP, we obtained a transduction rate of 23.0 ± 6.6% (mean ± SD) in freshly-isolated human NK-cells (FI-NK) and 83.4 ± 10.1% (mean ± SD) in NK-cells obtained from the NK-cell Activation and Expansion System (NKAES), with a sustained transgene expression for at least 21 days. BaEV-LVs outperformed Vesicular Stomatitis Virus type-G (VSV-G)-, RD114- and Measles Virus (MV)- pseudotyped LVs (p < 0.0001). mRNA expression of both BaEV receptors, ASCT1 and ASCT2, was detected in FI-NK and NKAES, with higher expression in NKAES. Transduction with BaEV-LVs encoding for CAR-CD22 resulted in robust CAR-expression on 38.3 ± 23.8% (mean ± SD) of NKAES cells, leading to specific killing of NK-resistant pre-B-ALL-RS4;11 cell line. Using a larger vector encoding a dual CD19/CD22-CAR, we were able to transduce and re-expand dual-CAR-expressing NKAES, even with lower viral titer. These dual-CAR-NK efficiently killed both CD19KO- and CD22KO-RS4;11 cells. Our results suggest that BaEV-LVs may efficiently enable NK-cell biological studies and translation of NK-cell-based immunotherapy to the clinic.

Education-dependent activation of glycolysis promotes the cytolytic potency of licensed human natural killer cells.

BACKGROUND: The mechanism by which natural killer (NK) cell education results in licensed NK cells with heightened effector function against missing self-targets is not known. OBJECTIVE: We sought to identify potential mechanisms of enhanced function in licensed human NK cells. METHODS: We used expanded human NK cells from killer immunoglobulin-like receptor (KIR)/HLA-genotyped donors sorted for single-KIR+ cells to generate pure populations of licensed and unlicensed NK cells. We performed proteomic and gene expression analysis of these cells before and after receptor cross-linking and performed functional and metabolic analysis before and after interference with selected metabolic pathways. We verified key findings using freshly isolated and sorted NK cells from peripheral blood. RESULTS: We confirmed that licensed human NK cells are greater in number in peripheral blood and proliferate more in vitro than unlicensed NK cells. Using high-throughput protein analysis, we found that unstimulated licensed NK cells have increased expression of the glycolytic enzyme pyruvate kinase muscle isozyme M2 and after KIR cross-linking have increased phosphorylation of the metabolic modulators p38-α and 5' adenosine monophosphate-activated protein kinase α. After cytokine expansion and activation, unlicensed NK cells depended solely on mitochondrial respiration for cytolytic function, whereas licensed NK cells demonstrated metabolic reprogramming toward glycolysis and mitochondrial-dependent glutaminolysis, leading to accumulation of glycolytic metabolites and depletion of glutamate. As such, blocking both glycolysis and mitochondrial-dependent respiration was required to suppress the cytotoxicity of licensed NK cells. CONCLUSIONS: Collectively, our data support an arming model of education in which enhanced glycolysis in licensed NK cells supports proliferative and cytotoxic capacity.

TGFß Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion.

Transforming growth factor-beta (TGFß) is a potent immunosuppressive cytokine that inhibits the anti-tumor responses of NK cells and T cells. However, the stimulation of natural killer (NK) cells with pro-inflammatory cytokines decreases NK cell sensitivity to TGFß. Herein, we sought to determine if TGFß imprinting (TGFßi) during NK cell activation and expansion would decrease NK cell sensitivity to TGFß suppression. To this end, we demonstrate that the activation of NK cells during chronic IL-2 stimulation and TGFßi potently induces NK cell hypersecretion of interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) in response to tumor targets which persists for at least one month in vitro after the removal of TGFß. TGFßi NK cell cytokine hypersecretion is induced following both cytokine and tumor activation. Further, TGFßi NK cells have a marked suppression of SMAD3 and T-bet which is associated with altered chromatin accessibility. In contrast to their heightened cytokine secretion, TGFßi NK cells downregulate several activating receptors, granzyme and perforin, and upregulate TRAIL, leading to cell-line-specific alterations in cytotoxicity. These findings may impact our understanding of how TGFß affects NK cell development and anti-tumor function.

