using-quantitative-real-time-pcr-to-determine-donor-cell-engraftment

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

Aktivierung der G-Protein-Signalisierung, dass 3 (AGS3) ist eines der neun S&auml;ugetierproteine, enth&auml;lt mindestens ein G-Protein-regulatorischen (GPR) Motive, die die BIP-gebundenen Gestaltung des Galphai zu stabilisieren. Solche Proteine haben unerwartete Funktionsvielfalt ergeben, f&uuml;r die &quot;G-Switch&quot; bei der Kontrolle der Ereignisse innerhalb der Zelle, die unabh&auml;ngig von der Rolle der trimeren G-Proteine als Wandler f&uuml;r G-Protein-gekoppelter Rezeptoren an der Zelloberfl&auml;che. Eine wichtige Frage bez&uuml;glich dieser Klasse von Proteinen ist, was ihre subzellulare Positionierung und Interaktion mit G-Proteinen steuert. Wir haben eine Reihe von Hefe-zwei-Hybrid-Bildschirmen zur Identifizierung von Proteinen, die Interaktion mit der Tetratricopeptide Wiederholung (TPR) durchgef&uuml;hrt, der AGS3, die spielt eine wichtige Rolle bei der subzellul&auml;ren Positionierung des Proteins. Wir berichten &uuml;ber die Identifizierung von Frmpd1 (FERM und PDZ-Dom&auml;nen 1 enth&auml;lt) als rechtliche Bindung Partner von AGS3. Frmpd1 bindet an die TPR-Dom&auml;ne der AGS3 und Coimmunoprecipitates mit AGS3 aus Zelllysate. Zelle Fraktionierung zeigten, dass Frmpd1 AGS3 in Sekundenbruchteilen Membrane stabilisiert. Nach Cotransfection der COS7 Zellen mit Frmpd1-GFP und AGS3-mRFP AGS3-mRFP wird in Regionen der Zelle Rinde beobachtet und auch in Membran Erweiterungen oder Prozesse, wo es scheint, mit Frmpd1-GFP colocalized werden, basierend auf die zusammengef&uuml;hrten fluoreszierende Signale. Frmpd1 Niederschlag (SiRNA) in Cath.a-differenzierte Nervenzellen sanken um etwa 50 % die H&ouml;he der endogenen AGS3 in Membrane Br&uuml;che und verbesserte die alpha2-adrenergene Rezeptor-vermittelte Hemmung der Forskolin-induzierten Anstieg der Lager. Die Coimmunoprecipitation des Frmpd1 mit AGS3 geht verloren, wie der Galphai3 in der Zelle erh&ouml;ht wird und AGS3 offenbar seine Bindung-Partner von Frmpd1 wechselt bis Galphai3, dass das Zusammenspiel von AGS3 mit Frmpd1 und Galphai3 gegenseitig ist. Mechanistisch, kann Frmpd1 positionieren AGS3 in einer Membran-Umgebung wo interagiert dann mit Galphai in einer geregelten Art und Weise.

Die PDZ und Band 4.1 enth&auml;lt Protein Frmpd1 regelt die subzellulare Position der Aktivator der G-Protein-Signalisierung 3 und dessen Interaktion mit G-Proteinen.

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gialpha-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gialpha and mammalian Inscuteable (mInsc). Gialpha rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.

Distribution of activator of G-protein signaling 3 within the aggresomal pathway: role of specific residues in the tetratricopeptide repeat domain and differential regulation by the AGS3 binding partners Gi(alpha) and mammalian inscuteable.

AGS3, ein Rezeptor-unabh&auml;ngige-Aktivator der G-Protein-Signalisierung, ist unerwartete Funktionsvielfalt f&uuml;r G-Protein-Signalling-Systeme beteiligt. AGS3 hat sieben Tetratricopeptide (TPR) Motive stromaufw&auml;rts der vier G-Protein-regulatorischen (GPR)-Motive, die als docking-Websites f&uuml;r Gialpha-BIP dienen. Die Positionierung der AGS3 innerhalb der Zelle und die intramolekulare Dynamik zwischen verschiedenen Bereichen der Proteine sind wahrscheinlich wichtigsten Determinanten ihrer F&auml;higkeit, G-Protein-Signalisierung zu beeinflussen. Wir berichten, dass die Aggresome-Pathway AGS3 eingeht und Verteilung des Proteins durch die AGS3-Bindung-Partner Gialpha und S&auml;ugetier-Inscuteable (mInsc) geregelt ist. Gialpha rettet AGS3 aus der Aggresome, w&auml;hrend mInsc die Aggresome-&auml;hnliche Verteilung der AGS3 erweitert. Die Verteilung der AGS3 auf die Aggresome ist abh&auml;ngig von der TPR-Dom&auml;ne, und es beschleunigt durch St&ouml;rung im TPR Organisationsstruktur oder die Einf&uuml;hrung einer nonsynonymous Einzel-Nukleotid Polymorphie. Diese Daten pr&auml;sentieren AGS3, G-Proteinen und mInsc als Kandidat Proteine regulieren zellul&auml;re Belastung Protein-Verarbeitung Pathologien zugeordnet.

