Dormant Stem Research Articles

Increased Retinoic Acid Catabolism in Olfactory Sensory Neurons Activates Dormant Tissue-Specific Stem Cells and Accelerates Age-Related Metaplasia.

The cellular and molecular basis of metaplasia and declining neurogenesis in the aging olfactory epithelium (OE) remains unknown. The horizontal basal cell (HBC) is a dormant tissue-specific stem cell presumed to only be forced into self-renewal and differentiation by injury. Here we analyze male and female mice and show that HBCs also are activated with increasing age as well as non-cell-autonomously by increased expression of the retinoic acid-degrading enzyme CYP26B1. Activating stimuli induce HBCs throughout OE to acquire a rounded morphology and express IP3R3, which is an inositol-1,4,5-trisphosphate receptor constitutively expressed in stem cells of the adjacent respiratory epithelium. Odor/air stimulates CYP26B1 expression in olfactory sensory neurons mainly located in the dorsomedial OE, which is spatially inverse to ventrolateral constitutive expression of the retinoic acid-synthesizing enzyme (RALDH1) in supporting cells. In ventrolateral OE, HBCs express low p63 levels and preferentially differentiate instead of self-renewing when activated. When activated by chronic CYP26B1 expression, repeated injury, or old age, ventrolateral HBCs diminish in number and generate a novel type of metaplastic respiratory cell that is RALDH- and secretes a mucin-like mucus barrier protein (FcγBP). Conversely, in the dorsomedial OE, CYP26B1 inhibits injury-induced and age-related replacement of RALDH- supporting cells with RALDH1+ ciliated respiratory cells. Collectively, these results support the concept that inositol-1,4,5-trisphosphate type 3 receptor signaling in HBCs, together with altered retinoic acid metabolism within the niche, promote HBC lineage commitment toward two types of respiratory cells that will maintain epithelial barrier function once the capacity to regenerate OE cells ceases.SIGNIFICANCE STATEMENT Little is known about signals that activate dormant stem cells to self-renew and regenerate odor-detecting neurons and other olfactory cell types after loss due to injury, infection, or toxin exposure in the nose. It is also unknown why the stem cells do not prevent age-dependent decline of odor-detecting neurons. We show that (1) stem cells are kept inactive by the vitamin A derivative retinoic acid, which is synthesized and degraded locally by olfactory cells; (2) old age as well as repeated injuries activate the stem cells and exhaust their potential to produce olfactory cells; and (3) exhausted stem cells alter the local retinoic acid metabolism and maintain the epithelial tissue barrier by generating airway cells instead of olfactory cells.

Alternatively Polarized Macrophages Regulate the Growth and Differentiation of Ependymal Stem Cells through the SIRT2 Pathway.

Ependymal stem cells (EpSCs) are dormant stem cells in the adult spinal cord that proliferate rapidly and migrate to the site of injury after spinal cord injury (SCI). Although they can differentiate into neurons under appropriate conditions in vitro, EpSCs mainly differentiate into astrocytes in vivo. Our previous study confirmed that alternatively polarized macrophages (M2) facilitate the differentiation of EpSCs towards neurons, but the detailed mechanism remains elusive. In the present study, we found that M2 conditioned medium could upregulate the expression of Sirtuin 2 (SIRT2) in EpSCs in vitro through the BDNF/TrkB-MEK/ERK signaling pathway. As an important deacetylase, SIRT2 deacetylated stable Ac-α-tubulin (Acetyl alpha Tubulin) in microtubules and thus promoted EpSC differentiation into neurons. The present study provides a theoretical basis and a new way to improve neural recovery, such as regulating the growth and differentiation of EpSCs by increasing the proportion of M2 cells in the local microenvironment or upregulating the expression of SIRT2 in EpSCs.

The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission.

Chronic myeloid leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the BM of patients having achieved remission under treatment. We demonstrate by single-cell RNASequencing analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38– leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their BM microenvironment.

