Nature communications, 2026
Gopal, Aparna, Tam, Derek, Mey, Franziska, Lin, Diana, May, Christina, Jiang, Jihong, Bridgers, Joshua, Caswell, Brett, O'Neill, Kieran, Vizeacoumar, Frederick S, MacAuley, Mackenzie, Haniak, Emilie, Craddock, Charles, Vyas, Paresh, Malcovati, Luca, Buckstein, Rena, Moksa, Michelle, Hirst, Martin, Vizeacoumar, Franco J, Medvedev, Nadia, Heuser, Michael, Stubbins, Ryan J, Deng, Yu, Karsan, Aly
Hypomethylating agents (HMAs) are a mainstay of therapy for myeloid cancers, but genetic biomarkers do not predict who will respond to treatment. Using a variety of single-cell sequencing approaches to define the epigenomic state of responder and nonresponder leukemic cells, we demonstrate that leukemic stem cells (LSC) exist in at least two different epigenomic states: a hematopoietic stem cell (HSC)-or multipotent progenitor (MPP)-like state that is sensitive to HMAs, independent of genetic mutations, or a lymphoid-primed MPP (LMPP)-like nonresponder state. Hypomethylation and chromatin accessibility at ZNF143- and CTCF-binding sites results in activation of HOXB4, which defines the HSC/MPP-like state and HMA-sensitivity. Our study provides evidence that the epigenomic state of the LSC is a major determinant of response to HMAs, and demonstrates that a routine clinical assay can identify patients who will respond.
The Journal of clinical investigation, 2026
Kincross, Hayle, Mo, Ya-Chi Angela, Wang, Xuan, Chang, Linda, Duns, Gerben, Mey, Franziska, Jiang, Jihong, Zhu, Zurui, Isak, Naomi, Kwan, Harwood, Lau, Tammy Ty, Docking, T Roderick, Garg, Pranav, Tran, Jessica, Colborne, Shane, Cheng, Se-Wing Grace, Huang, Shujun, Gharaee, Nadia, Willie, Elijah, Parker, Jeremy Dk, Bridgers, Joshua, Wood, Davis, Klein Geltink, Ramon I, Morin, Gregg B, Karsan, Aly
Acute myeloid leukemia (AML) is an aggressive cancer with very poor outcomes. To identify additional drivers of leukemogenesis, we analyzed sequencing data from 1,727 unique individual patients with AML, which revealed mutations in ubiquitin ligase family genes in 11.2% of samples from adult patients with AML with mutual exclusivity. The SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex gene, FBXO11, was the most significantly downregulated gene of the SCF complex in AML. We found that FBXO11 interacts with and catalyzes K63-linked ubiquitination of LONP1 in the cytosol, to promote LONP1 entry into mitochondria. We show that depletion of FBXO11 or LONP1 reduced mitochondrial respiration through impaired LONP1 chaperone activity to assemble electron transport chain Complex IV. Reduced mitochondrial respiration secondary to FBXO11 or LONP1 depletion imparted myeloid-biased stem cell properties in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro. In a human xenograft model, depletion of FBXO11 cooperated with AML1-ETO and mutant KRASG12D to generate serially transplantable AML. Our findings suggest that reduced FBXO11 cooperates to initiate AML by priming HSPC for myeloid-biased self renewal through attenuation of LONP1-mediated regulation of mitochondrial respiration.
Leukemia, 2025
Gharaee, Nadia, Wegrzyn-Woltosz, Joanna, Jiang, Jihong, Akhade, Vijay Suresh, Bridgers, Joshua, Stubbins, Ryan J, Hiwase, Devendra, Kutyna, Monika M, Chan, Onyee, Komrokji, Rami, Padron, Eric, Deng, Yu, Cole, Gary, Umlandt, Patricia, Fuller, Megan, Kim, Ada, Karsan, Aly
Myelodysplastic neoplasms (MDS) are stem cell disorders characterized by ineffective hematopoiesis and risk of transformation to acute myeloid leukemia (AML). Chromosomal alterations are frequent in MDS, with interstitial deletion of chromosome 5q (del(5q)) being the most common. Lenalidomide is the current first-line treatment for del(5q) MDS and its efficacy relies on degradation of CK1α which is encoded by the CSNK1A1 gene located in the commonly deleted region (CDR) of chromosome 5q. However, lenalidomide-resistance is common, often secondary to loss-of-function mutations in TP53 or RUNX1. The CDR in del(5q) harbors several genes, including noncoding miRNAs, the loss of which contribute to disease phenotypes. miR-143 and miR-145 are located within the del(5q) CDR, but precise understanding of their role in human hematopoiesis and in the pathogenesis of del(5q) MDS is lacking. Here we provide evidence that deficiency of miR-143 and miR-145 plays a role in clonal expansion of del(5q) MDS. We show that insulin-like growth factor 1 receptor (IGF-1R) is a direct target of both miR-143 and miR-145. Our data demonstrate that IGF-1R inhibition reduces proliferation and viability of del(5q) cells in vitro and in vivo, and that lenalidomide-resistant del(5q) MDS cells depleted of either TP53 or RUNX1 are sensitive to IGF-1R inhibition. Resistant del(5q) MDS-L cells, as well as primary MDS marrow cells, are also sensitive to targeting of IGF-1R-related dependencies in del(5q) MDS, which include the Abl and MAPK signaling pathways. This work thus provides potential new therapeutic avenues for lenalidomide-resistant del(5q) MDS.