Verteilung der Aktivator der G-Protein-Signalisierung 3 innerhalb der Aggresomal Weg: Rolle bestimmte R&uuml;ckst&auml;nde in der Tetratricopeptide Wiederholung Dom&auml;ne und differenzielle Verordnung durch die Bindung AGS3 Partner Gi(alpha) und S&auml;ugetier-Inscuteable.

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind GÎ±(i) free of GÎ²Î³ providing an unusual scaffold for the "G-switch" and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3Â·GÎ±(i) signaling module by a cell surface, seven-transmembrane receptor. AGS3 and GÎ±(i1) tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of Î±(2)-adrenergic receptors or Î¼-opioid receptors reduced AGS3-RLucÂ·GÎ±(i1)-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by GÎ²Î³ sequestration with the carboxyl terminus of GRK2. GÎ±(i)-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to GÎ±(i) and/or GÎ±(o) indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both GÎ±Î²Î³ and GPR-GÎ±(i) offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

Regulation of the AGS3Â·G{alpha}i signaling complex by a seven-transmembrane span receptor.

G-Protein-Signalisierung Modulatoren (GPSM) spielen diverse Funktionsrollen durch ihre Interaktion mit G-Protein-Untereinheiten. AGS3 (GPSM1) enth&auml;lt vier G-Protein-regulatorische Motive (GPR), die direkt G&alpha;(i) frei von G&beta;&gamma; vorsieht, die &quot;G-Switch&quot; eine ungew&ouml;hnliche Ger&uuml;st und Signalisierung von komplexen, aber der Mechanismus binden, durch welche Signale Spur in diesem Ger&uuml;st nicht gut verstanden. Wir berichten &uuml;ber die Regulierung der AGS3&middot;G&alpha;(i) Signalisierung Modul durch eine Zelloberfl&auml;che, sieben-Transmembran Rezeptor. AGS3 und G&alpha;(i1) die mit Renilla-Luciferase oder gelbes fluoreszierendes Protein ausgedr&uuml;ckt in den S&auml;ugetier-Zellen ausgestellt S&auml;ttigbare, spezifische Biolumineszenz-Resonanz-Energie-Transfer angibt, Komplexbildung in der Zelle. Aktivierung von &alpha; (2)-adrenerge Rezeptoren oder &mu;-Opioid-Rezeptoren verringert AGS3-RLuc&middot;G&alpha; (i1)-YFP Energie&uuml;bertragung um &uuml;ber 30 %. Der Agonist-vermittelte Effekte wurden durch Pertussis-Toxin und Koexpression von RGS4 gehemmt, sondern wurden von G&beta;&gamma;-Sequestrierung mit das Carboxyl-Terminus der GRK2 nicht ge&auml;ndert. G&alpha; (i)-abh&auml;ngige und Agonist-Sensitive Biolumineszenz-Resonanz-Energie-Transfer wurde auch beobachtet, zwischen AGS3 und Zelloberfl&auml;che Rezeptoren in der Regel gekoppelt mit G&alpha;(i) und/oder G&alpha;(o) dass AGS3 Teil einer gr&ouml;&szlig;eren signalisieren Komplex ist. Nach der Aktivierung des Rezeptors distanziert AGS3 umkehrbar von dieses Komplexes an der Zelle Kortex. Rezeptor-Ankopplung an G&alpha;&beta;&gamma; und GPR-G&alpha;(i) bieten zus&auml;tzliche Flexibilit&auml;t f&uuml;r Systeme reagieren und Herausforderungen anpassen und komplexe Verhaltensweisen zu orchestrieren.