BEX2 Suppresses Mitochondrial Activity and Is Required for Dormant Cancer Stem Cell Maintenance in Intrahepatic Cholangiocarcinoma

Cancer stem cells (CSCs) define a subpopulation of cancer cells that are resistant to therapy. However, little is known of how CSC characteristics are regulated. We previously showed that dormant cancer stem cells are enriched with a CD274low fraction of cholangiocarcinoma cells. Here we found that BEX2 was highly expressed in CD274low cells, and that BEX2 knockdown decreased the tumorigenicity and G0 phase of cholangiocarcinoma cells. BEX2 was found to be expressed predominantly in G0 phase and starvation induced the USF2 transcriptional factor, which induced BEX2 transcription. Comprehensive screening of BEX2 binding proteins identified E3 ubiquitin ligase complex proteins, FEM1B and CUL2, and a mitochondrial protein TUFM, and further demonstrated that knockdown of BEX2 or TUFM increased mitochondria-related oxygen consumption and decreased tumorigenicity in cholangiocarcinoma cells. These results suggest that BEX2 is essential for maintaining dormant cancer stem cells through the suppression of mitochondrial activity in cholangiocarcinoma. Funding Statement: This research was supported in part by the Biomedical Research Core of Tohoku University Graduate School of Medicine. This research was supported in part by JSPS KAKENHI grant numbers JP: 19K08430, 17K10716, and 16K07132, and Practical Research for Innovative Cancer Control from AMED by grant number JP17ck0106197, The Cooperative Research Project Program of Joint Usage/Research Center at The Institute of Development, Aging and Cancer, Tohoku University, and Takeda Medical Foundation. Declaration of Interests: There are no financial conflicts of interest. Ethics Approval Statement: This study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Ethics Committees at Miyagi Cancer Center (Natori, Japan) and Tohoku University Graduate School of Medicine (Sendai, Japan). The protocol of animal experiments was approved by the Miyagi Cancer Center Animal Care and Use Committee (permit number: MCC-AE-2019-8).

Disseminated Tumor Cells and Dormancy in Breast Cancer Progression.

Hematogenous dissemination of single cancer cells is a common phenomenon in patients with solid tumors. These cells may experience different fates: most will die during the process; some will grow into metastasis and some will persist in secondary homing sites for many years in a state referred to as dormancy. The mechanisms of this state are still not clear; single cancer cells can survive either by completely withdrawing from the cell cycle or by continuing to proliferate at a slow rate that is counterbalanced by cell death. Another hypothesis assumes that at least some of dormant tumor cells feature stem cell-like characteristics that may contribute to their extremely long half-lives and enhance chemotherapy resistance. Breast cancer is particularly known for prolonged periods of clinical freedom of disease (sometimes up to 20-30years), followed by a distant relapse. In this chapter, we explore the relationship between the clinical phenomenon of tumor dormancy and the disseminated tumor cells and discuss the potential implications for treatment.

Discrimination of Dormant and Active Hematopoietic Stem Cells by G0 Marker Reveals Dormancy Regulation by Cytoplasmic Calcium

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between "dormant" and "active" HSCs remains unresolved. Here we generate a G0 marker (G0M) mouse line that visualizes quiescent cells and identify a small population of active HSCs (G0Mlow), which are distinct from dormant HSCs (G0Mhigh), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of thesepopulations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca2+]c) are linked to dormancy of HSCs. These findings indicate that G0M separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca2+]c. This G0M mouse line represents a useful resource for investigating physiologically important stem cell subpopulations.

1021 - ENDOTHELIAL NICHE ADHESIVE REGULATION OF NORMAL AND MALIGNANT HAEMATOPOIETIC STEM CELLS

Our laboratory previously identified a novel role for vascular cell adhesion molecules in promoting the awakening of dormant Haematopoietic Stem Cells (HSCs) in the bone marrow (BM) (Nat Med 2012). Now we show vascular niche adhesion can trigger a cascade of intracellular signalling events that can metabolically and transcriptionally reprogram a cell. For bone marrow (BM) Haematopoietic Stem and Progenitor Cells (HSPC) these open new avenues to tailor immune response and recovery during stress. Malignant cells also take advantage of these pathways. In malignant cells, aberrant glycosylation associated with oncogenic transformation promotes expression of surface ligands that utilise these pro-survival pathways. The discovery of how cellular adhesion acts to prime leukocytes for appropriate inflammatory response and how this evolutionary-ancient signalling network is hijacked by malignant cells for growth and survival, are the subject of this talk.