Journal of clinical pathology, 2025
Sabatini, Peter J B, Bridgers, Josh, Huang, Shujun, Downs, Gregory, Zhang, Tong, Sheen, Clare, Park, Nicole, Kridel, Robert, Marra, Marco A, Steidl, Christian, Scott, David W, Karsan, Aly
Genomic sequencing of lymphomas is under-represented in routine clinical testing despite having prognostic and predictive value. Clinical implementation is challenging due to a lack of consensus on reportable targets and a paucity of reference samples. We organised a cross-validation study of a lymphoma-tailored next-generation sequencing panel between two College of American Pathologists (CAP)-accredited clinical laboratories to mitigate these challenges.
The Journal of molecular diagnostics : JMD, 2024
Bridgers, Joshua, Alexander, Kenyon, Karsan, Aly
Quality assurance (QA) is essential for precision oncology workflows, in particular in the clinical setting. However, because of numerous variations in laboratory and bioinformatics pipelines, QA practices remain non-standardized, are often ad hoc, and are lacking longitudinal tracking. A selected review of existing software was performed for quality control of Illumina next-generation sequencing data, focusing specifically on generalizable tools that can be integrated into any bioinformatics workflow to easily develop a QA workflow with longitudinal tracking. Although all implementations need to be integrated, validated, and iterated upon to suit individual operations, providing a base suite of options will enable better validation and use of QA in clinical somatic mutation testing for workflows using Illumina next-generation sequencing and beyond.
Clinical chemistry, 2024
Grants, Jennifer M, May, Christina, Bridgers, Josh, Huang, Shujun, Gillis, Sierra, Meissner, Barbara, Boyle, Merrill, Ben-Neriah, Susana, Hung, Stacy, Duns, Gerben, Hilton, Laura, Gerrie, Alina S, Marra, Marco, Kridel, Robert, Sabatini, Peter J B, Steidl, Christian, Scott, David W, Karsan, Aly
Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays.
Nature communications, 2023
Coulombe, Patrick, Cole, Grace, Fentiman, Amanda, Parker, Jeremy D K, Yung, Eric, Bilenky, Misha, Degefie, Lemlem, Lac, Patrick, Ling, Maggie Y M, Tam, Derek, Humphries, R Keith, Karsan, Aly
Hematopoietic stem and progenitor cells (HSPCs) originate from an endothelial-to-hematopoietic transition (EHT) during embryogenesis. Characterization of early hemogenic endothelial (HE) cells is required to understand what drives hemogenic specification and to accurately define cells capable of undergoing EHT. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), we define the early subpopulation of pre-HE cells based on both surface markers and transcriptomes. We identify the transcription factor Meis1 as an essential regulator of hemogenic cell specification in the embryo prior to Runx1 expression. Meis1 is expressed at the earliest stages of EHT and distinguishes pre-HE cells primed towards the hemogenic trajectory from the arterial endothelial cells that continue towards a vascular fate. Endothelial-specific deletion of Meis1 impairs the formation of functional Runx1-expressing HE which significantly impedes the emergence of pre-HSPC via EHT. Our findings implicate Meis1 in a critical fate-determining step for establishing EHT potential in endothelial cells.
