The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Myeloma Intergroup conducted a workshop on Immune and Cellular Therapy in Multiple Myeloma on January 7, 2022. This workshop included ...presentations by basic, translational, and clinical researchers with expertise in plasma cell dyscrasias. Four main topics were discussed: platforms for myeloma disease evaluation, insights into pathophysiology, therapeutic target and resistance mechanisms, and cellular therapy for multiple myeloma. Here we provide a comprehensive summary of these workshop presentations.
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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: Human bone marrow aging is typified by decreased cellularity, stem cell exhaustion and myeloid lineage bias that may set the stage for development of myeloid malignancies. ...Secondary AML (sAML) is a malignancy that has been associated with alterations in RNA processing genes and currently has few effective treatment options available. A central goal of future therapeutic strategies is to prevent disease relapse and therapeutic resistance by selectively targeting unique gene products that are essential to LSC but not normal HSC function. Therefore, we established whole gene, long non-coding RNA (lncRNA), splice isoform, and RNA editing signatures of benign versus malignant bone marrow progenitor cell aging, and evaluated the therapeutic efficacy of splicing-targeted agents in pre-clinical humanized in vitro and in vivo model systems.
Methods: Whole transcriptome sequencing (RNA-Seq) was performed on FACS-purified hematopoietic stem (CD34+CD38-Lin-) and progenitor cells (CD34+CD38+Lin-) from aged (average age = 65.9 ± 6.8 years old) versus young (average age = 25.8 ± 3.0 years old) adult healthy bone marrow samples, and in leukemia stem cells (LSC) from patients with sAML (average age = 71.4 ± 7.9 years old). Comparative gene set enrichment analyses (GSEA), splice isoform, lncRNA, and RNA editing profiles were identified for normal and malignant progenitor cell aging. Then, we evaluated the spliceosome modulatory agent 17S-FD-895 in splicing reporter activity, PCR, and functional in vitro hematopoietic progenitor and in vivo LSC primagraft assays.
Results: Disruption of pre-mRNA splicing activity has recently been implicated as a therapeutic vulnerability in some types of cancer. Comparative whole transcriptome RNA sequencing (RNA-seq) analyses revealed pre-mRNA splicing factor gene expression was significantly disrupted in human AML LSC compared with age-matched normal progenitors. Comparative splice isoform RNA-seq and qRT-PCR validation revealed recurrent intron retention and exon skipping in expressed transcripts, such as PTK2B and several protein phosphatase gene products. Notably, transcription factor profiling of AML LSC demonstrated downregulation of key tumor suppressor genes, such as IRF8 and TP53. We then investigated the LSC inhibitory efficacy of a stable and potent splicing modulatory agent, 17S-FD-895, in humanized stromal co-culture and AML LSC primagraft assays. Pharmacological spliceosome modulation disrupted AML LSC maintenance in vivo by altering splicing of stem cell survival and AML-associated transcripts at doses that spared normal hematopoietic progenitors.
Conclusions: Detection and targeted modulation of aberrant RNA processing provides an innovative strategy for AML LSC eradication with implications for treatment of a variety of human malignancies and other age-related disorders.
Citation Format: Leslie A. Crews, Larisa Balaian, Heather S. Leu, Nathaniel P. Delos Santos, Angela C. Court, Anil Sadarangani, Maria A. Zipeto, James J. La Clair, Reymundo Villa, Sheldon R. Morris, Rainer Storb, Anna Kulidjian, Edward D. Ball, Michael D. Burkart, Catriona H.M. Jamieson. RNA processing signatures of normal versus malignant progenitor cell aging predict leukemia stem cell sensitivity to RNA splicing modulation. 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 915.
