Elisa Lazzari

Alu-dependent RNA editing of GLI1 promotes malignant regeneration in multiple myeloma

Despite novel therapies, relapse of multiple myeloma (MM) is virtually inevitable. Amplification of chromosome 1q, which harbors the inflammation-responsive RNA editase adenosine deaminase acting on RNA (ADAR)1 gene, occurs in 30–50% of MM patients and portends a poor prognosis. Since adenosine-to-inosine RNA editing has recently emerged as a driver of cancer progression, genomic amplification combined with inflammatory cytokine activation of ADAR1 could stimulate MM progression and therapeutic resistance. Here, we report that high ADAR1 RNA expression correlates with reduced patient survival rates in the MMRF CoMMpass data set. Expression of wild-type, but not mutant, ADAR1 enhances Alu-dependent editing and transcriptional activity of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist, and promotes immunomodulatory drug resistance in vitro. Finally, ADAR1 knockdown reduces regeneration of high-risk MM in serially transplantable patient-derived xenografts. These data demonstrate that ADAR1 promotes malignant regeneration of MM and if selectively inhibited may obviate progression and relapse.The treatment of multiple myeloma is challenging due to high relapse rates. Here the authors show that expression of ADAR1 correlates with poor patient outcomes, and that ADAR1-mediated editing of GLI1 is a mechanism relevant in the context of multiple myeloma progression and drug resistance.

Multiple myeloma-derived Jagged ligands increases autocrine and paracrine interleukin-6 expression in bone marrow niche

Multiple myeloma cell growth relies on intrinsic aggressiveness, due to a high karyotypic instability, or on the support from bone marrow (BM) niche. We and other groups have provided evidences that Notch signaling is related to tumor cell growth, pharmacological resistance, localization/recirculation in the BM and bone disease. This study indicates that high gene expression levels of Notch signaling members (JAG1, NOTCH2, HES5 and HES6) correlate with malignant progression or high-risk disease, and Notch signaling may participate in myeloma progression by increasing the BM levels of interleukin-6 (IL-6), a major player in myeloma cell growth and survival. Indeed, in vitro results, confirmed by correlation analysis on gene expression profiles of myeloma patients and immunohistochemical studies, demonstrated that Notch signaling controls IL-6 gene expression in those myeloma cells capable of IL-6 autonomous production as well as in surrounding BM stromal cells. In both cases Notch signaling activation may be triggered by myeloma cell-derived Jagged ligands. The evidence that Notch signaling positively controls IL-6 in the myeloma-associated BM makes this pathway a key mediator of tumor-directed reprogramming of the bone niche. This work strengthens the rationale for a novel Notch-directed therapy in multiple myeloma based on the inhibition of Jagged ligands.

PI3K/AKT signaling inhibits NOTCH1 lysosome‐mediated degradation

The pathways of NOTCH and PI3K/AKT are dysregulated in about 60% and 48% of T‐cell acute lymphoblastic leukemia (T‐ALL) patients, respectively. In this context, they interact and cooperate in controlling tumor cell biology. Here, we propose a novel mechanism by which the PI3K/AKT pathway regulates NOTCH1 in T‐ALL, starting from the evidence that the inhibition of PI3K/AKT signaling induced by treatment with LY294002 or transient transfection with a dominant negative AKT mutant downregulates NOTCH1 protein levels and activity, without affecting NOTCH1 transcription. We showed that the withdrawal of PI3K/AKT signaling was associated to NOTCH1 phosphorylation in tyrosine residues and monoubiquitination of NOTCH1 detected by Ubiquitin capture assay. Co‐immunoprecipitation assay and colocalization analysis further showed that the E3 ubiquitin ligase c‐Cbl interacts and monoubiquitinates NOTCH1, activating its lysosomal degradation. These results suggest that the degradation of NOTCH1 could represent a mechanism of control by which NOTCH1 receptors are actively removed from the cell surface. This mechanism is finely regulated by the PI3K/AKT pathway in physiological conditions. In pathological conditions characterized by PI3K/AKT hyperactivation, such as T‐ALL, the excessive AKT signaling could lead to NOTCH1 signaling dysregulation. Therefore, a therapeutic strategy directed to PI3K/AKT in T‐ALL could contemporaneously inhibit the dysregulated NOTCH1 signaling.