1030 - REGULATION OF HEMATOPOIETIC AND IMMUNE ESCAPE OF AML STEM CELLS

Dormant hematopoietic stem cells (HSCs) are forming the top of the hematopoietic hierarchy. Here we identify Neogenin, a cell surface receptor for neural guidance molecules, to be specifically expressed in dormant HSCs. Loss of Neogenin leads to an initial increase in HSC proliferation followed by premature HSC exhaustion. The Neogenin ligand Netrin-1 induces Egr1 expression in HSC and maintains HSC engraftment potential in vitro in a Neogenin-dependent manner. Conditional deletion of Netrin-1 in bone marrow niche cells leads to a depletion of HSCs associated to signs of activation and premature differentiation. Netrin-1 is produced by arteriolar endothelial and peri-arteriolar stromal cells within the bone marrow niche and Its expression becomes diminished upon aging. Our study identifies with Netrin-1-Neogenin a novel ligand-receptor pair regulating HSC quiescence and long-term self-renewal, which is altered during aging. Patients with acute myeloid leukaemia (AML) often achieve remission after therapy yet subsequently die of relapse driven by chemotherapy resistant leukaemic stem cells (LSCs). Using human AML xenografts and syngeneic mouse leukaemia models, we show that ligands of the danger detector NKG2D, a critical mediator of anti-tumour immunity by cytotoxic lymphocytes such as natural killer (NK) cells, are generally expressed on bulk AML cells but not on LSCs. These NKG2DLneg AML cells display molecular and functional stemness characteristics and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant (PDX) models. Mechanistically, PARP1 represses NKG2DL expression and inhibition of PARP1 induces NKG2DL on the LSC surface. Treatment with PARPi followed by transfer of polyclonal NK cells suppresses leukaemogenesis in PDX models (Paczulla et al., Nature 2019). In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition which renders them amenable to NK cell control in vivo.

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ASIA-PACIFIC – A microRNA study might cast new light on ALS treatment. ASIA-PACIFIC – New standard of care for advanced nose cancer patients. ASIA-PACIFIC – Dormant neural stem cells in fruit flies activate to generate new brain cells. REST OF THE WORLD – A wearable that’s not to be laughed at. REST OF THE WORLD – Junk food could be responsible for the food allergy epidemic.

A specialized post‐transcriptional program in chemoresistant, quiescent cancer cells

Quiescent (G0) cells are a relevant fraction in cancers, which include dormant stem cells, and resist clinical therapy. G0 cells reveal changes in gene expression at the protein and translation levels. We previously identified that the translation mechanism is altered in G0 cancer cells. MicroRNAs—noncoding RNAs that target mRNAs to alter gene expression—associate with a key RNA‐binding protein, and recruit non‐canonical translation factors to mediate specific mRNA translation in G0. We find that G0 leukemic cells show similar proteome and translatome to cells isolated post‐chemotherapy. These data suggest that specialized post‐transcriptional mechanisms in G0 leukemic cells produce a distinct translatome to facilitate chemoresistance.To understand post‐transcriptional regulation in chemoresistance, we compared global transcriptome, translatome and proteome in chemoresistant G0 acute monocytic leukemic (AML) cells. We find that chemotherapy or G0 leads to DNA damage responsive ATM and stress signaling, which alter post‐transcriptional and translational mechanisms. ATM and stress activated p38 MAPK/MK2 increase AU‐rich‐element (ARE) bearing pro‐inflammatory cytokine and immune gene mRNAs, by regulating a key ARE RNA binding protein and modifying canonical translation. AREs are present on 3′UTRs of tightly regulated cytokines to post‐transcriptionally control their expression. Both rate limiting steps—tRNA recruitment and mRNA cap recognition—in canonical translation are altered. Stress signaling activates kinases that reduce canonical tRNA recruitment. This signaling also reduces mTOR activity in G0 and chemoresistant cells, which activates the cap complex inhibitor, eIF4EBP to impair canonical translation, leading to non‐canonical translation of specific mRNAs with specialized cap binding and ribosome recruitment factors.These changes permit translation of ARE bearing pro‐inflammatory cytokine TNFα, and immune modulators that promote survival. Co‐inhibiting p38 MAPK and TNFα that promote anti‐apoptosis— prior to or along with chemotherapy—decreases chemoresistance in AML cells, in vivo, and in patient samples, without affecting normal cells. Our studies reveal a pro‐inflammatory subpopulation in AML that mediates resistance, enabled by DNA damage‐ and stress‐regulated post‐transcriptional and translational mechanisms that are mediated by AU‐rich‐elements and a critical ARE RNA binding protein. Disrupting ARE regulation reduces TNFα and chemoresistance, revealing AREs, the ARE RNA binding protein and non‐canonical translation as regulators of chemoresistance. These studies reveal the importance of post‐transcriptional regulation of pro‐inflammatory genes in chemoresistance.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CARDIAC TAMPONADE: AN UNCOMMON PRESENTATION OF COMMON INFLUENZA INFECTION