Verordnung &uuml;ber die AGS3&middot;G {Alpha} ich signalisieren Komplex durch eine sieben-Transmembran-Span-Element-Rezeptor.

Activation of the IKK-NFÎºB pathway increases the resistance of cancer cells to ionizing radiation (IR). This effect has been largely attributed to the induction of anti-apoptotic proteins by NFÎºB. Since efficient repair of DNA double strand breaks (DSBs) is required for the clonogenic survival of irradiated cells, we investigated if activation of the IKK-NFÎºB pathway also regulates DSB repair to promote cell survival after IR. We found that inhibition of the IKK-NFÎºB pathway with a specific IKKÎ² inhibitor significantly reduced the repair of IR-induced DSBs in MCF-7 cells. The repair of DSBs was also significantly inhibited by silencing IKKÎ² expression with IKKÎ² shRNA. However, down-regulation of IKKÎ± expression with IKKÎ± shRNA had no significant effect on the repair of IR-induced DSBs. Similar findings were also observed in IKKÎ± and/or IKKÎ² knockout mouse embryonic fibroblasts (MEFs). More importantly, inhibition of IKKÎ² with an inhibitor or down-regulation of IKKÎ² with IKKÎ² shRNA sensitized MCF-7 cells to IR-induced clonogenic cell death. DSB repair function and resistance to IR were completely restored by IKKÎ² reconstitution in IKKÎ²-knockdown MCF-7 cells. These findings demonstrate that IKKÎ² can regulate the repair of DSBs, a previously undescribed and important IKKÎ² kinase function; and inhibition of DSB repair may contribute to cance cell radiosensitization induced by IKKÎ² inhibition. As such, specific inhibition of IKKÎ² may represents a more effective approach to sensitize cancer cells to radiotherapy.

IKKÎ² regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells.

Aktivierung des Signalweges IKK-NF&kappa;B erh&ouml;ht die Resistenz von Krebszellen durch ionisierende Strahlung (IR). Dieser Effekt wurde weitgehend auf die Induktion von Anti-apoptotischen Proteine von NF&kappa;B zur&uuml;ckgef&uuml;hrt. Da effiziente Reparatur von Br&uuml;chen der DNA-Doppelstrang (DSBs) f&uuml;r das Clonogenic &Uuml;berleben der bestrahlten Zellen erforderlich ist, untersuchten wir wenn Aktivierung des Signalweges IKK-NF&kappa;B auch DSB-Reparatur zur F&ouml;rderung der Zelle &Uuml;berleben nach dem IR. reguliert Wir fanden, dass die Hemmung des Signalweges IKK-NF&kappa;B mit einem bestimmten IKK&beta; Inhibitor die Reparatur von IR-induzierte DSBs in MCF-7 Zellen deutlich reduziert. Die Reparatur des DSBs wurde auch deutlich an, indem IKK&beta; Ausdruck mit IKK&beta; ShRNA gehemmt. Herunterregulation IKK&alpha; Ausdruck mit IKK&alpha; ShRNA hatte jedoch keinen signifikanten Einfluss auf die Reparatur von IR-induzierte DSBs. &auml;hnliche Ergebnisse auch IKK&alpha; bzw. IKK&beta; ko M&auml;useembryonalen (MEFs) beobachtet wurden. Noch wichtiger ist, sensibilisiert die Hemmung der IKK&beta; mit einem Inhibitor oder Herunterregulation IKK&beta; mit IKK&beta; ShRNA MCF-7 Zellen induziert durch IR Clonogenic Zelltod. DSB-Reparatur-Funktion und Best&auml;ndigkeit gegen IR wurden von IKK&beta; Rekonstitution in IKK&beta;-Niederschlag MCF-7 Zellen vollst&auml;ndig restauriert. Diese Ergebnisse zeigen, dass IKK&beta; die Reparatur des DSBs, eine bisher unbeschriebene und wichtige IKK&beta; Kinase Funktion regulieren kann; und Hemmung der DSB-Reparatur darf Cance Zelle Radiosensitization induziert durch Hemmung von IKK&beta; beitragen. Als solche kann spezifische Hemmung der IKK&beta; ist ein effektiveren Ansatz, Krebszellen zu Strahlentherapie zu sensibilisieren.

