Tracking Human Myeloma Cell Therapeutic Resistance Using a Novel Diagnostic Assay for Detection of RNA Editing Biomarkers of Cancer Stem Cell Generation
Authors: Lazzari Elisa , Crews Leslie A , Zipeto Maria Anna , Ali Shawn , Jamieson Catriona HM 
Published: October 2019

Abstract
INTRODUCTION
Multiple myeloma is a heterogeneous hematopoietic malignancy that represents 10% of all blood cancers and is characterized by malignant plasma cell expansion in the bone marrow, which is rich in pro-inflammatory cytokines such as interleukin-6 (IL-6). The emergence of therapeutically recalcitrant disease even following intensive treatment suggests the existence of a drug-resistant myeloma-initiating population. These myeloma-initiating cells are thought to co-opt stem cell pathways that enhance their capacity to self-renew and become dormant in protective niches. Recently, we and others showed that aberrant RNA editing plays a key role in malignant transformation through activation of the inflammation-responsive RNA editase ADAR1. In human leukemia stem cells, ADAR1 activation is associated with enhanced self-renewal of dormant progenitors, while lentiviral-shRNA ADAR1 knockdown reduces malignant stem cell self-renewal in vivo. Notably, in 30% of multiple myeloma cases copy number amplification of the ADAR locus on chromosome 1q21, which portends a poor prognosis. Thus, the goal of this study was to investigate whether ADAR1-mediated RNA editing in multiple myeloma occurs as a result of chromosome amplification and pro-inflammatory signaling, and to evaluate the effects of prolonged immunomodulatory therapy on ADAR1 activation leading to the emergence of drug-resistant myeloma-initiating cells.
METHODS AND RESULTS
Increased ADAR1 expression in myeloma patient samples
To determine whether gene expression of ADAR1 correlates with chromosome 1q21 amplification, we evaluated existing microarray datasets (Multiple Myeloma Genomics Initiative) from well-characterized patient samples harboring 2 to ≥4 copies of the CKS1B gene, which is located within 500kB adjacent to ADAR as well as the interleukin-6 (IL-6) receptor locus IL6R on chromosome 1q21. Notably, gain of 1q21 copy number is associated with more proliferative disease and poor-risk cytogenetics, which could also be related to ADAR activation. Interestingly, our analysis of ADAR1 gene expression showed increased ADAR1 levels in patients with a CKS1B score of 4 or greater (n=6) compared with patients with a score of 2 (n=18).
IL-6 mediated induction of RNA editing activity
To evaluate the role of pro-inflammatory cytokine stimulation of ADAR1 in the chromosome 1q21-amplified human myeloma cell line H929, cells were exposed to increasing doses (5-20ng/mL) of recombinant human IL-6. ADAR1 activity, evaluated using a novel diagnostic RNA editing site-specific qPCR (RESSq-PCR) assay to detect cancer stem cell generation, showed that IL-6-treated H929 harbored increased ADAR1 activity.
ADAR1 activation after prolonged lenalidomide exposure
To investigate the effects of immunomodulatory drug treatment on ADAR1-dependent RNA editing, drug-resistant human myeloma cell lines were experimentally derived by low-dose treatment (1 mM) of H929 cells over 10 weeks. Further incubation with lenalidomide at 10mM selected a robustly drug-resistant population. These cells showed a marked increase in ADAR1 expression and RNA editing activity, as measured by direct sequencing and RESSq-PCR. Long-term lenalidomide treatment of H929 also expanded the CD138-negative fraction, suggesting the emergence of a previously-identified myeloma-initiating cell population. Stromal co-culture of H929 cells with a 1:1 mixture of previously inactivated human bone marrow stromal cell lines (HS-5 and HS-27a) secreting IL-6 and other cytokines also enriched for CD138-negative and CD138-dim populations.
CONCLUSION
Considering the recently described role for ADAR1 in malignant transformation and cancer stem cell generation, myeloma cells harboring 1q21 amplification could gain a self-renewal advantage through ADAR1 activation. Using RESSq-PCR to detect aberrant RNA editing, we observed increased ADAR1 activity during the evolution of 1q21-amplified human myeloma cells under prolonged drug treatment, coupled with an expansion of the CD138-negative fraction, which was also observed following bone marrow stromal co-culture. Together these data suggest that ADAR1 represents a novel diagnostic and therapeutic target for multiple myeloma. Moreover, this multiple myeloma niche model represents a valuable tool for evaluating novel methods to inhibit aberrant ADAR1 activation in drug-resistant malignancies.

Disclosures
Jamieson: Sanofi: Honoraria; Roche: Honoraria.