Abstract Quiescent (G0) cells are transient cell-cycle-arrested subpopulations in cancers that include dormant cancer stem cells. While leukemic stem cells/minimal-residual-disease were studied transcriptionally, their translation profile and post-transcriptional mechanisms that control their proteome—and thereby, survival—are unknown. We find G0 leukemic cells are chemoresistant, with altered canonical translation and similar proteome and translatome—rather than transcriptome—to cells isolated post-chemotherapy, implicating post-transcriptional regulation in chemoresistance. Mechanistically, we find DNA-damage-responsive-ATM and stress-activated-p38-MAPK/MK2 alter post-transcriptional mechanisms, regulating mRNA-decay-factor, TTP, to increase AU-rich-element-bearing mRNAs. This permits translation of AU-rich-element-bearing pro-inflammatory-cytokine TNFα, and immune modulators (CD47) that promote survival. Co-inhibiting p38-MAPK/MK2 and TNFα—prior to/alongwith chemotherapy—decreases chemoresistance in vivo and in patient samples. Disrupting TTP regulation reduces TNFα and chemoresistance, revealing TTP as a regulator of inflammation-mediated chemoresistance. These studies uncover a pro-inflammatory subpopulation in cancer that enables chemoresistance via DNA-damage- and stress-regulated post-transcriptional mechanisms, and develop a new combination therapy against chemo-survival. Statement of Significance These studies reveal the significance of post-transcriptional regulation of inflammatory/immune response in chemoresistance, mediated by AU-rich-elements, mRNA-decay-regulator TTP, and non-canonical translation. These results reveal DNA-damage/stress-induced TTP as a key regulator of inflammation-mediated chemoresistance and developed a new combination therapy to reduce resistance in cell lines, patient samples and in vivo.

Discrimination of Dormant and Active Hematopoietic Stem Cells by G <sub>0</sub> Markers Reveals Dormancy Regulation by Basal Cytoplasmic Calcium

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between “dormant” and “active” HSCs remains unresolved. Here we generated a G0 marker mouse line that visualizes quiescent cells. The G0 marker identifies a population of active HSCs (G0 markerlow) which are rare and distinct from dormant HSCs (G0 markerhigh) within the conventional quiescent HSC fraction. Single-cell RNA-sequencing analyses showed that the gene expression profiles of these populations were nearly identical, yet differed in their Cdk4/6 activity. Furthermore high-throughput small molecule screening revealed that high levels of basal cytoplasmic calcium (Ca2+) protected the dormancy of HSCs from cell cycle activation induced by calcium influx. These findings indicate that the G0 marker separates dormant and active adult HSCs, which are regulated by Cdk4/6 and Ca2+ levels. This G0 marker mouse line represents a useful new resource for investigating physiologically important stem cell subpopulations.

Vasculogenic Stem and Progenitor Cells in Human: Future Cell Therapy Product or Liquid Biopsy for Vascular Disease.

New blood vessel formation in adults was considered to result exclusively from sprouting of preexisting endothelial cells, a process referred to angiogenesis. Vasculogenesis, the formation of new blood vessels from endothelial progenitor cells, was thought to occur only during embryonic life. Discovery of adult endothelial progenitor cells (EPCs) in 1997 opened the door for cell therapy in vascular disease. Endothelial progenitor cells contribute to vascular repair and are now well established as postnatal vasculogenic cells in humans. It is now admitted that endothelial colony-forming cells (ECFCs) are the vasculogenic subtype. ECFCs could be used as a cell therapy product and also as a liquid biopsy in several vascular diseases or as vector for gene therapy. However, despite a huge interest in these cells, their tissue and molecular origin is still unclear. We recently proposed that endothelial progenitor could come from very small embryonic-like stem cells (VSELs) isolated in human from CD133 positive cells. VSELs are small dormant stem cells related to migratory primordial germ cells. They have been described in bone marrow and other organs. This chapter discusses the reported findings from in vitro data and also preclinical studies that aimed to explore stem cells at the origin of vasculogenesis in human and then explore the potential use of ECFCs to promote newly formed vessels or serve as liquid biopsy to understand vascular pathophysiology and in particular pulmonary disease and haemostasis disorders.

