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.
Abstract
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
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.
Transcription factors (TFs) mediate transcriptional responses, allowing cells to respond to changing internal or external stimuli, including infection. The DNA sequences bound by TFs in regulatory ...regions are called motifs. Researchers employ Motif Enrichment Analysis (MEA) methods to study transcriptional regulation, which analyzes DNA sequences from regulatory regions and determines the statistical overrepresentation of motifs in those sequences, allowing inference of relevant TFs for that set of regulatory regions. However, most MEA tools feature oversimplifications of one or more pertinent axes of the data, obscuring potential insights into transcriptional regulation.For example, most MEA require thresholding of DNA sequences into two sets in order to determine motif enrichment, thus oversimplifying the underlying biological scores that determine those sets, which can prevent biological discovery. We introduce Motif Enrichment In Ranked Lists of Peaks (MEIRLOP), a score-based MEA method that allows researchers to determine the enrichment of motifs within a dataset of scored regulatory region DNA sequences. MEIRLOP uniquely utilizes a logistic regression model that also accounts for lower order levels of sequence bias and other covariates. We demonstrate its utility on multiple ChIP-seq datasets, where it proves more capable (relative to other methods) of finding the enrichment of key transcription factor binding motifs, including the enrichment of binding sites key to immune response.An overlooked axis in most MEA is the position of motifs relative to anchor features such as transcription start sites (TSS), which can be characterized at high positional resolution using capped short RNA-sequencing (csRNA-seq). We introduce Motif Enrichment Positional Profiling (MEPP), which uses specialized convolutional neural networks to create a profile that characterizes motif enrichment at different motif positions over a dataset of scored sequences. We also introduce Learning Motifs from Positional Priors (LMPP), which uses machine learning to perform the opposite of MEPP: Learning a motif whose positional enrichment resembles a target profile. We use both methods to analyze multiple TSS from csRNA-seq datasets, revealing the positional preferences of transcription factors key to antibacterial and antiviral responses.Overall, this dissertation presents novel methods by which researchers may analyze transcriptional regulation in and beyond immune response.
Compelling murine studies demonstrate that adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase associated with RNA1 (ADAR1) is vital for both fetal and adult hematopoiesis. ...While genetic ablation of ADAR1 editase leads to murine embryonic lethality due to severe defects in erythropoiesis, conditional deletion in the hematopoietic system impairs maintenance indicative of cell type and context specific roles for ADAR1 in cell fate specification and self-renewal. By regulating mRNA and microRNA (miRNA) stability, ADAR1 exhibit wide-ranging effects on embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 plays important roles in both stem cell differentiation and self-renewal in CML (chronic myeloid leukemia) disease progression. Here, we describe a novel function of ADAR1 in cell cycle regulation of normal hematopoietic stem cell and progenitors (HSPC) by regulation of miRNA biogenesis.
Our results demonstrated that ADAR1 induces G0 to G1 phase transition in normal cord blood HSPCs, as demonstrated by elevated expression of Ki67, reduced DiR signal, and enhanced in vivo cord blood engraftment. Cell cycle qRT-qPCR microarray of 84 cell cycle transcripts and whole transcriptome RNA-sequencing analysis of KEGG cell cycle pathway indicate that several cell cycle genes (CDKN1A, CDKN2A, CCNC, CCND1, BRCA2, etc) are differentially expression upon overexpression of ADAR1 WT or an A-to-I editing deficient ADAR1 mutant (ADAR1E912A). We previously demonstrated that impaired biogenesis of let-7 miRNAs by ADAR1 WT induces enhanced self-renewal in cord blood CD34+ HSPCs. To determine the miRNA targets of ADAR1-mediated RNA editing, we performed miRNome miScript PCR array of 1008 miRNA candidates in cord blood CD34+ HSPCs overexpressing ADAR1 WT or ADAR1E912A. Total of 263 miRNAs were differentially expressed (142 upregulated and 121 downregulated) by comparing ADAR1 WT to the backbone control. Interestingly, ADAR1E912A mutant also exhibit A-to-I editing independent regulation of miRNAs (307 upregulated and 59 downregulated). We found that the expression of miR-26a-5p, a miRNA frequently downregulated in leukemia, is inhibited by ADAR1-mediated RNA editing. ADAR1 directly binds and edits the DROSHA cleavage site of primary miR-26a transcript, thereby prevent miR26a-5p maturation. Moreover, lentiviral expression of mature miR26-5p reverses the effect of ADAR1 WT, including enhanced CDKN1A expression, inhibition of cord blood proliferation in vivo, as well as reduced HSC self-renewal as measured by colony-formation assay.
Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell proliferation in HSC. For future study, it will be interesting to investigate if the elevated expression of ADAR1 in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells.
Jamieson:CTI Biopharma: Research Funding; GlaxoSmithKline: Research Funding; Johnson & Johnson: 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
Background
In advanced human malignancies, RNA sequencing (RNA-seq) has uncovered deregulation of adenosine deaminase acting on RNA (ADAR) editases that promote therapeutic resistance and leukemia ...stem cell (LSC) generation. Chronic myeloid leukemia (CML), an important paradigm for understanding LSC evolution, is initiated by BCR-ABL1 oncogene expression in hematopoietic stem cells (HSCs) but undergoes blast crisis (BC) transformation following aberrant self-renewal acquisition by myeloid progenitors harboring cytokine-responsive ADAR1 p150 overexpression. Emerging evidence suggests that adenosine to inosine editing at the level of primary (pri) or precursor (pre)-microRNA (miRNA), alters miRNA biogenesis and impairs biogenesis. However, relatively little is known about the role of inflammatory niche-driven ADAR1 miRNA editing in malignant reprogramming of progenitors into self-renewing LSCs.
Methods
Primary normal and CML progenitors were FACS-purified and RNA-Seq analysis as well as qRT-PCR validation were performed according to published methods (Jiang, 2013). MiRNAs were extracted from purified CD34+ cells derived from CP, BC CML and cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human JAK2, let-7a, wt-ADAR1 and mutant ADAR1, which lacks a functional deaminase domain. Because STAT signaling triggers ADAR1 transcriptional activation and both BCR-ABL1 and JAK2 activate STAT5a, nanoproteomics analysis of STAT5a levels was performed. Engrafted immunocompromised RAG2-/-γc-/- mice were treated with a JAK2 inhibitor, SAR302503, alone or in combination with a potent BCR-ABL1 TKI Dasatinib, for two weeks followed by FACS analysis of human progenitor engraftment in hematopoietic tissues and serial transplantation.
Results
RNA-seq and qRT-PCR analysis in FACS purified BC CML progenitors revealed an over-representation of inflammatory pathway activation and higher levels of JAK2-dependent inflammatory cytokine receptors, when compared to normal and chronic phase (CP) progenitors. Moreover, RNA-seq and qRT-PCR analysis showed decreased levels of mature let-7 family of stem cell regulatory miRNA in BC compared to normal and CP progenitors. Lentiviral human JAK2 transduction of CD34+ progenitors led to an increase of ADAR1 transcript levels and to a reduction in let-7 family members. Interestingly, lentiviral human JAK2 transduction of normal progenitors enhanced ADAR1 activity, as revealed by RNA editing-specific qRT-PCR and RNA-seq analysis. Moreover, qRT-PCR analysis of CD34+ progenitors transduced with wt-ADAR1, but not mutant ADAR1 lacking functional deaminase activity, reduced let-7 miRNA levels. These data suggested that ADAR1 impairs let-7 family biogenesis in a RNA editing dependent manner. Interestingly, RNA-seq analysis confirmed higher frequency of A-to-I editing events in pri- and pre-let-7 family members in CD34+ BC compared to CP progenitors, as well as normal progenitors transduced with human JAK2 and ADAR1-wt, but not mutant ADAR1. Lentiviral ADAR1 overexpression enhanced CP CML progenitor self-renewal and decreased levels of some members of the let-7 family. In contrast, lentiviral transduction of human let-7a significantly reduced self-renewal of progenitors. In vivo treatments with Dasatinib in combination with a JAK2 inhibitor, significantly reduced self-renewal of BCR-ABL1 expressing BC progenitors in the bone marrow thereby prolonging survival of serially transplanted mice. Finally, a reduction in ADAR1 p150 transcripts was also noted following combination treatment only suggesting a role for ADAR1 in CSC propagation.
Conclusion
This is the first demonstration that intrinsic BCR-ABL oncogenic signaling and extrinsic cytokines signaling through JAK2 converge on activation of ADAR1 that drives LSC generation by impairing let-7 miRNA biogenesis. Targeted reversal of ADAR1-mediated miRNA editing may enhance eradication of inflammatory niche resident cancer stem cells in a broad array of malignancies, including JAK2-driven myeloproliferative neoplasms.