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins.

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGFß. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Detection of a MicroRNA molecular signature of ultraviolet radiation in the superficial regions of melanocytic nevi on sun-exposed skin.

How melanocytes transform into melanoma cells remains largely unknown. However, prolonged ultraviolet radiation exposure is linked with melanoma, and the DNA of melanomas arising in chronically sun-exposed skin is characterized by an elevated number of pyrimidine transitions, mainly C>T (predominantly caused by ultraviolet B), and transversions of GC>TA or AT>CG (caused by ultraviolet A over indirect mechanisms). Since ultraviolet penetrates mostly only the superficial dermis, we sought to determine the extent to which superficial and deep melanocytes of nevi in sun-exposed skin differ in their miRNA expression and consider the changes as likely secondary to ultraviolet radiation-induced damage. The differentially expressed miRNAs were analyzed for known potential oncomiRs or linked to potential oncogenes or tumor suppressors. Superficial and deep melanocytes were microdissected from the nevi of 14 patients. The suspensions were processed for hybridization to a ribonucleotide protection system with 2280 total probes, including 2256 miRNA probes targeting 2083 human miRNAs. A comprehensive analysis of all human miRNAs registered in miRBase 11.0 was performed using the HTG Molecular Diagnostic database. Statistical analysis of these data yielded for 14 samples a statistically relevant profile of 39 miRNA species at FDR<0.1 that were differentially expressed between superficial and deep melanocytes. Ingenuity Pathway Analysis based on the expression data of these 39 miRNAs suggested the gene transcripts AR, MDM2, SMAD2/3, and YBX1 as the most probable miRNA targets, which were validated at the protein level. Our findings suggest that superficial ultraviolet radiation-damaged melanocytes can be differentiated from deep melanocytes on the basis of the expression of 39 miRNAs, the most probable gene transcript and protein targets of which are AR, MDM2, SMAD2/3, and YBX1, with YBX1 expression validating the best the molecular signature in immunohistochemical analysis.

Summary of expression of SPARC protein in cutaneous vascular neoplasms and mimickers.

BACKGROUND: Serum protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein, which regulates cell proliferation and facilitates intracellular transport of albumin bound particles including chemotherapeutic agents such as Nab-paclitaxel/ABI-007. Therefore the presence of SPARC may achieve higher intra-tumoral drug concentration with lower dosage and thus reduce systemic side-effects. Several trials of ABI-007, in melanoma, show promising clinical activity. DESIGN: Fifty-four cases of dermal based neoplasms were retrieved including 24 angiosarcomas (AS), 10 hemangiomas, 9 nodular melanomas, 4 Kaposi sarcomas (KS), 3 leiomyosarcomas (LMS), 3 atypical fibroxanthomas (AFX) and 1 spindle cell squamous cell carcinoma (SSCC). SPARC immunohistochemistry (IHC) was performed with a mouse monoclonal antibody. RESULTS: SPARC expression was detected in a majority of AS (17/24), melanomas (8/9), AFX (3/3), LMS (3/3) and KS (4/4) with some expression in hemangiomas (3/10), while being negative in SSCC (0/1); and was significantly associated with tumor group (p = 0.017). Although a significant difference in overall survival was observed between SPARC expression groups (positive vs. negative) for all patients, there was no significant difference noted among angiosarcoma patients. CONCLUSION: We have confirmed the presence of SPARC expression in melanoma, KS, LMS and AS and also detected it for the first time in AFX. Since paclitaxel has shown some effectiveness in AS, melanoma and KS, ABI-007 could also be beneficial in these patients.

Diminished microRNA-29b level is associated with BRD4-mediated activation of oncogenes in cutaneous T-cell lymphoma.