Synergistic Effect of Imatinib and Ruxolitinib in a Patient with JAK2V617F positive Myelofibrosis and Concomitant BCR-ABL1 positive Chronic Myeloid Leukemia

Introduction:JAK2V617Fmutation is present in majority of polycythemia vera (PV) and in 50-60% of patients with primary myelofibrosis (MF). JAK1/2 inhibitor ruxolitinib (RUX) is approved for treatment of both PV and intermediate/high risk MF. Two independent randomized studies showed the benefit of RUX in MF with significant reduction in spleen volume and improvement in constitutional symptoms compared to best supportive care. Clonal evolution may occur in PV and MF upon acquisition of new mutations and transformation to acute myeloid leukemia. Rarely, patients with JAK2V617Fpositive PV or MF have also concomitant BCR-ABL1 fusion gene with phenotype of chronic myelogenous leukemia (CML). Imatinib, a BCR-ABL1 inhibitor, induces long-term cytogenetic and molecular remissions in CML. Single agent imatinib has shown no clinical benefit in MF patients despite in vitro efficacy (Gaikwad et al Exp. Hematol. 2007). We describe here a patient with JAK2V617F positive post-PV MF who later developed concomitant BCR-ABL+ CML. Splenomegaly was initially resolved by RUX but returned coincidently with the diagnosis of CML and was again normalized by imatinib alone. Massive splenomegaly reappeared along with elevated hematocrit on imatinib and was resolved by the addition of low dose RUX.Case report: A 71-year old woman initially presented in September 2011 with isolated erythrocytosis and splenomegaly. A diagnosis of PV was established based on the presence of the JAK2V617Fmutation and hypercellular marrow. Bone marrow karyotype was normal (Table 1). The patient was randomized to pegylated interferon arm in the MPD Consortium study. A complete hematologic response was achieved after nearly 2 years on maximal dose 180 mcg weekly. In early 2015, she developed constitutional symptoms, and progressive splenomegaly. Progression to post-PV MF was confirmed on bone marrow evaluation. The patient was taken off study and started on RUX 20 mg BID. She developed severe anemia 4 months later requiring dose reduction to 10 mg BID. The anemia markedly improved, while JAK2V617F allele burden remained high at 88% with normal cytogenetics. In December 2017, the patient developed anemia, fatigue and rapid regrowth of spleen. Marrow karyotype showed Philadelphia (Ph) chromosome in 25% of the cells analyzed, PCR was positive for BCR-ABL1 p210, and JAK2V617F allele burden was reduced to 56.4%. In January 2018, she started imatinib 400 mg daily and RUX was discontinued due to toxicity concerns. In April 2018, her hematocrit increased and massive splenomegaly returned. Restart of initial RUX 20 mg BID was followed by severe pancytopenia which normalized when RUX was reduced to 5 mg BID with continuing imatinib regimen. After 8 weeks, splenomegaly completely resolved (Figure 1). A complete cytogenetic response and major molecular response were achieved after 3 months and 6 months of combination therapy respectively.Methods: Mononuclear cells from peripheral blood collected in June 2018 were plated in methylcellulose without erythropoietin or other cytokines. After 14 days, colonies were plucked, with ½ of each colony used for JAK2V617F allele-specific PCR and ½ prepared as cytospins for BCR-ABL fluorescent in situ hybridization (FISH) with the Vysis LSI BCR/ABL Dual Color, Dual Fusion Translocation Probe from Abbott Laboratories.Results: Analysis of 3-6 EPO-independent colonies of each CFU-E, G, M, GM, GEMM, BFU-E showed no BCR-ABL1 positive cells, while a single CFU-GEMM colony was positive and also homozygous for JAK2V617F. Frozen marrow cells from the time of the original diagnosis are being analyzed.Conclusion: The recurrence of splenomegaly in this patient following single agent RUX indicates emergence of novel BCR-ABL1+ clone. While single agent imatinib temporarily normalized the splenomegaly and leukocytosis for several months, MF phenotype reoccurred. Splenomegaly resolved completely in 8 weeks with the addition of very low dose RUX to standard dose imatinib, providing the first clinical observation of in vivo synergism of RUX and imatinib. We are investigating serial samples during her course, which will be presented at the meeting. The analysis of a single clone demonstrate that BCR-ABL1 mutation did not occur in previously normal dormant stem cell but in JAK2V617F positive progenitors and represent in this pts a novel subclone of previous PV/MF stem cell. DisclosuresDeininger:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Identification of Chemotherapy-Induced Leukemic-Regenerating Cells Reveals a Transient Vulnerability of Human AML Recurrence

