Nathaniel Delos Santos

ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.

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.

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.

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.