Veronique Voisin

Attenuation of miR-126 Activity Expands HSC In Vivo without Exhaustion

Summary Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.

A Comparative Transcriptomic Analysis Reveals Conserved Features of Stem Cell Pluripotency in Planarians and Mammals

Many long‐lived species of animals require the function of adult stem cells throughout their lives. However, the transcriptomes of stem cells in invertebrates and vertebrates have not been compared, and consequently, ancestral regulatory circuits that control stem cell populations remain poorly defined. In this study, we have used data from high‐throughput RNA sequencing to compare the transcriptomes of pluripotent adult stem cells from planarians with the transcriptomes of human and mouse pluripotent embryonic stem cells. From a stringently defined set of 4,432 orthologs shared between planarians, mice and humans, we identified 123 conserved genes that are ≥5‐fold differentially expressed in stem cells from all three species. Guided by this gene set, we used RNAi screening in adult planarians to discover novel stem cell regulators, which we found to affect the stem cell‐associated functions of tissue homeostasis, regeneration, and stem cell maintenance. Examples of genes that disrupted these processes included the orthologs of TBL3, PSD12, TTC27, and RACK1. From these analyses, we concluded that by comparing stem cell transcriptomes from diverse species, it is possible to uncover conserved factors that function in stem cell biology. These results provide insights into which genes comprised the ancestral circuitry underlying the control of stem cell self‐renewal and pluripotency. STEM Cells2012;30:1734–1745

Quiescent Sox2+ Cells Drive Hierarchical Growth and Relapse in Sonic Hedgehog Subgroup Medulloblastoma

Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2(+) cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2(+) cells produce rapidly cycling doublecortin(+) progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2(+) cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2(+) cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2(+) cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2(+) cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma.

miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells

Summary To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.

Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

Tracing the origins of relapse in acute myeloid leukaemia to stem cells

In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.

Combined deletion of Pten and p53 in mammary epithelium accelerates triple‐negative breast cancer with dependency on eEF2K

The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary‐specific deletion of Pten via WAP‐Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV‐Cre, which targets basal/luminal progenitors. Combined Pten‐p53 mutations accelerated formation of claudin‐low, triple‐negative‐like breast cancer (TNBC) that exhibited hyper‐activated AKT signaling and more mesenchymal features relative to Pten or p53 single‐mutant tumors. Twenty‐four genes that were significantly and differentially expressed between WAP‐Cre:Pten/p53 and MMTV‐Cre:Pten/p53 tumors predicted poor survival for claudin‐low patients. Kinome screens identified eukaryotic elongation factor‐2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53‐deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53‐deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro. Our results identify a prognostic signature for claudin‐low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling.

Seventeen-gene signature from enriched Her2/Neu mammary tumor-initiating cells predicts clinical outcome for human HER2+:ERα− breast cancer

Human Epidermal Growth Factor Receptor 2-positive (HER2+) breast cancer (BC) is a highly aggressive disease commonly treated with chemotherapy and anti-HER2 drugs, including trastuzumab. There is currently no way to predict which HER2+ BC patients will benefit from these treatments. Previous prognostic signatures for HER2+ BC were developed irrespective of the subtype or the hierarchical organization of cancer in which only a fraction of cells, tumor-initiating cells (TICs), can sustain tumor growth. Here, we used serial dilution and single-cell transplantation assays to identify MMTV-Her2/Neu mouse mammary TICs as CD24+:JAG1− at a frequency of 2–4.5%. A 17-gene Her2-TIC-enriched signature (HTICS), generated on the basis of differentially expressed genes in TIC versus non-TIC fractions and trained on one HER2+ BC cohort, predicted clinical outcome on multiple independent HER2+ cohorts. HTICS included up-regulated genes involved in S/G2/M transition and down-regulated genes involved in immune response. Its prognostic power was independent of other predictors, stratified lymph node+ HER2+ BC into low and high-risk subgroups, and was specific for HER2+:estrogen receptor alpha-negative (ERα−) patients (10-y overall survival of 83.6% for HTICS− and 24.0% for HTICS+ tumors; hazard ratio = 5.57; P = 0.002). Whereas HTICS was specific to HER2+:ERα− tumors, a previously reported stroma-derived signature was predictive for HER2+:ERα+ BC. Retrospective analyses revealed that patients with HTICS+ HER2+:ERα− tumors resisted chemotherapy but responded to chemotherapy plus trastuzumab. HTICS is, therefore, a powerful prognostic signature for HER2+:ERα− BC that can be used to identify high risk patients that would benefit from anti-HER2 therapy.

Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells

Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRβ, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.

Musashi-2 attenuates AHR signalling to expand human haematopoietic stem cells

Umbilical cord blood-derived haematopoietic stem cells (HSCs) are essential for many life-saving regenerative therapies. However, despite their advantages for transplantation, their clinical use is restricted because HSCs in cord blood are found only in small numbers. Small molecules that enhance haematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified, but in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular circuitry that underpins the self-renewal of human HSCs will facilitate the development of targeted strategies that expand HSCs for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs, the post-transcriptional mechanisms that guide HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2–RNA interactions, we show that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signalling through post-transcriptional downregulation of canonical AHR pathway components in cord blood HSPCs. Our study gives mechanistic insight into RNA networks controlled by RNA-binding proteins that underlie self-renewal and provides evidence that manipulating such networks ex vivo can enhance the regenerative potential of human HSCs.