Introduction
Representing ten percent of hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to therapy-resistant ...plasma cell leukemia (PCL). Despite many novel therapies, relapse rates remain high as a result of malignant regeneration (self-renewal) of MM cells in inflammatory microenvironments. In addition to recurrent DNA mutations and epigenetic deregulation, inflammatory cytokine-responsive adenosine deaminase associated with RNA (ADAR1) mediated adenosine to inosine (A-to-I) RNA editing has emerged as a key driver of cancer relapse and progression. In MM, copy number amplification of chromosome 1q21, which contains both ADAR1 and interleukin-6 receptor (IL-6R) gene loci, portends a poor prognosis. Thus, we hypothesized that ADAR1 copy number amplification combined with inflammatory cytokine activation of ADAR1 stimulate malignant regeneration of MM and therapeutic resistance.
Methods and Results
Analysis of MMRF CoMMpass RNA sequencing (RNA-seq) data revealed that high ADAR1 expression (n=162 patients) correlated with significantly reduced progression-free and overall survival compared with a low ADAR1 subset (n=159 patients). In contrast to lentiviral ADAR1 shRNA knockdown and overexpression of an editase defective ADAR1 mutant (ADAR1E912A), lentiviral wild-type ADAR1 overexpression enhanced editing of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist. Editing of GLI1 transcripts enhanced GLI transcriptional activity in luciferase reporter assays, and promoted lenalidomide resistance in vitro . Finally, lentiviral shRNA ADAR1 knockdown reduced regeneration of high-risk MM in humanized serial transplantation mouse models indicative of reduced malignant self-renewal capacity. These data demonstrate that ADAR1 promotes malignant self-renewal of MM and if selectively inhibited may prevent progression and relapse.
Conclusions
Deregulated RNA editing, driven by aberrant ADAR1 activation, represents a unique source of transcriptomic and proteomic diversity, resulting in self-renewal of MM cells in inflammatory microenvironments. In summary, both genetic (1q21 amplification) and microenvironmental factors (inflammatory cytokines, IMiDs) combine to drive GLI1-dependent malignant regeneration in MM. Thus, ADAR1 represents both a vital prognostic biomarker and therapeutic target in MM.
Stewart:Amgen: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Janssen: Consultancy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Introduction
Multiple myeloma (MM) is a plasma cell malignancy that accounts for more than 10% of all blood cancers and may progress to plasma cell leukemia (PCL). Despite treatment, ...virtually all patients become unresponsive to treatment. RNA editing is a post-transcriptional pre-mRNA processing activity that represents an unexplored potential source of clonal molecular heterogeneity contributing to therapeutic resistance. In particular, adenosine deaminase acting on RNA (ADAR) 1, which exists in two isoforms, one constitutive and one inflammation-responsive, has been associated with disease progression and cancer stem cell (CSC) maintenance. The aim of this study was to investigate whether enhanced ADAR1 expression and activity contributed to therapeutic resistance of MM and PCL.
Procedures
1) ADAR Quantification: Whole gene and isoform-specific qRT-PCR was used to detect ADAR1 expression in PCL and MM primary samples and in human MM cell lines (HMCL).
2) RNA Editing Detection: We developed a RNA editing site-specific qPCR (RESS-qPCR) assay to detect RNA editing in cancer stem-cell associated transcripts.
3) Therapeutic Resistance Assay. A MM cell line was exposed to lenalidomide continuously in vitro to establish a model of therapeutic resistance.
4) Development of a humanized PCL mouse model: We established novel in vivo PCL primagrafts by intrahepatic transplantation of primary total mononuclear cells into neonatal RAG2-/-gc-/- mice.
Results
Approximately, 30% of MM patients in the MM Genomic Initiative dataset harbor copy number amplifications of the ADAR locus on chromosome 1q21, which portends a poor prognosis. We observed significantly increased ADAR1 expression in primary PCL samples and aberrant RNA editing of the stem cell transcription factor GLI1 and the DNA cytidine deaminase APOBEC3D. Notably, high-ADAR1-expressing PCL cells successfully engrafted in RAG2-/-gc-/- mice. As the inflammation-responsive isoform of ADAR1 was upregulated in primary samples, we sought to explore the effects of the anti-MM agent and immunomodulatory drug lenalidomide on ADAR1 expression and activity. Continuous in vitro exposure to lenalidomide led to increased ADAR1 mRNA and protein level and a potent induction of RNA editing activity. Increased RNA editing was detected in several cancer and stem cell-associated transcripts, including GLI1, APOBEC3D, AZIN1 and MDM2. Notably, this aberrant RNA editing activity was associated with increased self-renewal capacity in vitro and a cancer stem cell phenotype.