Jamieson:J&J: Research Funding; GSK: 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
Introduction
Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are generated from progenitors that have aberrantly activated self-renewal pathways thereby resulting in tyrosine kinase ...inhibitor (TKI) resistance. The telomerase complex, consisting of a reverse transcriptase subunit (TERT), an RNA template subunit (TERC), and a protective shelterin scaffold, transcriptionally modulates the Wnt/b-catenin self-renewal pathway. Many malignancies, including BCR-ABL TKI resistant blast crisis CML (BC CML), exhibit robust telomerase activity thereby prompting the development of imetelstat, a competitive inhibitor of telomerase enzymatic activity. Imetelstat is a covalently lipidated 13-mer oligonucleotide that binds with high affinity to the TERC subunit. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms. However, the role of imetelstat in selective self-renewing LSC inhibition in CML had not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized LSC primagraft studies to investigate the capacity of imetelstat to selectively inhibit LSC self-renewal and to determine the mechanism of action.
Methods and Results
Cytoscape analysis of RNA-seq data derived from FACS-purified progenitors from human blast crisis (BC; n=9) compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples revealed transcriptional upregulation of b-catenin, LEF1, TCF7L1, ABL1 and other key genes within the TERT interactome suggesting a role for TERT activation in human BC LSC generation. Human progenitor LSC-supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 mM significantly inhibited (p<0.001, ANOVA) in vitro self-renewal of BC CML (n=5) compared with age-matched normal bone marrow progenitors. Treatment of primagraft mouse models of human BC CML with 30 mg/kg of imetelstat three times a week for four weeks resulted in a significant reduction in bone marrow progenitor LSC burden compared with mismatch treated controls (p=0.04). Furthermore, qRT-PCR showed decreased β-catenin transcript levels in imetelstat compared with vehicle-treated samples. Finally, FACS analysis revealed a significant reduction in activated b-catenin protein levels in engrafted human myeloid progenitors following imetelstat treatment in the TKI resistant bone marrow niche.
Conclusions
Niche responsive interactions between the telomerase complex and the Wnt/b-catenin self-renewal pathway sensitize b-catenin activated LSC to imetelstat in both in vitro and in vivo humanized pre-clinical BC CML models thereby providing a strong rationale for LSC eradication trials involving imetelstat.
Huang:Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Jamieson:UC San Diego: Other: I received funding from Janssen Research & Development, LLC.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is ...involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established.
Methods
Whole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013).
Results
We performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance.
Conclusion
In summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication.
Jamieson:Johnson & Johnson: Research Funding; GlaxoSmithKline: 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
Malignant reprogramming of progenitors into self-renewing cancer stem cells (CSCs) that have a predisposition for dormancy in protective niches has been implicated in therapeutic resistance of ...chronic myeloid leukemia (CML) and other CSC-driven malignancies. An unmet medical need for developing therapies that target niche dependent dormant human CSCs provides a compelling rationale for identifying key differences in gene expression at different cell cycle phases between normal and malignant progenitors in a CSC-supportive stromal co-culture system. Currently, few methods exist for quantifying cell cycle kinetics in live human leukemia stem cells (LSC). To date, efficient cell cycle transit time analysis in single live human leukemic progenitors derived from primary patient samples has been hampered by 1) decreased cell viability following transfection or transduction, 2) limited sample size, 3) dormancy of primitive progenitor populations thereby necessitating lentiviral rather than retroviral transduction and 4) increased apoptosis in the absence of a supportive microenvironment. To alleviate these challenges and improve transduction efficiency, we generated Fucci2BL, a lentiviral bicistronic reporter vector. Fucci2BL expresses mVenus-hGem(1/110) fused to mCherry-hCdt1(30/120) by the T2A peptide using an EF1 promoter that generates optimal levels of gene expression in progenitors. Initially, the reporter fidelity was characterized in 293A cells using flow cytometry and time-lapse confocal fluorescence microscopy. Time-lapse confocal fluorescence microscopy revealed normal cell morphology and distinct nuclear staining of either green or red fluorescence depending on the cell cycle stage. Once the fidelity of the Fucci2BL reporter was characterized, differences in gene expression levels between normal and malignant progenitors were analyzed. Whole transcriptome RNA-seq analysis revealed both cell cycle and DNA replication pathways were enriched in chronic phase CP (CML) compared to normal progenitors. Cell cycle kinetics between normal and chronic phase (CML) progenitors co-cultured in a niche were also analyzed using the Fucci2BL reporter. Normal progenitor cells on average transited the cell cycle within 26 hours while CP progenitor cells demonstrated a prolongation of transit through G1. In summary, the Fucci2BL system enables single transduction and single cell cycle tracking as well as gene expression changes in live primary progenitors in response to a niche. This robust lentiviral reporter can reproducibly distinguish cell cycle phases thereby providing an opportunity to quantitatively study the contribution of cell cycle kinetics to single cancer stem cell therapeutic resistance and to relapse.
Jamieson:J&J: Research Funding; GSK: 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
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