MicroRNA (miRNA) dysregulation is a hallmark of cutaneous T-cell lymphoma (CTCL), an often-fatal malignancy of skin-homing CD4+ T cells for which there are few effective therapies. The role of microRNAs (miRs) in controlling epigenetic modifier-dependent transcriptional regulation in CTCL is unknown. In this study, we characterize a novel miR dysregulation that contributes to overexpression of the epigenetic reader bromodomain-containing protein 4 (BRD4). We used patient CD4+ T cells to show diminished levels of miR-29b compared with healthy donor cells. Patient cells and miR-29b-/- mouse cells revealed an inverse relationship between miR-29b and BRD4, the latter of which is overexpressed in these cells. Chromatin immunoprecipitation and sequencing analysis revealed increased genome-wide BRD4 occupancy at promoter and enhancer regions in CD4+ T cells from CTCL patients. The cumulative result of BRD4 binding was increased expression of tumor-associated genes such as NOTCH1 and RBPJ, as well as the interleukin-15 (IL-15) receptor complex, the latter enhancing IL-15 autocrine signaling. Furthermore, we confirm the in vivo relevance of this pathway in our IL-15 transgenic mouse model of CTCL by showing that interference with BRD4-mediated pathogenesis, either by restoring miR-29b levels via bortezomib treatment or by directly inhibiting BRD4 binding via JQ1 treatment, prevents progression of CTCL. We describe a novel oncogenic pathway featuring IL-15, miR-29b, and BRD4 in CTCL and suggest targeting of these components as a potentially effective therapy for CTCL patients.

High cytotoxic T-lymphocyte-associated antigen 4 and phospho-Akt expression in tumor samples predicts poor clinical outcomes in ipilimumab-treated melanoma patients.

Ipilimumab, a fully human monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), is the first immune checkpoint inhibitor approved for the treatment of unresectable melanoma on the basis of its overall survival (OS) benefit. However, ipilimumab is associated with significant immune-related adverse events. We hypothesized that biomarker exploration of pretreatment tumor samples and correlation with clinical outcome would enable patient selection with an increased benefit/risk ratio for ipilimumab therapy. At the University of Texas MD Anderson Cancer Center, a total of 81 advanced melanoma patients were treated on the Ipilimumab Expanded Access Program from 2007 to 2008. Using immunohistochemistry, we analyzed the expression of immune checkpoint (CTLA-4, PD-1, PD-L1) and Akt-pathway proteins in formalin-fixed tumor tissue. Associations between these biomarkers and progression-free survival (PFS) and OS were analyzed with univariate and multivariate Cox proportional-hazards models. There was a significant correlation between high CTLA-4 protein expression levels in tumor cells and risk of death (P=0.02) and decreased PFS (P=0.023). In addition, high expression of CTLA-4 in peritumoral lymphocytes correlated with poor OS (P=0.023). In multivariate analysis, patients with high CTLA-4 and phospho-Akt (p-Akt) expression correlated with poor OS (log-rank test, P=0.039) and PFS (log-rank test, P=0.014). High levels of CTLA-4 and p-Akt expression in pretreatment tumor cells in melanoma patients were associated with poor clinical outcomes. Immunohistochemistry analysis of CTLA-4 and p-Akt in pretreatment tumor samples provides useful biomarkers that may enable improved patient selection for ipilimumab therapy. Prospective clinical studies are warranted to investigate the predictive value of these biomarkers.

NFAT1 Directly Regulates IL8 and MMP3 to Promote Melanoma Tumor Growth and Metastasis.

Nuclear factor of activated T cell (NFAT1, NFATC2) is a transcription factor that binds and positively regulates IL2 expression during T-cell activation. NFAT1 has important roles in both innate and adaptive immune responses, but its involvement in cancer is not completely understood. We previously demonstrated that NFAT1 contributes to melanoma growth and metastasis by regulating the autotaxin gene (Enpp2). Here, we report a strong correlation between NFAT1 expression and metastatic potential in melanoma cell lines and tumor specimens. To elucidate the mechanisms underlying NFAT1 overexpression during melanoma progression, we conducted a microarray on a highly metastatic melanoma cell line in which NFAT1 expression was stably silenced. We identified and validated two downstream targets of NFAT1, IL8, and MMP3. Accordingly, NFAT1 depletion in metastatic melanoma cell lines was associated with reduced IL8 and MMP3 expression, whereas NFAT1 overexpression in a weakly metastatic cell line induced expression of these targets. Restoration of NFAT1 expression recovered IL8 and MMP3 expression levels back to baseline, indicating that both are direct targets of NFAT1. Moreover, in vivo studies demonstrated that NFAT1 and MMP3 promoted melanoma tumor growth and lung metastasis. Collectively, our findings assign a new role for NFAT1 in melanoma progression, underscoring the multifaceted functions that immunomodulatory factors may acquire in an unpredictable tumor microenvironment. Cancer Res; 76(11); 3145-55. ©2016 AACR.