Secondary acute myeloid leukemia (sAML) is the most therapeutically recalcitrant form of AML with a life expectancy of less than 12 months. Secondary AML evolves from relatively prevalent ...myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS), or after chemotherapy, radiation therapy, or hematopoietic cell transplantation (HCT) that together confer a 14% risk of sAML at 15 years. Cumulative sequencing studies show that human splicing factor mutations, epigenetic spliceosome deregulation, RNA editing-induced splicing alterations, and pro-survival splice isoform switching drive dormant leukemia stem cell (LSC) generation and sAML resistance to chemotherapy and molecularly targeted agents resulting in high rates of relapse. LSC are immunologically silent in part because they activate adenosine deaminase acting on dsRNA (ADAR1), which attenuates the innate immune response. In addition, therapeutic splicing modulation has the potential to induce neoepitope formation and augment checkpoint inhibitor therapy. Thus, there is a pressing need for clinical development of splicing modulatory agents that eradicate therapy resistant LSC and reduce sAML drug resistance and relapse. Rebecsinib (17 S-FD-895) is a pharmacologically stable, potent, and selective small molecule splicing modulator that targets the SF3B core of the spliceosome at the interface of SF3B1, SF3B3 and PHF5A. We previously showed that Rebecsinib inhibits human LSC maintenance in sAML models at doses that spare normal hematopoietic stem and progenitor cells (HSPCs). In IND-enabling studies, we now demonstrate that splicing modulation with this potent agent is a pre-clinical tox-proven strategy to eradicate LSC with the potential to overcome immune checkpoint resistance via inhibition of ADAR1 splicing and activity. We further describe targeted LSC eradication that correlates with detection of unique intron-retained and exon-skipped transcripts that can be quantified by splice isoform-specific qRT-PCR and RNA-sequencing analyses and can be used as predictive biomarkers to monitor molecular responses to Rebecsinib treatment. Mechanistically, the therapeutic effects were accompanied by on-target splicing modulatory effects, including reductions in pro-survival MCL1L transcripts and splicing factor gene products such as SF3B1 and SF3B3, which form part of the splicing modulator binding pocket as well as alterations in self-renewal promoting ADAR1 and STAT3beta transcripts. In multi-species toxicology and pharmacokinetic/pharmacodynamic studies, Rebecsinib induced splicing modulation and was well-tolerated over a broad range of doses. Because of disrupted spliceosome function, SF3B1 overexpression and increased dependence on pro-survival splice isoform expression, Rebecsinib-mediated induction of pro-survival to pro-apoptotic splice isoform switching inhibits sAML LSC survival and self-renewal at doses that spare normal HSPCs in vitro and in humanized mouse models commensurate with dose-dependent changes in splicing reporter exon skipping and SF3B1, MCL1, BCL2 and CD44 isoform levels. Together, this potent and selective agent along with biomarkers of response to splicing modulation provide a sensitive method of detecting activity and mechanism of action of Rebecsinib, and demonstrate its LSC selectivity in humanized stromal co-cultures and humanized mouse models, which will have utility in future clinical development of this novel therapeutic agent.
Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Introduction
As the second most common blood cancer in the U.S., 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
To evaluate the contribution of ADAR1 modulation to global RNA editing changes and differential gene expression, we performed whole transcriptome sequencing of primary MM and PCL samples that were lentivirally transduced with shRNA targeting human ADAR1 compared to vector control shRNA. Analysis at the single nucleotide level revealed that 20-50% knockdown of ADAR1 was sufficient to induce substantial downregulation of A-to-I editing rates throughout the transcriptome, particularly at 3'UTR loci, along with specific modulation of extracellular matrix and inflammasome-associated gene expression patterns. Moreover, 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. Notably, in human myeloma cells analyzed using NanoString nCounter assays, overexpression of wild-type versus edited GLI1 elicited distinct gene expression changes also in extracellular matrix and immune response genes. These data demonstrate that ADAR1 promotes malignant self-renewal of MM and if selectively inhibited may prevent progression and relapse through modulation of global A-to-I RNA editing and extracellular and immune response gene expression.
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, immunomodulatory drugs) combine to drive A-to-I RNA editing-dependent malignant regeneration in MM through inflammasome and extracellular matrix gene pathway remodeling. These effects can be down-modulated even with small reductions in ADAR1 expression and activity. Thus, ADAR1 represents both a vital prognostic biomarker and therapeutic target in MM.