Leukemia, 2023
Akhade, Vijay Suresh, Liu, Tian, Docking, T Roderick, Jiang, Jihong, Gopal, Aparna, Karsan, Aly
We recently described a 16-gene expression signature for improved risk stratification of acute myeloid leukemia (AML) patients called the AML Prognostic Score (APS). A subset of APS-high-risk AML patients showed increased levels of focal adhesion kinase (FAK), encoded by the Protein Tyrosine Kinase 2 (PTK2) gene, which was correlated with RUNX1 mutations. RUNX1 mutant cells are more sensitive to PTK2 inhibitors. As we were not able to detect RUNX1-binding sites in the PTK2 promoter, we hypothesized that RUNX1 might regulate micro(mi)RNAs that repress PTK2, such that loss-of-function RUNX1 mutations would result in reduced miRNA expression and derepression of PTK2. Examination of paired RNA-seq and miRNA-seq data from 301 AML cases revealed two miRNAs that positively correlated with RUNX1 expression, contained RUNX1-binding sites in their promoters and were predicted to target PTK2. We show that the hsa-let7a-2-3p and hsa-miR-135a-5p promoters are regulated by RUNX1, and that PTK2 is a direct target of both miRNAs. Even in the absence of RUNX1 mutations, hsa-let7a-2-3p and hsa-miR-135a-5p regulate PTK2 expression, and reduced expression of these two miRNAs sensitizes AML cells to PTK2 inhibition. These data explain how RUNX1 regulates PTK2, and identify potential miRNA biomarkers for targeting AML with PTK2 inhibitors.
JCO precision oncology, 2022
Kwon JS, Tinker AV, Santos J, Compton K, Sun S, Schrader KA, Karsan A.
PURPOSE: In 2020, ASCO recommended that all women with epithelial ovarian cancer have germline testing for BRCA1/2 mutations, and those without a germline pathogenic variant (PV) should have somatic tumor testing to determine eligibility for a poly (ADP-ribose) polymerase inhibitor. Consequently, the majority of patients with ovarian cancer will have both germline testing and somatic testing. An alternate strategy is tumor testing first and then germline testing if there is a PV in the tumor and/or significant family history. The objective was to conduct a cost-effectiveness analysis comparing the two testing strategies. METHODS: The Markov model compared the costs (US dollars) and benefits of two testing strategies for newly diagnosed ovarian cancer: (1) ASCO strategy and (2) tumor testing triage for germline testing. Data were applied from SOLO-1, and costs were from wholesale acquisition prices, Medicare, and published sources. Sensitivity analyses accounted for uncertainty around various parameters. Monte Carlo simulation estimated the number tested and identified with germline and somatic BRCA PV for olaparib maintenance treatment annually in the US population. RESULTS: The ASCO strategy was more effective but more costly than tumor testing triage in identifying patients for olaparib, with an incremental cost-effectiveness ratio of $281,296 US dollars per progression-free life year gained. Assuming 10,000 eligible patients with ovarian cancer annually, Monte Carlo simulation yielded comparable numbers of patients with BRCA PV in the germline and tumor with the ASCO and tumor testing triage strategies (2,080 v 2,062, respectively), but substantially higher number of patients tested using the ASCO strategy (8,052 v 3,076). CONCLUSION: The ASCO strategy may identify more BRCA PVs but is not cost-effective. Tumor testing in epithelial ovarian cancer as triage for germline testing is the favored strategy in this health care system.
Leukemia, 2022
Xiang, Ping, Yang, Xining, Escano, Leo, Dhillon, Ishpreet, Schneider, Edith, Clemans-Gibbon, Jack, Wei, Wei, Wong, Jasper, Wang, Simon Xufeng, Tam, Derek, Deng, Yu, Yung, Eric, Morin, Gregg B, Hoodless, Pamela A, Hirst, Martin, Karsan, Aly, Kuchenbauer, Florian, Humphries, R Keith, Rouhi, Arefeh
Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is a critical factor in the pathogenesis of a large subset of acute myeloid leukemia (AML). Despite the clinical relevance of MEIS1, its regulation is largely unknown. To understand the transcriptional regulatory mechanisms contributing to human MEIS1 expression, we created a knock-in green florescent protein (GFP) reporter system at the endogenous MEIS1 locus in a human AML cell line. Using this model, we have delineated and dissected a critical enhancer region of the MEIS1 locus for transcription factor (TF) binding through in silico prediction in combination with oligo pull-down, mass-spectrometry and knockout analysis leading to the identification of FLI1, an E-twenty-six (ETS) transcription factor, as an important regulator of MEIS1 transcription. We further show direct binding of FLI1 to the MEIS1 locus in human AML cell lines as well as enrichment of histone acetylation in MEIS1-high healthy and leukemic cells. We also observe a positive correlation between high FLI1 transcript levels and worse overall survival in AML patients. Our study expands the role of ETS factors in AML and our model constitutes a feasible tool for a more detailed understanding of transcriptional regulatory elements and their interactome.