Abstract 3351: Aberrant RNA editing of GLI1 promotes malignant regeneration in multiple myeloma

Introduction: Despite novel therapies, most of multiple myeloma (MM) patients relapse as a result of clonal evolution in inflammatory microenvironments. Adenosine-to-inosine (A-to-I) RNA editing, driven by inflammatory cytokine-responsive adenosine deaminase acting on RNA1 (ADAR1), promotes cancer progression by enhancing survival and self-renewal of malignant progenitor cells. Amplifications of chromosome 1q21, containing IL-6R and ADAR1 loci, occur frequently in high-risk MM patients, who frequently develop secondary plasma cell leukemia (PCL) and have shorter survival. While increased IL-6 signaling has been linked to relapse and A-to-I editing contributes to therapeutic resistance in a broad array of malignancies, the role of ADAR1 in MM pathogenesis has not been elucidated. This study aimed to investigate whether pro-inflammatory cues in MM activate ADAR1 editing thereby promoting malignant regeneration. Procedures: Publicly available primary patient datasets were analyzed and validated in a separate cohort of biobanked primary samples and human myeloma cell lines. Lentiviral vector-mediated activation or knockdown of ADAR1, or treatment with extrinsic pro-inflammatory stimuli, was utilized to probe the functional impact of RNA editing activity in MM models. Site-specific qPCR was used to quantify RNA editing in specific cancer stem cell-associated loci. Functional effects of ADAR1 activity were assessed in in vitro survival and self-renewal assays, and in novel in vivo PCL xenografts. Results: Patients harboring 1q21 amplification showed significant and stage-dependent increases in ADAR1 expression. In a set of separate primary PCL samples, aberrant RNA editing in the coding region of the Hedgehog (Hh) pathway transcription factor GLI1 was observed in high ADAR1-expressing samples. Notably, increased GLI1 editing, previously reported to have increased capacity to activate its transcriptional targets, was detected in serially transplantable, patient-derived xenograft models. Furthermore, abolition of ADAR1 editase activity impaired GLI1 editing. Lastly, in vitro pro-inflammatory IL-6 stimulation, or continuous exposure to the immunomodulatory drug lenalidomide led to increased ADAR1 mRNA and protein levels, with a concomitant induction of RNA editing activity. Conclusions: In MM, 1q21 amplification has been linked to progression. We provide new evidence linking expression and activity of ADAR1, located on 1q21, and disease stage. Because ADAR1 induces transcript recoding, A-to-I editing could contribute to the marked transcriptomic diversity typical of advanced MM. While the Hh pathway has been linked to cancer stem cell generation in human MM, here we identified a primate-specific mechanism of Hh pathway activation in MM through RNA editing-dependent stabilization of GLI1. Together, both genetic and microenvironmental factors modulate epitranscriptomic deregulation of cancer stem cell pathways in MM. Citation Format: Elisa Lazzari, Nathaniel Delos Santos, Christina Wu, Heather Leu, Gabriel Pineda, Shawn Ali, Caitlin Costello, Mark Minden, Raffaella Chiaramonte, Leslie Crews, Catriona Jamieson. Aberrant RNA editing of GLI1 promotes malignant regeneration in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3351. doi:10.1158/1538-7445.AM2017-3351

Abstract 4437: Down-modulation of ADAR1-mediated GLI1 editing alters extracellular and immune response genes in multiple myeloma

Introduction: Representing 10% 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 stimulates 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 (ADAR1 E912A ), 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. Whole-transcriptome RNA-sequencing of primary samples after lentiviral shRNA knockdown of ADAR1 revealed specific modulation of extracellular and immune response genes, while overexpression of wild-type versus edited GLI1 elicited distinct gene expression changes in human myeloma cells analyzed using NanoString nCounter assays. These data demonstrate that ADAR1 promotes malignant self-renewal of MM and, if selectively inhibited, may prevent progression and relapse through modulation of extracellular and immune response genes. 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. Citation Format: Leslie A. Crews, Elisa Lazzari, Phoebe K. Mondala, Nathaniel Delos Santos, Amber Miller, Gabriel Pineda, Qingfei Jiang, Anusha-Preethi Ganesan, Christina Wu, Caitlin Costello, Mark Minden, Raffaella Chiaramonte, A. Keith Stewart, Catriona H. M. Jamieson. Down-modulation of ADAR1-mediated GLI1 editing alters extracellular and immune response genes in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4437.