Despite successful remission induction, recurrence of acute myeloid leukemia (AML) remains a clinical obstacle thought to be caused by the retention of dormant leukemic stem cells (LSCs). Using chemotherapy-treated AML xenografts and patient samples, we have modeled patient remission and relapse kinetics to reveal that LSCs are effectively depleted via cell-cycle recruitment, leaving the origins of relapse unclear. Post-chemotherapy, invivo characterization at the onset of disease relapse revealed a unique molecular state of leukemic-regenerating cells (LRCs) responsible for disease re-growth. LRCs are transient, can only be detected invivo, and are molecularly distinct from therapy-naive LSCs. We demonstrate that LRC features can be used as markers of relapse and are therapeutically targetable to prevent disease recurrence.

Biochemical re-programming of human dermal stem cells to neurons by increasing mitochondrial membrane potential.

Stem cells are generally believed to contain a small number of mitochondria, thus accounting for their glycolytic phenotype. We demonstrate here, however, that despite an indispensable glucose dependency, human dermal stem cells (hDSCs) contain very numerous mitochondria. Interestingly, these stem cells segregate into two distinct subpopulations. One exhibits high, the other low-mitochondrial membrane potentials (Δψm). We have made the same observations with mouse neural stem cells (mNSCs) which serve here as a complementary model to hDSCs. Strikingly, pharmacologic inhibition of phosphoinositide 3-kinase (PI3K) increased the overall Δψm, decreased the dependency on glycolysis and led to formation of TUJ1 positive, electrophysiologically functional neuron-like cells in both mNSCs and hDSCs, even in the absence of any neuronal growth factors. Furthermore, of the two, it was the Δψm-high subpopulation which produced more mitochondrial reactive oxygen species (ROS) and showed an enhanced neuronal differentiation capacity as compared to the Δψm-low subpopulation. These data suggest that the Δψm-low stem cells may function as the dormant stem cell population to sustain future neuronal differentiation by avoiding excessive ROS production. Thus, chemical modulation of PI3K activity, switching the metabotype of hDSCs to neurons, may have potential as an autologous transplantation strategy for neurodegenerative diseases.

Inverse and correlative relationships between TRIBBLES genes indicate non-redundant functions during normal and malignant hemopoiesis

TRIBBLES pseudokinases (TRIB1, TRIB2, and TRIB3) are important regulators of normal and malignant hemopoiesis. The relative abundance of each TRIBBLES family member may be important for distinct oncogenic or tumor suppressor functions. We map the expression profiles of TRIB1, TRIB2, and TRIB3 in human and murine hemopoietic stem, progenitor and mature cells, and in human leukemia datasets. Our data show that TRIB1-TRIB2 have an inverse expression relationship in normal hemopoiesis, whereas TRIB1-TRIB3 have a positive correlation. We reveal that TRIB3 expression is high in the dormant hemopoietic stem cell (HSC) population, implicating a novel role for TRIB3 in stem cell quiescence. These analyses support a non-redundant role for each TRIBBLES member during normal hemopoietic differentiation. We show that TRIB1-TRIB2 display a significant negative correlation in myelodysplastic syndrome and acute myeloid leukemia (AML) subtypes, but not in acute lymphoid leukemia. This inverse relationship is specific to certain subtypes of AML. A positive correlation exists in different leukemia subtypes between TRIB1-TRIB3. The TRIB1-TRIB2 and TRIB1-TRIB3 correlations are consistent with a correlative relationship with C/EBP transcription factor family members. Our results have implications for the development of strategies to therapeutically target these genes in different types of leukemia.

Abstract 4443: A post-transcriptional program of chemoresistance regulators in quiescent cancer cells