The Journal of experimental medicine, 2022
Gopal, Aparna, Ibrahim, Rawa, Fuller, Megan, Umlandt, Patricia, Parker, Jeremy, Tran, Jessica, Chang, Linda, Wegrzyn-Woltosz, Joanna, Lam, Jeffrey, Li, Jenny, Lu, Melody, Karsan, Aly
Inflammation is associated with bone marrow failure syndromes, but how specific molecules impact the bone marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS) and suppresses all three major hematopoietic lineages. TIRAP expression promotes up-regulation of Ifnγ, leading to myelosuppression through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the bone marrow endothelial niche. Deletion of Ifnγ blocks Hmgb1 release and is sufficient to reverse the endothelial defect and restore myelopoiesis. Contrary to current dogma, TIRAP-activated Ifnγ-driven bone marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of MDS to acute leukemia. These findings reveal novel, noncanonical roles of TIRAP, Hmgb1, and Ifnγ in the bone marrow microenvironment and provide insight into the pathophysiology of preleukemic syndromes.
Nature communications, 2021
Docking, T Roderick, Parker, Jeremy D K, Jädersten, Martin, Duns, Gerben, Chang, Linda, Jiang, Jihong, Pilsworth, Jessica A, Swanson, Lucas A, Chan, Simon K, Chiu, Readman, Nip, Ka Ming, Mar, Samantha, Mo, Angela, Wang, Xuan, Martinez-Høyer, Sergio, Stubbins, Ryan J, Mungall, Karen L, Mungall, Andrew J, Moore, Richard A, Jones, Steven J M, Birol, İnanç, Marra, Marco A, Hogge, Donna, Karsan, Aly
As more clinically-relevant genomic features of myeloid malignancies are revealed, it has become clear that targeted clinical genetic testing is inadequate for risk stratification. Here, we develop and validate a clinical transcriptome-based assay for stratification of acute myeloid leukemia (AML). Comparison of ribonucleic acid sequencing (RNA-Seq) to whole genome and exome sequencing reveals that a standalone RNA-Seq assay offers the greatest diagnostic return, enabling identification of expressed gene fusions, single nucleotide and short insertion/deletion variants, and whole-transcriptome expression information. Expression data from 154 AML patients are used to develop a novel AML prognostic score, which is strongly associated with patient outcomes across 620 patients from three independent cohorts, and 42 patients from a prospective cohort. When combined with molecular risk guidelines, the risk score allows for the re-stratification of 22.1 to 25.3% of AML patients from three independent cohorts into correct risk groups. Within the adverse-risk subgroup, we identify a subset of patients characterized by dysregulated integrin signaling and RUNX1 or TP53 mutation. We show that these patients may benefit from therapy with inhibitors of focal adhesion kinase, encoded by PTK2, demonstrating additional utility of transcriptome-based testing for therapy selection in myeloid malignancy.
Blood, 2020
Grants, Jennifer M, Wegrzyn, Joanna, Hui, Tony, O'Neill, Kieran, Shadbolt, Marion, Knapp, David J H F, Parker, Jeremy, Deng, Yu, Gopal, Aparna, Docking, T Roderick, Fuller, Megan, Li, Jenny, Boldin, Mark, Eaves, Connie J, Hirst, Martin, Karsan, Aly
Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging ("inflammaging") has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a-/- myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a-/- HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a-/- mice. miR-146a-/- HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.