Conclusions
ADAR1 overexpression and deregulated RNA editing represents a unique source of RNA and proteomic diversity, and may confer a survival and self-renewal advantage to MM cells. This research identifies ADAR1 as a new diagnostic and therapeutic target in MM, and establishes a robust humanized PCL primagraft model for future pre-clinical testing of ADAR1 modulatory agents.
Citation Format: Elisa Lazzari, Leslie A. Crews, Christina Wu, Heather Leu, Shawn Ali, Raffaella Chiaramonte, Mark Minden, Caitlin Costello, Catriona H.M. Jamieson. ADAR1-dependent RNA editing is a mechanism of therapeutic resistance in human plasma cell malignancies. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2414.
Notch history begins in 1919 with Thomas Hunt Morgan studies on fruit fly mutants. From then, this gene aroused lively interest in the scientific community since it is involved in a wide variety of ...processes, including morphogenesis, tissue homeostasis, and stem cell maintenance. Deregulation of Notch signaling characterizes several human tumors. Hematopoietic system is affected by mutations of Notch receptors, Notch ligands, and proteins controlling their stability. Approximately 60% T acute lymphoblastic leukemia (T-ALL) patients carry activating Notch1 mutations prompting blasts growth. In addition, multiple myeloma is characterized by Notch signaling hyper-activation due to an abnormal expression of the Jagged2 ligand; this affects not only myeloma cells, but also their interaction with bone marrow microenvironment, influencing tumor burden and bone disease. These findings make Notch a rational target of a therapeutic approach. Inhibitors of the Notch activating enzyme, ?-Secretase, have been successfully used in vitro and in vivo and are currently under clinical trials for T-ALL and breast cancer. Yet a wide use of these inhibitors is prevented by frequently occurring drug resistance. To elucidate the mechanism underlying this phenomenon, a number of pathways have been identified mediating Notch biological effects: AKT and c-Myc are frequently deregulated in leukemic patients and account for resistance to ?-Secretase inhibitors by acting downstream Notch receptor. Therefore, the interaction of Notch with other cancer-associated proteins should be clarified to predict the biological outcome of a Notch targeted therapy and possibly, to exploit combined treatments against the key deregulated elements in Notch-associated cancers.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Breast carcinoma is a major health issue for millions of women. Current therapies have serious side effects, and are only partially effective in patients with metastatic tumors. Thus, the need for ...novel and less toxic therapies is urgent. Moreover, hormonal and antibody therapies effective in other subtypes are not effective in Triple Negative Breast Cancer (TNBC). Immunotherapeutic strategies directed against specific tumor-associated antigens (TAAs) and mediated by specific cytotoxic T lymphocytes (CTL) have been largely underexplored in this disease. Cancer-testis antigens (CTA) are a group of TAAs displaying the ideal characteristics of promising vaccine targets,
strong immunogenicity and cancer specificity. The CTA, Sperm Protein 17 (SP17), has been found to be aberrantly expressed in different neoplasms, including ovarian and esophageal cancers, nervous system tumors and multiple myeloma, and has been suggested as a candidate target for immunotherapy. Here, we evaluated SP17 expression levels in breast cancer cell lines, invasive ductal breast carcinoma, including patients with TNBC, and adjacent non-neoplastic breast tissue, and determined whether SP17 was capable of generating SP17-specific cytotoxic T lymphocytes
. We showed that SP17 is expressed in breast cancer cell lines and primary breast tumors and importantly in TNBC subtype, but not in adjacent non-tumoral breast tissue or unaffected tissues, except in male germinal cells. Furthermore, we detected specific anti-SP17 antibodies in patients' sera and we generated SP17-specific, HLA class I-restricted, cytotoxic T lymphocytes capable of efficiently killing breast cancer cells.