Costello:Celgene: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Takeda: Consultancy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
INTRODUCTION
Because acute myeloid leukemia (AML) is the leading cause of pediatric leukemia relapse-related mortality, there is a desperate need for developing new therapeutics and providing ...mechanistic insights into the cell type and molecular context specific causes of relapse. Myeloid malignancies, including AML, have a propensity to disrupt the spliceosome either through acquisition of splicing factor gene mutations or epigenetic spliceosome disruption in leukemia stem cells (reviewed in Chua, Van Der Werf, Jamieson, Signer. Cancer Cell 2020;26:138-159). While leukemia stem cells (LSCs) promote relapse in adult AML as a result of their inherent capacity to become dormant and resist therapies that target dividing cells, the role of alternative splicing (AS) deregulation in relapse and therapeutic vulnerability of LSCs has not been thoroughly studied in pediatric AML.
METHODS
Recently, we completed pre-IND enabling studies with a selective splicing modulator, 17S-FD-895. Previously, we showed that 17S-FD-895 selectively eliminated adult AML LSC (Crews...Burkart, Jamieson. Cell Stem Cell 2016;19:599-612). As a stable pladienolide-derived small molecule splicing modulator, 17S-FD-895 targets a key component of the spliceosome, SF3B1, thereby modulating mRNA splicing. To investigate the role of splicing deregulation in pediatric AML LSC maintenance, we developed a sensitive in vitro and in vivo lentiviral splicing reporter assay, whole transcriptome RNA sequencing (RNA-seq) stem and progenitor cell splicing analysis pipelines, qRT-PCR splice isoform specific biomarkers of response, as well as stromal co-culture, hematopoietic progenitor colony survival and replating assays to assay LSC eradication.
RESULTS
Because splicing regulation is cell type and context dependent, we generated a comprehensive transcriptome expression map of FACS-purified hematopoietic stem cells (HSCs; CD34+CD38-Lin-) and hematopoietic progenitor cells (HPCs;CD34+CD38+Lin). By utilizing a splice variant-specific alignment algorithm, we evaluated genome wide alternative splicing events and uncovered widespread exon skipping in pediatric AML compared to non-leukemic donors. More than 2000 exon skipping events were identified in pediatric AML HSCs and HPCs. In addition, both pediatric AML HSC and HPC demonstrated a downregulation of the splicing regulator RNA-Binding fox 2 (RBFOX2), which has been linked to embryonic stem cell splice variant signatures that are vital for leukemia cell survival (Holm...Jamieson. PNAS 2015;112:15444-15449; Denichenko et al Nat Commun 2019;10:1590). Treatment with 17S-FD-895 induced a dose-dependent reduction in clonogenicity (p=0.001; Student's t-test) and self-renewal of CD34+ cells (p=0.001; Student's t-test) isolated from pediatric AML samples. Pediatric AML samples were significantly more sensitive to splicing modulation than adult de novo or adult secondary AML. Moreover, normal cord blood HSC and HPC samples were unaffected by splicing modulator treatment. Our lentiviral splicing reporter assays demonstrated a dose dependent increase in MAPT intron retention in in pediatric leukemia cell lines as measured by a switch from GFP to RFP. Finally, splice isoform specific RT-PCR demonstrated a dose-dependent increase in SF3B1 intron retention following treatment as well as MCL1 exon 2 skipping, producing pro-apoptotic MCL1-S transcripts.
CONCLUSIONS
Cumulatively, our data indicate that spliceosome modulation via 17S-FD-895-mediated targeting of SF3B1 constitutes a novel potential therapeutic strategy for pediatric patients with AML.
Kaspers:Boehringer Ingelheim: Membership on an entity’s Board of Directors or advisory committees; AbbVie: Ended employment in the past 24 months; Janssen R&D: Ended employment in the past 24 months; Helsinn Healthcare: Ended employment in the past 24 months. Crews:Ionis Pharmaceuticals: Research Funding. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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Currently, the limited capacity of pediatric acute myeloid leukemia (AML) therapies to prevent recurrence has contributed to high mortality rates. While dormant self-renewing leukemia stem cells ...(LSCs) contribute to adult AML relapse, their role in pediatric AML therapeutic resistance has not been clearly elucidated and thus was investigated in the context of this study.