Abstract Quiescent (G0) cells are a clinically relevant fraction in several cancers, which include dormant cancer stem cells, and resist clinical therapy. G0 cells reveal extensive changes in gene expression at the protein and translation levels. We previously identified that the translation mechanism is altered in G0 cancer cells. MicroRNAs, noncoding RNAs that target distinct mRNAs to alter gene expression—were found to associate with an important RNA-binding protein, and enable specialized functions in G0—where they recruit non-canonical translation factors to regulate specific mRNA translation. We find that G0 leukemic cells show similar proteome and translatome to cells isolated post-chemotherapy. These data suggest that specialized post-transcriptional and translational mechanisms in G0 leukemic cells regulate a distinct translatome to mediate chemoresistance. To understand the role of post-transcriptional and translational regulation in chemoresistance, we compared global RNA, translational and proteome profiling in chemoresistant G0 acute monocytic leukemic (AML) cells. We find that chemotherapy or G0 induction leads to DNA damage responsive ATM and stress signaling, which alter post-transcriptional and translational mechanisms. ATM and stress activated p38 MAPK/MK2 increase AU-rich-element (ARE) bearing pro-inflammatory cytokine and immune gene mRNAs by regulating a key ARE RNA binding protein, and by activating STAT1/interferon pathway to alter canonical translation. AREs are present on 3'UTRs of critical, tightly regulated oncogenes and cytokines to post-transcriptionally control their expression. These changes permit translation of ARE bearing pro-inflammatory cytokine TNFα, and immune and cell-migration modulators that promote survival. Co-inhibiting p38 MAPK and TNFα that promote anti-apoptosis—prior to or alongwith chemotherapy—decreases chemoresistance in AML cell lines, in vivo, and in patient samples, without affecting normal cells. These studies reveal a pro-inflammatory subpopulation in AML that mediates chemoresistance, enabled by DNA damage- and stress-regulated post-transcriptional and translational mechanisms that are mediated by AU-rich-elements and a critical ARE RNA binding protein. Disrupting ARE regulation reduces TNFα and chemoresistance, revealing AREs and an important ARE RNA binding protein as key regulators of inflammation-mediated chemoresistance. These studies reveal the significance of post-transcriptional regulation of inflammation/immune gene-mediated chemoresistance. Correspondence: vasudevan.shobha@mgh.harvard.edu Citation Format: Sooncheol Lee, Samuel S. Truesdell, Syed I. Bukhari, Myriam Boukhali, Dongjun Lee, Maria A. Mazzola, Radhika Raheja, Adam Langenbucher, Nicholas J. Haradhvala, Michael Lawrence, Roopali Gandhi, David A. Sweetser, Wilhelm Haas, Shobha Vasudevan. A post-transcriptional program of chemoresistance regulators in quiescent cancer cells [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 4443.

A Circular RNA Protects Dormant Hematopoietic Stem Cells from DNA Sensor cGAS-Mediated Exhaustion

Disrupting the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) leads to bone marrow failure or hematologic malignancy. However, how HSCs sustain their quiescent state and avoid type I interferon (IFN)-mediated exhaustion remains elusive. Here we defined a circular RNA that we named cia-cGAS that was highly expressed in the nucleus of long-term (LT)-HSCs. Cia-cGAS deficiency in mice caused elevated expression of type I IFNs in bone marrow and led to decreased numbers of dormant LT-HSCs. Under homeostatic conditions, cia-cGAS bound DNA sensor cGAS in the nucleus to block its synthase activity, thereby protecting dormant LT-HSCs from cGAS-mediated exhaustion. Moreover, cia-cGAS harbored a stronger binding affinity to cGAS than self-DNA did and consequently suppressed cGAS-mediated production of type I IFNs in LT-HSCs. Our findings reveal a mechanism by which cia-cGAS inhibits nuclear cGAS by blocking its enzymatic activity and preventing cGAS from recognizing self-DNA to maintain host homeostasis.

Reservoirs for repair? Damage-responsive stem cells and adult tissue regeneration in Drosophila.

Adult stem cells in mammals are important for normal tissue renewal (homeostasis) and regeneration after injury. In the past ten years, different types of homeostatic adult stem cells have also been identified in the genetically accessible fruit fly (Drosophila melanogaster), among which intestinal stem cells have taken centre stage. Recent studies provide evidence that adult fly tissues may also harbor quiescent stem cells, which can enter cell cycle upon injury to regenerate compromised tissue. Such damage-responsive stem cells have been described in flight muscles, the adult brain and in a narrow region of the fly hindgut. Strikingly, many mammalian tissues have also been shown to maintain quiescent, but regeneration-competent, stem cells. However, little is known about the injury-induced signals that lead to their activation. Here, we provide a brief overview of active and damage-responsive adult stem cells in the fruit fly and focus on injury-dependent signalling events. We highlight the potential of Drosophila to model damage-induced stem cell activation to deepen our molecular understanding of how dormant stem cells can be efficiently recruited for tissue repair after injury.