Interactions of multiple myeloma (MM) cells with endothelial cells (ECs) enhance angiogenesis and MM progression. Here, we investigated the role of Notch signaling in the cross talk between ECs and ...MM cells enabling angiogenesis. MMECs showed higher expression of Jagged1/2 ligands, of activated Notch1/2 receptors, and of Hes1/Hey1 Notch target genes than ECs from monoclonal gammopathy of undetermined significance patients, suggesting that homotypic activation of Notch pathway occurs in MM. MM cells co-cultured with MMECs triggered Notch activation in these cells through a cell-to-cell contact-dependent way
via
Jagged1/2, resulting in Hes1/Hey1 overexpression. The angiogenic effect of Notch pathway was analyzed through Notch1/2·siRNAs and the γ-secretase inhibitor MK-0752 by
in vitro
(adhesion, migration, chemotaxis, angiogenesis) and
in vivo
(Vk12598/C57B/6 J mouse model) studies. Activated Notch1/2 pathway was associated with the overangiogenic MMEC phenotype: Notch1/2 knockdown or MK-0752 treatment reduced Hes1/Hey1 expression, impairing
in vitro
angiogenesis of both MMECs alone and co-cultured with MM cells. MM cells were unable to restore angiogenic abilities of treated MMECs, proving that MMEC angiogenic activities closely rely on Notch pathway. Furthermore, Notch1/2 knockdown affected VEGF/VEGFR2 axis, indicating that the Notch pathway interferes with VEGF-mediated control on angiogenesis. MK-0752 reduced secretion of proangiogenic/proinflammatory cytokines in conditioned media, thus inhibiting blood vessel formation in the CAM assay. In the Vk12598/C57B/6 J mouse, MK-0752 treatment restrained angiogenesis by reducing microvessel density. Overall, homotypic and heterotypic Jagged1/2-mediated Notch activation enhances MMECs angiogenesis. Notch axis inhibition blocked angiogenesis
in vitro
and
in vivo
, suggesting that the Notch pathway may represent a novel therapeutic target in MM.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Notch receptors are transmembrane proteins critically determining cell fate and maintenance of progenitor cells in many developmental systems. Notch signaling is involved in stem cell self-renewal ...and regulates the main functions of cell life at different levels of development: cell proliferation, differentiation and apoptosis. By virtue of its involvement in the regulation of cell physiology, it is not surprising that a deregulation of the Notch pathway leads to the development of different tumors. In this review, we critically discuss the latest findings concerning Notch roles in hematologic oncology, with a special focus on T-cell acute lymphoblastic leukemia and B-cell malignancies. We also describe the molecular mediators of Notch-driven oncogenic effects and the current pharmacological approaches targeting Notch signaling.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Lung cancer is the main cause of cancer mortality worldwide. This is mainly due to the fact that it is diagnosed in advanced stage patients, which are no more surgically curable. Consequently, ...searching for novel treatments and new modalities for early diagnosis offers great promise to improve the clinical outcome. Recently, a new group of antigens, the cancer testis antigens, have been described as possible early diagnostic tools and therapeutic targets in cancer therapy.This review will report emerging evidences of cancer testis antigens deregulation in lung cancer and explore the state of the art of their currently known role and potential as markers for early diagnosis and disease progression and targets of an immunotherapeutic approach aiming to improve the cure rate of this tumor.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
NOTCH1 is involved in the pathogenesis of T-acute lymphoblastic leukemia (T-ALL) carrying the very rare translocation t(7;9)(q34;q34.3).
We analyzed the expression of genes belonging to NOTCH ...pathway, in acute leukemia primary samples and lymphoblastoid cell lines. NOTCH1 pathway activation represents a common feature of T-ALL when compared to acute myelogenous leukemia (AML) and B-cell precursor acute lymphoblastic leukemia. The contemporary expression of
NOTCH1 and its ligands on cell surface contributes to high levels of pathway activity. AML primary samples show high levels of
JAGGED1 expression despite the low NOTCH1 pathway activation, consistent with an autonomous JAGGED1 signaling in myeloid leukemogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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