Through whole transcriptome sequencing (RNA-seq) analyses of FACS-purified human hematopoietic stem cells (HSCs; CD34 +CD38 -Lineage -) and progenitor cells (HPCs; CD34 +CD38 + Lineage -) from pediatric AML (n=10) compared with adult de novo (n=5) and secondary AML (n=6) as well as non-leukemic pediatric bone marrow samples (n=6), we identified widespread splicing alterations in pediatric AML compared to non-leukemic donors, indicative of a disruption in splicing regulation. In this study, we identified 2,000 exon skipping events in pediatric AML HSCs and HPCs. Moreover, we detected increased exon skipping and intron retention in stem cell self-renewal and survival transcripts in pediatric AML stem and progenitor cells. Specifically, the pro-survival isoform of MCL1, MCL1 long, was significantly increased in comparison to its pro-apoptotic counterpart, MCL1 short. In addition, self-renewal, RNA editing and splice isoform altering adenosine deaminase RNA specific 1 (ADAR1) p150 isoform levels were significantly (p=0.05) upregulated in pediatric AML progenitors suggesting that splicing and RNA editing deregulation could fuel pediatric AML stem and progenitor cell propagation.
After successful completion of pre-IND development of a pharmacologically stable, potent, and selective small molecule splicing modulator, Rebecsinib (17S-FD-895) (Crews, Balain et al Cell Stem Cell 2016; Chan et al Cell Reports 2020), we developed a dual fluorescence lentiviral splicing reporter that assays the on target anti-leukemic efficacy of Rebcsinib and to assess the therapeutic index between LSCs and normal hematopoietic stem and progenitor cells. In hematopoietic progenitor assays, we observed a dose-dependent reduction in clonogenicity and replating of CD34 + cells isolated from pediatric AML samples following treatment with Rebecsinib. While pediatric AML samples were more sensitive to splicing modulation than adult de novo or adult secondary AML samples, normal cord blood progenitor samples were unaffected by splicing modulator treatment. In addition, we identified dose-dependent alterations in lentiviral splicing reporter activity in pediatric leukemia cells engrafted in a humanized AML mouse xenograft model following intravenous treatment with one dose of 10mg/kg and 20mg/kg of Rebecsinib. Finally, we observed a reduction in ADAR1 p150 transcripts by RNA-seq analysis of hematopoietic tissues in serially transplanted patient derived AML xenografts after Rebecsinib treatment suggesting that inhibition of ADAR1 splicing prevents LSC self-renewal.
Cumulatively, these data demonstrate that stem and progenitor cell specific deregulation of pre-mRNA splicing and ADAR1 activation represent a therapeutic vulnerability to splicing modulation, which provides a strong rationale for developing Rebecsinib for preventing pediatric AML recurrence.
Cloos: Astellas: Speakers Bureau; DC-One: Other, Research Funding; Genentech: Research Funding; Helsinn: Other; Janssen: Research Funding; Merus: Other, Research Funding; Navigate: Patents & Royalties; Novartis: Consultancy, Other, Research Funding; Takeda: Research Funding. Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
In the development of Myelofibrosis (MF), disease which is often associated with aging, deregulation of stem cell activity by somatic mutations occurs in the setting of deregulation of the ...bone marrow microenvironment. While somatic mutations in stem and progenitor cells have been shown to promote pathological myeloproliferation, functional involvement of stromal cells in MF pathogenesis is not well defined. Chronic inflammation has long been regarded as a driving force for MF development, which is intensified by continuous release of pro-inflammatory cytokines and chemokines. We have recently reported that inflammatory cytokine signaling in the aging bone marrow niche via aberrant RNA editing and splicing may predispose patients to leukemia stem cell generation. However, little is known about the functional effects of stromal cell driven inflammasome signaling in MF pathogenesis. Here we investigate the impact of the inflammasome on aging of the BM niche and how changes in the activity of BM niche cells contribute to MF pathogenesis.
Methods
Stromal monolayers were established from CD34-negative bone marrow cells obtained from young (<3 y/o; y-NBM), aged (> 60 y/o; a-NBM) healthy donors and myelofibrosis (MF) patient samples. Conditioned media from those stromal cultures was collected after 7 d post confluence and was analyzed for secreted factors by Human multi-analyte (MAP) Luminex-based analysis (Myriad). Some stromal cultures were used for RNA transcriptome analysis. The other stromal cultures were utilized for the co-culture experiments with CD34+ cells (hematopoietic stem cells, HSC) selected from y-NBM, a-NBM and cord blood (CB), which were subsequently subjected to survival and self-renewal assays.