IKK&beta; regelt die Reparatur von Br&uuml;chen von DNA-Doppel-Strang durch ionisierende Strahlung in MCF-7 Brustkrebs-Zellen induziert.

ABSTRACT: BACKGROUND: Pim (proviral insertion in murine lymphoma) kinases are a small family of constitutively active, highly conservative serine/threonine oncogenic kinases and have 3 members: Pim1, Pim2, and Pim3. Pim kinases are also implicated in the regulation of B- and T- cell responses to cytokines and hematopoietic growth factors. The roles of Pim kinases in the regulation of primitive hematopoietic stem cells (HSCs) are largely unknown. METHODS: In the current study, Pim1-/-2-/-3-/- triple knockout (TKO) mice were used to determine the role of Pim kinases in hematopoiesis. Peripheral blood hematological parameters were measured in Pim TKO mice and age-matched wild-type (WT) controls. Primary, secondary, and competitive transplantations were performed to assay the long-term repopulating HSCs in Pim TKO mice. In vivo BrdU incorporation assay and ex vivo Ki67 staining and caspase 3 labeling were performed to evaluate the proliferation and apoptosis of HSCs in Pim TKO mice. RESULTS: Compared to age-matched WT controls, Pim TKO mice had lower peripheral blood platelet count and exhibited erythrocyte hypochromic microcytosis. The bone marrow cells from Pim TKO mice demonstrated decreased hematopoietic progenitor colony-forming ability. Importantly, Pim TKO bone marrow cells had significantly impaired capacity in rescuing lethally irradiated mice and reconstituting hematopoiesis in primary, secondary and competitive transplant models. In vivo BrdU incorporation in long-term HSCs was reduced in Pim TKO mice. Finally, cultured HSCs from Pim TKO mice showed reduced proliferation evaluated by Ki67 staining and higher rate of apoptosis via caspase 3 activation. CONCLUSIONS: Pim kinases are not only essential in the hematopoietic lineage cell development, but also important in HSC expansion, self-renewal, and long-term repopulation.

Abnormal hematopoietic phenotypes in Pim kinase triple knockout mice: Pim kinase regulates hematopoiesis.

ABSTRACT: BACKGROUND: Pim (proviral Einf&uuml;gung in murinen Lymphom) Kinasen sind eine Familie der konstitutiv aktiv, h&ouml;chst konservativen Serin/Threonin Onkogene Kinasen und haben 3 Mitglieder: Pim1, Pim2 und Pim3. PIM-Kinasen sind auch bei der Regulierung der B - und T-Zell-Antworten zu Zytokine und h&auml;matopoetische Wachstumsfaktoren beteiligt. Die Rollen der Pim-Kinasen bei der Regulierung der primitiven h&auml;matopoetischen Stammzellen (HSCs) sind weitgehend unbekannt. Methoden: In der aktuellen Studie wurden Pim1-/-2-/-3-/-dreifach Knockout (TKO) M&auml;use zum bestimmen, der Rolle der Pim-Kinasen in Blutbildung. Auf Pim TKO M&auml;use und altersbezogenen Wildtyp (WT)-Steuerelemente wurden peripherem Blut h&auml;matologischen Parameter gemessen. Prim&auml;re, sekund&auml;re und wettbewerbsf&auml;hige Transplantationen wurden durchgef&uuml;hrt, um die langfristige repopulating HSCs Pim TKO M&auml;usen Brutversuch. In Vivo BrdU Einbeziehung Assay und Ex Vivo Ki67 wurden F&auml;rbe- und Caspase 3 Kennzeichnung durchgef&uuml;hrt, um die Proliferation und Apoptose von HSCs Pim TKO M&auml;usen zu bewerten. Ergebnisse: Im Vergleich zu altersbezogenen WT Steuerelemente, Pim TKO M&auml;use hatten niedrigere peripheren Blut Thrombozytenzahl und Erythrozyten hypochrome Microcytosis ausgestellt. Die Knochenmarkzellen von Pim TKO-M&auml;usen gezeigt verringert h&auml;matopoetischen Vorl&auml;uferzellen kolonienbildende F&auml;higkeit. Wichtigsten Pim TKO Knochenmarkzellen hatte Kapazit&auml;t Rettung lethally bestrahlten M&auml;usen erheblich beeintr&auml;chtigt und Rekonstituierung der Blutbildung in prim&auml;r-, Sekund&auml;r- und wettbewerbsf&auml;hige Modelle zu verpflanzen. In-vivo BrdU-Aufnahme in langfristige HSCs wurde Pim TKO M&auml;usen reduziert. Schlie&szlig;lich ergab kultivierten HSCs Pim TKO M&auml;use geringere Verbreitung von Ki67 F&auml;rbe- und h&ouml;here Rate der Apoptose durch die Aktivierung der Caspase 3 ausgewertet. Schlussfolgerungen: Pim-Kinasen sind nicht nur in der h&auml;matopoetischen Linie Zelle Entwicklung wesentlich, aber auch wichtig im HSC Ausbau, Selbsterneuerung und langfristige Wiederbelegung.