Nature cell biology, 2020
Martinez-Høyer, Sergio, Deng, Yu, Parker, Jeremy, Jiang, Jihong, Mo, Angela, Docking, T Roderick, Gharaee, Nadia, Li, Jenny, Umlandt, Patricia, Fuller, Megan, Jädersten, Martin, Kulasekararaj, Austin, Malcovati, Luca, List, Alan F, Hellström-Lindberg, Eva, Platzbecker, Uwe, Karsan, Aly
Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common structural genomic variant in myelodysplastic syndromes (MDS){{sup}}1{{/sup}}. Lenalidomide (LEN) is the treatment of choice for patients with del(5q) MDS, but half of the responding patients become resistant{{sup}}2{{/sup}} within 2 years. TP53 mutations are detected in ~20% of LEN-resistant patients{{sup}}3{{/sup}}. Here we show that patients who become resistant to LEN harbour recurrent variants of TP53 or RUNX1. LEN upregulated RUNX1 protein and function in a CRBN- and TP53-dependent manner in del(5q) cells, and mutation or downregulation of RUNX1 rendered cells resistant to LEN. LEN induced megakaryocytic differentiation of del(5q) cells followed by cell death that was dependent on calpain activation and CSNK1A1 degradation{{sup}}4,5{{/sup}}. We also identified GATA2 as a LEN-responsive gene that is required for LEN-induced megakaryocyte differentiation. Megakaryocytic gene-promoter analyses suggested that LEN-induced degradation of IKZF1 enables a RUNX1-GATA2 complex to drive megakaryocytic differentiation. Overexpression of GATA2 restored LEN sensitivity in the context of RUNX1 or TP53 mutations by enhancing LEN-induced megakaryocytic differentiation. Screening for mutations that block LEN-induced megakaryocytic differentiation should identify patients who are resistant to LEN.
The Journal of molecular diagnostics : JMD, 2020
Moore, Richard A, Zeng, Thomas, Docking, T Roderick, Bosdet, Ian, Butterfield, Yaron S, Munro, Sarah, Li, Irene, Swanson, Lucas, Starks, Elizabeth R, Tse, Kane, Mungall, Andrew J, Holt, Robert A, Karsan, Aly
Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (<30 bases) and contain no long homopolymer (>7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.
Cell reports, 2019
Coulombe, Patrick, Paliouras, Grigorios N, Clayton, Ashley, Hussainkhel, Angela, Fuller, Megan, Jovanovic, Vida, Dauphinee, Shauna, Umlandt, Patricia, Xiang, Ping, Kyle, Alistair H, Minchinton, Andrew I, Humphries, R Keith, Hoodless, Pamela A, Parker, Jeremy D K, Wright, Joanne L, Karsan, Aly
The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1{{sup}}-/-{{/sup}} mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with β-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash1 in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy.
Nature communications, 2018
Lam, Jeffrey, van den Bosch, Marion, Wegrzyn, Joanna, Parker, Jeremy, Ibrahim, Rawa, Slowski, Kate, Chang, Linda, Martinez-Høyer, Sergio, Condorelli, Gianluigi, Boldin, Mark, Deng, Yu, Umlandt, Patricia, Fuller, Megan, Karsan, Aly
Expression of miR-143 and miR-145 is reduced in hematopoietic stem/progenitor cells (HSPCs) of myelodysplastic syndrome patients with a deletion in the long arm of chromosome 5. Here we show that mice lacking miR-143/145 have impaired HSPC activity with depletion of functional hematopoietic stem cells (HSCs), but activation of progenitor cells (HPCs). We identify components of the transforming growth factor β (TGFβ) pathway as key targets of miR-143/145. Enforced expression of the TGFβ adaptor protein and miR-145 target, Disabled-2 (DAB2), recapitulates the HSC defect seen in miR-143/145{{sup}}-/-{{/sup}} mice. Despite reduced HSC activity, older miR-143/145{{sup}}-/-{{/sup}} and DAB2-expressing mice show elevated leukocyte counts associated with increased HPC activity. A subset of mice develop a serially transplantable myeloid malignancy, associated with expansion of HPC. Thus, miR-143/145 play a cell context-dependent role in HSPC function through regulation of TGFβ/DAB2 activation, and loss of these miRNAs creates a preleukemic state.
The Journal of experimental medicine, 2015
Kulic, Iva, Robertson, Gordon, Chang, Linda, Baker, Jennifer H E, Lockwood, William W, Mok, Winnie, Fuller, Megan, Fournier, Michèle, Wong, Nelson, Chou, Vennie, Robinson, Mark D, Chun, Hye-Jung, Gilks, Blake, Kempkes, Bettina, Thomson, Thomas A, Hirst, Martin, Minchinton, Andrew I, Lam, Wan L, Jones, Steven, Marra, Marco, Karsan, Aly
Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.
Proceedings of the National Academy of Sciences of the United States of America, 2012
Chang, Linda, Noseda, Michela, Higginson, Michelle, Ly, Michelle, Patenaude, Alexandre, Fuller, Megan, Kyle, Alastair H, Minchinton, Andrew I, Puri, Mira C, Dumont, Daniel J, Karsan, Aly
Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.
