Angela C. Court

ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.

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.

NOTCH1 Signaling Promotes Human T-Cell Acute Lymphoblastic Leukemia Initiating Cell Regeneration in Supportive Niches

Background Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated. Methodology and Principal Findings We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34+ cells from NOTCH1Mutated T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1Wild-type CD34+ cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1Mutated CD34+ fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1Mutated T-ALL LIC-engrafted mice and resulted in depletion of CD34+CD2+CD7+ cells that harbor serial transplantation capacity. Conclusions These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.

GLI2 inhibition abrogates human leukemia stem cell dormancy.

BackgroundDormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.MethodsTo test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment.ResultsNotably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.ConclusionIn summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.

Abstract 375: A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction The adenosine deaminase acting on RNA (ADAR) family of RNA editases has been linked to the pathogenesis of diverse malignancies, including leukemia, breast cancer and hepatocellular carcinoma. We previously showed that human leukemia stem cells (LSC) from blast crisis (BC) chronic myeloid leukemia (CML) patients harbor increased ADAR1 expression compared with normal and chronic phase (CP) progenitors. Whole transcriptome RNA sequencing (RNA-Seq) revealed increased adenosine to inosine (A-to-I) RNA editing during CML progression concentrated within primate specific Alu-containing transcripts. However, detection of RNA editing by RNA-Seq in rare cell populations can be technically challenging, costly and requires PCR validation. Thus, the objectives of this study were to validate RNA editing of a subset of these LSC-associated transcripts in the context of lentivirally enforced ADAR1 expression, and to develop an RNA editing reporter reporter assay in human leukemia cells and a qPCR-based diagnostic test to rapidly detect CSC-associated aberrant RNA editing. Methods The BCR-ABL+ human leukemia cell line K562 was stably transduced with lentiviral human ADAR1 or vector. FACS-purified K562-ADAR1 cells were transfected with a luciferase-based reporter vector to confirm RNA editing activity. Two genes, MDM2 and APOBEC3D, were selected from our previous RNA-Seq studies of BC progenitors (Jiang et al, 2013). Targeted sequencing was performed on high fidelity PCR products using primers flanking each of 2 editing sites in each gene. RNA editing-specific qPCR primers were designed for each editing site using an allele-specific strategy that detects cDNA containing either an A or G(I) representing an RNA editing event. Both targeted sequencing and qPCR were used to detect RNA editing in K562-ADAR1 and primary cord blood-derived hematopoietic stem cells (HSC) lentivirally transduced with ADAR1. Results Lentivirally enforced ADAR1 expression promoted RNA editing activity as measured by luciferase reporter activity. Increased A-to-I changes in MDM2 and APOBEC3D were confirmed by targeted sequencing. In independent experiments, RNA editing site-specific qRT-PCR accurately detected RNA editing in K562-ADAR1 cells (n=3) and in primary HSC overexpressing ADAR1 (n=4). Site-specific primers distinguished G(I) bases at RNA editing sites in cDNA and as predicted gave no signal in gDNA. Relative A-to-I RNA editing ratios were increased by 2 to 3 fold in ADAR1-expressing cells at all four sites. Conclusions These results set the stage for development of primate-specific RNA editing as a novel diagnostic strategy for clinical LSC detection and identify ADAR1 as a potential therapeutic target in LSC. These data shed new light on the mechanisms of ADAR1-mediated generation of malignant progenitors that drive therapeutic resistance, disease progression and relapse in CML and may be applicable to other CSC-driven malignancies. Citation Format: Leslie A. Crews, Qingfei Jiang, Maria A. Zipeto, Angela C. Court, Christian L. Barrett, Marco A. Marra, Kelly A. Frazer, Catriona H. M. Jamieson. A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 375. doi:10.1158/1538-7445.AM2014-375

Abstract 1011: NOTCH1 signaling is essential for leukemia initiating cell self-renewal in T-ALL