Predictive factors of activity of anti-programmed death-1/programmed death ligand-1 drugs: immunohistochemistry analysis.

Anti-programmed death-1 (anti-PD1)/programmed death ligand-1 (PD-L1) therapeutic antibodies targeting regulatory pathways in T cells have recently shown to promising clinical effectiveness in several solid tumors by enhancing antitumor immune response. Immune checkpoint therapy has propelled therapeutic efforts opening a new field in cancer treatment. However, durable clinical response has been educed only in a fraction of patients, underlining the need to predictively select those patients most likely to respond, e.g., by detecting predictive biomarkers. Immunohistochemistry (IHC) detection of PD-L1 in tumor cells has been used in various trials of anti-PD-1/PD-L1 agents to try to select those patients most likely to respond. However, since there are different techniques and scoring systems, results have not been conclusive. Thus efforts are needed to develop standardized IHC assays as well as to explore additional biomarkers to evaluate and predict immune responses elicited by anti-PD-1/PD-L1 therapies.

Molecular profiling of patient-matched brain and extracranial melanoma metastases implicates the PI3K pathway as a therapeutic target.

PURPOSE: An improved understanding of the molecular pathogenesis of brain metastases, one of the most common and devastating complications of advanced melanoma, may identify and prioritize rational therapeutic approaches for this disease. In particular, the identification of molecular differences between brain and extracranial metastases would support the need for the development of organ-specific therapeutic approaches. EXPERIMENTAL DESIGN: Hotspot mutations, copy number variations (CNV), global mRNA expression patterns, and quantitative analysis of protein expression and activation by reverse-phase protein array (RPPA) analysis were evaluated in pairs of melanoma brain metastases and extracranial metastases from patients who had undergone surgical resection for both types of tumors. RESULTS: The status of 154 previously reported hotspot mutations, including driver mutations in BRAF and NRAS, were concordant in all evaluable patient-matched pairs of tumors. Overall patterns of CNV, mRNA expression, and protein expression were largely similar between the paired samples for individual patients. However, brain metastases demonstrated increased expression of several activation-specific protein markers in the PI3K/AKT pathway compared with the extracranial metastases. CONCLUSIONS: These results add to the understanding of the molecular characteristics of melanoma brain metastases and support the rationale for additional testing of the PI3K/AKT pathway as a therapeutic target in these highly aggressive tumors.

MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth.

Some solid tumors have reduced posttranscriptional RNA editing by adenosine deaminase acting on RNA (ADAR) enzymes, but the functional significance of this alteration has been unclear. Here, we found the primary RNA-editing enzyme ADAR1 is frequently reduced in metastatic melanomas. In situ analysis of melanoma samples using progression tissue microarrays indicated a substantial downregulation of ADAR1 during the metastatic transition. Further, ADAR1 knockdown altered cell morphology, promoted in vitro proliferation, and markedly enhanced the tumorigenicity in vivo. A comparative whole genome expression microarray analysis revealed that ADAR1 controls the expression of more than 100 microRNAs (miRNAs) that regulate many genes associated with the observed phenotypes. Importantly, we discovered that ADAR1 fundamentally regulates miRNA processing in an RNA binding­dependent, yet RNA editing­independent manner by regulating Dicer expression at the translational level via let-7. In addition, ADAR1 formed a complex with DGCR8 that was mutually exclusive with the DGCR8-Drosha complex that processes pri-miRNAs in the nucleus. We found that cancer cells silence ADAR1 by overexpressing miR-17 and miR-432, which both directly target the ADAR1 transcript. We further demonstrated that the genes encoding miR-17 and miR-432 are frequently amplified in melanoma and that aberrant hypomethylation of the imprinted DLK1-DIO3 region in chromosome 14 can also drive miR-432 overexpression.