Results
MF and a-NBM derived stroma severely impaired survival and self-renewal of CB (n=5) and y-NBM (n=5) HSC in co-culture models. In similar experiments HSC from a-NBM (n=6) demonstrated significantly higher survival and self-renewal capacity when co-cultured with y-NBM stroma (n=3) compared to a-NBM stroma (n=3). Conditioned media (CM) from both a-NBM and MF stroma by itself also greatly reduced HSC survival and self-renewal. Notably, co-culture conditions resulted in a greater reduction in survival and self-renewal capacity than CM alone, suggesting that cell-cell contact or unstable secreted factors exacerbate the effects.
Focused analysis of 45 inflammation-associated secreted factors in 3 y-NBM and 4 a-NBM stromal CM samples revealed no substantial alterations in inflammatory factors However, a-NBM stroma demonstrated a significant reduction in production of a variety of HSC-regulatory factors (BDNF, IL-17, IL-12p40, SCF, ICAM-1,VEGF, Eotaxin-1, RANTES). Moreover, RNA transcriptome analysis of 3 y-NBM, 4 a-NBM and 3 MF stromal monolayers revealed significant variations in differentially expressed genes (DEG) between y-NBM and a-NBM. Specifically, aged stroma was typified by upregulated expression of inflammation and immune response genes and of genes associated with the lysosome (ELANE, CLU, DEFA1, LBP). Downregulation of collagen genes and energy metabolism genes, particularly mitochondrial genes, as well as alterations in the expression of apoptosis, cell cycle and cellular senescence biomarkers (IL1B, IDS, TPST2, SERPINB2) was observed.
Comparisons in gene expression between a-NBM and MF stroma revealed upregulated inflammatory response(PLIN2, IL1B,PDK4,) and TGF-β signaling pathways(PTGDS,) and downregulated ability to support HSC (RARRES2, FGF7, FGF23, and IGF2) Notably, some transcripts (RUNX3, BCL2) were identified within the MF stromal niche, while they were absent in a-NBM stroma or HSC from a-NBM and MF samples.
Conclusions
These data indicate that MF and aged stroma exhibit a severely compromised ability to maintain normal hematopoiesis. Molecular inflammasome signatures of aging and MF reflect a combination of degenerative processes and transcriptional responses. Targeting this pathological interplay between the MF niche and MF stem cells could represent a novel avenue for the treatment of MF.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Innate immune anti-viral adenosine to inosine (A-to-I) base editing enzymes (editases) promote hematopoietic stem cell (HSC) self-renewal and protect the human genome from retroviral integration in ...response to inflammatory cytokine signaling. However, hyper-editing has been linked to therapeutic resistance and cancer progression. Because myeloproliferative neoplasm (MPN) progression is typified by increased JAK2/STAT-mediated cytokine signaling, we investigated the cell type and context specific role of adenosine deaminase acting on RNA1 (ADAR1) editaseactivity in MPN pre-leukemia stem cell (pre-LSC) evolution into acute myeloid leukemia stem cells (LSCs). Here we show by whole transcriptome sequencing (RNA-seq) of 113 FACS-purified hematopoietic stem cells and progenitors from 78 individuals, including 54 MPN and AML patients and 24healthy young and aged individuals, that anti-viral signaling pathway activation and splice isoform switching from ADAR1p110 to JAK2/STAT-inducible ADAR1p150 RNA editase activation contributes to MPN progression. Pre-LSC evolution to LSC was characterized by ADAR1p150 upregulation, distinctive RNA editome patterns, STAT3 hyper-editing, increased replating as a measure of self-renewal. Moreover, LSC generation was typified by beta-catenin self-renewal pathway upregulation, which was recapitulated by lentiviral ADAR1p150 overexpression and reversed by lentiviral ADAR1p150 shRNA knockdown. Our studyunderscores the importance of inflammatory-cytokine fueled enzymatic mutagenesis in human MPN pre-LSC evolution to LSC. Thus, this study sets the stage for developing predictive RNA editome biomarkers of LSC generation to guidetherapeutic strategies aimed at preventing progression of hematopoietic malignancies.
Crews:Ionis Pharmaceuticals: Research Funding.
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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: 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 (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. 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.
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.
Jamieson:Sanofi: Honoraria; Roche: Honoraria.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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