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Leukemia initiating cells (LIC) contribute to therapeutic resistance through mutations in cellular self-renewal and survival pathways. NOTCH1 mutations are common in T-cell acute lymphoblastic leukemia (T-ALL). However, the role of NOTCH1 activation in human LIC propagation has not been established. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2R gamma null (NSG) mice, was identified in primary adult T-ALL. To identify the molecularly characterized potential LIC populations in pediatric T-ALL without proceeding in vitro culture and examine the role of NOTCH1 activation in LIC propagation. 12 pediatric T-ALL samples were sequenced for NOTCH1 mutation examination. Humanized LIC mouse models were established and dosed with either NOTCH1 mAb or IgG1 mAb control at 10 mg/kg intraperitoneally every 4 days for 6 doses. Mice were sacrificed one day after the last dose, and hematopoietic organs were collected for FACS analysis. To further define the LIC populations in pediatric T-ALL, CD34+CD38+CD2+CD7+Lin− and CD34+CD38+CD2+CD7−Lin− cells were isolated from T-ALL primary patients’ blood by FACS sorting and transplanted into neonatal RAG2−/−γc−/− mice to determine their leukemic engraftment potential. Serial transplantations were done for testing the LIC self-renewal capacity. Mouse hematopoietic organs were collected for FACS analysis, mouse brains were sectioned for human cells examination by immunohistochemistry. NOTCH1 and its downstream gene expressions were examined by q-RT-PCR between the T-ALL CD34+ and CD34− populations. Six of 12 pediatric T-ALL patient samples were found NOTCH1 mutation. Mice transplanted with CD34+ and CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale BM and enlarged spleen, thymus and liver. Human CD34+ enriched cells from NOTCH1 mutated T-ALL maintained leukemic engraftment while an equivalent number of CD34+ cells from NOTCH1 wild type T-ALL did not. T-ALL CD34+ progenitors from NOTCH1 mutated T-ALL have a significant higher engraftment in BM when compared with those from NOTCH1 wild type T-ALL. CD34+CD2+CD7+ and CD34+CD2+CD7− populations are more prominent in NOTCH1 mutated samples. Both the human CD34+ and CD34+CD2+CD7+ populations were significantly reduced in BM when treated with hN1 mAb in vivo. NOTCH1 and its downstream genes expression were significantly reduced in NOTCH1 mutated CD34+ cells when compared with CD34− cells. Human T-ALL LIC have enhanced NOTCH1 expression; CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL; A selective hN1 mAb inhibits human T-ALL LIC survival and self-renewal in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1011. doi:1538-7445.AM2012-1011

Abstract 4313: Mouse model of human T-cell acute lymphoblastic leukemia stem cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC T-cell acute lymphoblastic leukemia (T-ALL) is a very common malignancy diagnosed in children, accounting for 15% of all pediatric ALL cases. In pediatric T-ALL, 50% of patients harbor a Notch1 activating mutation1. Patients with relapsed T-ALL have a poor prognosis and thus it is important to understand the molecular mechanisms. Leukemia stem cells (LSC) play a key role in cancer propagation and have the capacity to self-renew and differentiate. LSC have been reported in T-ALL where, following in vitro culture, CD34+/CD4—— and CD34+/CD7——subfractions of T-ALL marrow were enriched for LSC capable of engrafting leukemia in NOD/SCID mice2. However, difficulties in maintaining primary cultures of leukemia cells have hampered investigations into the biology of T-ALL underscoring the need for a direct xenotransplant model for screening candidate drugs that inhibit self-renew pathways. Candidate LSC from T-ALL patient samples (n=10) were sorted using a FACSAria. Notch1, Hes1 and c-myc expression were analyzed in sorted cells by Q-PCR and Notch1 levels were measured by FACS. In addition, key genes were sequenced from some of the samples. To develop a mouse model for human T-ALL, sorted candidate LSC were lentivirally transduced with GFP-Luciferase fusion protein (GLF) and transplanted intrahepatically into neonatal T, B, and NK cell deficient mice3. Leukemic engraftment was monitored by in vivo bioluminescence imaging. The mice were sacrificed 8 weeks after transplant; hematopoietic organs were collected for FACS analysis of human CD2, CD7, CD34 and CD45 engraftment. Finally, to assay LSC self-renewal, engrafted human CD34+ cells from the bone marrow or thymus were transplanted into secondary and tertiary recipients. While Q-PCR and FACS data showed that Notch1 levels varied among different T-ALL patients, Notch 1 expression correlated with level of engraftment. We transplanted 10 T-ALL patient samples with higher Notch1 expression and 9 of 10 samples engrafted immunocompromised mice. Transplanted LSC could be tracked 4 weeks after transplant byin vivo bioluminescent imaging and human CD34+/CD45+, CD2+/CD7+/CD45+ cells were found in hematopoietic organs of engrafted mice at 8 weeks post transplant. Importantly, the engraftment of CD34+/CD45+, CD2+/CD7+/CD45+ cell populations in hematopoietic organs derived from T-ALL patient samples correlates with the status of mutations. Finally, isolated human CD34 progenitor cells could engraft 2nd and 3rd recipients demonstrating their propensity for self-renewal and differentiation. 1. Enhanced Notch1 expression was found in T-ALL CD34+ cells. 2. Candidate T-ALL LSC (CD34+/CD4−/CD7−) give rise to CD34+/CD45+ and CD2+/CD7+/CD45+ cells in the hematopoietic organs of transplanted mice. 3. T-ALL LSC can be tracked in 1st and 2nd transplanted mice providing a robust in vivo model for testing novel self-renew pathway inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4313.