The Journal of clinical investigation, 2011
Starczynowski, Daniel T, Lockwood, William W, Deléhouzée, Sophie, Chari, Raj, Wegrzyn, Joanna, Fuller, Megan, Tsao, Ming-Sound, Lam, Stephen, Gazdar, Adi F, Lam, Wan L, Karsan, Aly
Somatic mutations and copy number alterations (as a result of deletion or amplification of large portions of a chromosome) are major drivers of human lung cancers. Detailed analysis of lung cancer-associated chromosomal amplifications could identify novel oncogenes. By performing an integrative cytogenetic and gene expression analysis of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines and tumors, we report here the identification of a frequently recurring amplification at chromosome 11 band p13. Within this region, only TNF receptor-associated factor 6 (TRAF6) exhibited concomitant mRNA overexpression and gene amplification in lung cancers. Inhibition of TRAF6 in human lung cancer cell lines suppressed NF-κB activation, anchorage-independent growth, and tumor formation. In these lung cancer cell lines, RAS required TRAF6 for its oncogenic capabilities. Furthermore, TRAF6 overexpression in NIH3T3 cells resulted in NF-κB activation, anchorage-independent growth, and tumor formation. Our findings show that TRAF6 is an oncogene that is important for RAS-mediated oncogenesis and provide a mechanistic explanation for the previously apparent importance of constitutive NF-κB activation in RAS-driven lung cancers.
Developmental cell, 2011
Chang, Alex C Y, Fu, YangXin, Garside, Victoria C, Niessen, Kyle, Chang, Linda, Fuller, Megan, Setiadi, Audi, Smrz, Justin, Kyle, Alastair, Minchinton, Andrew, Marra, Marco, Hoodless, Pamela A, Karsan, Aly
The heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC).
Blood, 2011
Starczynowski, Daniel T, Morin, Ryan, McPherson, Andrew, Lam, Jeff, Chari, Raj, Wegrzyn, Joanna, Kuchenbauer, Florian, Hirst, Martin, Tohyama, Kaoru, Humphries, R Keith, Lam, Wan L, Marra, Marco, Karsan, Aly
Cytogenetic alterations, such as amplifications, deletions, or translocations, contribute to myeloid malignancies. MicroRNAs (miRNAs) have emerged as critical regulators of hematopoiesis, and their aberrant expression has been associated with leukemia. Genomic regions containing sequence alterations and fragile sites in cancers are enriched with miRNAs; however, the relevant miRNAs within these regions have not been evaluated on a global basis. Here, we investigated miRNAs relevant to acute myeloid leukemia (AML) by (1) mapping miRNAs within leukemia-associated genomic alterations in human AML cell lines by high-resolution genome arrays and (2) evaluating absolute expression of these miRNAs by massively parallel small RNA sequencing. Seventy-seven percent (542 of 706) of miRNAs mapped to leukemia-associated copy-number alterations in the cell lines; however, only 18% (99 of 542) of these miRNAs are expressed above background levels. As evidence that this subset of miRNAs is relevant to leukemia, we show that loss of 2 miRNAs identified in our analysis, miR-145 and miR-146a, results in leukemia in a mouse model. Small RNA sequencing identified 28 putative novel miRNAs, 18 of which map to leukemia-associated copy-number alterations. This detailed genomic and small RNA analysis points to a subset of miRNAs that may play a role in myeloid malignancies.
Nature medicine, 2010
Starczynowski, Daniel T, Kuchenbauer, Florian, Argiropoulos, Bob, Sung, Sandy, Morin, Ryan, Muranyi, Andrew, Hirst, Martin, Hogge, Donna, Marra, Marco, Wells, Richard A, Buckstein, Rena, Lam, Wan, Humphries, R Keith, Karsan, Aly
5q- syndrome is a subtype of myelodysplastic syndrome characterized by severe anemia and variable neutropenia but normal or high platelet counts with dysplastic megakaryocytes. We examined expression of microRNAs (miRNAs) encoded on chromosome 5q as a possible cause of haploinsufficiency. We show that deletion of chromosome 5q correlates with loss of two miRNAs that are abundant in hematopoietic stem/progenitor cells (HSPCs), miR-145 and miR-146a, and we identify Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) as respective targets of these miRNAs. TIRAP is known to lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6 in mouse HSPCs resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow progressed either to marrow failure or acute myeloid leukemia. Thus, inappropriate activation of innate immune signals in HSPCs phenocopies several clinical features of 5q- syndrome.