Abstract LB-025: The role of NOTCH pathway in multiple myeloma associated drug resistance

The aim of this study was to investigate the role of Notch signaling in intrinsic and bone marrow stromal cells (BMSC)-mediated drug resistance in multiple myeloma (MM) and in MM-stem cell (SC) niche maintenance. MM is an incurable hematological malignancy due to intrinsic or BMSC-mediated drug resistance; the hyperexpression of two Notch ligands, Jag1 and 2 in MM increases Notch signaling in MM cells and BMSCs resulting in malignant cells survival and proliferation. Notch pathway supports stem cell maintenance and drug resistance is an intrinsic feature of cancer stem cells; MM stem cells (MM-SCs) have been characterized as CD138- subpopulation. MM-SCs are resistant to common drugs used in therapy and responsible for disease relapse. MM cell lines were cultured alone or co-cultured with NIH3T3 murine fibroblasts or HS5 human BMSC line. To detect apoptosis induced by Mitoxantrone, Bortezomib and Melphalan, AnnexinV+ cells were processed by flow cytometry (FC). Jag1 and Jag2 were transiently silenced in MM cells using specific siRNAs. The gene expression levels were analyzed by quantitative RT-PCR. Anti-apoptotic proteins were assessed by FC. Notch inhibition was obtained by γ-secretase inhibitor and the effect on MM cell stemness potential of was assessed by FC measure of CD138- MM cells or clonogenic serial replating in methylcellulose-based medium. Our results demonstrate that Jag1 and 2 silencing reduces anti-apoptotic genes expression, i.e. SDF1α, CXCR4, Bcl-XL, Bcl2, Survivin and ABCC1 and increases sensitivity of MM cells to the used drugs. MM cells and BMSCs reciprocally activate Notch signaling resulting in increased drug resistance due to: i) an elevated expression of the anti-apoptotic genes in MM cells; ii) BMSCs release of soluble factors, i.e. SDF1α and VEGF, relevant for MM cell growth and survival. Interestingly, Jag1 and 2 silencing in MM cells co-cultured with BMSCs could reverse all gene and protein expression changes as well as BMSCs protective effect increasing the apoptotic rate of MM cells. In addition, we show in MM cell lines that DAPT-mediated Notch inhibition decreases MM-SCs and reduces the clonogenic ability in serial replating. The evidence that Jag1 and 2 silencing affects the intrinsic and BMSC-induced drug resistance in MM cells also by affecting the MM-SC population supports the rationale for a Notch-tailored approach to overcome the unavoidable relapse pf MM patient. Citation Format: Silvia Garavelli, Elisa Lazzari, Michela Colombo, Natalia Platonova, Maria Teresa Palano, Francesco Baccianti, Serena Galletti, Antonino Neri, Leslie Crews, Catriona Jamieson, Raffaella Chiaramonte. The role of NOTCH pathway in multiple myeloma associated drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-025. doi:10.1158/1538-7445.AM2017-LB-025

Tracking of Normal and Malignant Progenitor Cell Cycle Transit in a Defined Niche.

While implicated in therapeutic resistance, malignant progenitor cell cycle kinetics have been difficult to quantify in real-time. We developed an efficient lentiviral bicistronic fluorescent, ubiquitination-based cell cycle indicator reporter (Fucci2BL) to image live single progenitors on a defined niche coupled with cell cycle gene expression analysis. We have identified key differences in cell cycle regulatory gene expression and transit times between normal and chronic myeloid leukemia progenitors that may inform cancer stem cell eradication strategies.

Abstract 2414: ADAR1-dependent RNA editing is a mechanism of therapeutic resistance in human plasma cell malignancies

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.