Abnormale h&auml;matopoetische Ph&auml;notypen im Pim-Kinase verdreifachen Knock-out-M&auml;use: Pim-Kinase regelt die Blutbildung.

One third of tumor suppressors are haploinsufficient transcriptional regulators, yet it remains unknown how a 50% reduction of a transcription factor is translated at the cis-regulatory level into a malignant transcriptional program. We studied CUX1, a haploinsufficient transcription factor that is recurrently mutated in hematopoietic and solid tumors. We determined CUX1 DNA-binding and target gene regulation in the wildtype and haploinsufficient states. CUX1 binds with transcriptional activators and cohesin at distal enhancers across three different human cell types. Haploinsufficiency of CUX1 altered the expression of a large number of genes, including cell cycle regulators, with concomitant increased cellular proliferation. Surprisingly, CUX1 occupancy decreased genome-wide in the haploinsufficient state, and binding site affinity did not correlate with differential gene expression. Instead, differentially expressed genes had multiple, low-affinity CUX1 binding sites, features of analog gene regulation. A machine-learning algorithm determined that chromatin accessibility, enhancer activity, and distance to the transcription start site are features of dose-sensitive CUX1 transcriptional regulation. Moreover, CUX1 is enriched at sites of DNA looping, as determined by Hi-C analysis, and these loops connect CUX1 to the promoters of regulated genes. We propose an analog model for haploinsufficient transcriptional deregulation mediated by higher order genome architecture.

The haploinsufficient tumor suppressor, CUX1, acts as an analog transcriptional regulator that controls target genes through distal enhancers that loop to target promoters.

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset.

Monosomy 7 (-7) and del(7q) are high-risk cytogenetic abnormalities common in myeloid malignancies. We previously reported that , a homeodomain-containing transcription factor encoded on 7q22, is frequently inactivated in myeloid neoplasms, and CUX1 myeloid tumor suppressor activity is conserved from humans to -inactivating mutations are recurrent in clonal hematopoiesis of indeterminate potential as well as myeloid malignancies, in which they independently carry a poor prognosis. To determine the role for CUX1 in hematopoiesis, we generated 2 short hairpin RNA-based mouse models with ∼54% (Cux1) or ∼12% (Cux1) residual CUX1 protein. Cux1 mice develop myelodysplastic syndrome (MDS) with anemia and trilineage dysplasia, whereas CUX1 mice developed MDS/myeloproliferative neoplasms and anemia. In diseased mice, restoration of CUX1 expression was sufficient to reverse the disease. CUX1 knockdown bone marrow transplant recipients exhibited a transient hematopoietic expansion, followed by a reduction of hematopoietic stem cells (HSCs), and fatal bone marrow failure, in a dose-dependent manner. RNA-sequencing after  knockdown in human CD34 cells identified a -7/del(7q) MDS gene signature and altered differentiation, proliferative, and phosphatidylinositol 3-kinase (PI3K) signaling pathways. In functional assays, CUX1 maintained HSC quiescence and repressed proliferation. These homeostatic changes occurred in parallel with decreased expression of the PI3K inhibitor, , and elevated PI3K/AKT signaling upon CUX1 knockdown. Our data support a model wherein CUX1 knockdown promotes PI3K signaling, drives HSC exit from quiescence and proliferation, and results in HSC exhaustion. Our results also demonstrate that reduction of a single 7q gene, , is sufficient to cause MDS in mice.

Article	Year
The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins. The Journal of biological chemistry\| PubMed ID: 18566450	2008
Distribution of activator of G-protein signaling 3 within the aggresomal pathway: role of specific residues in the tetratricopeptide repeat domain and differential regulation by the AGS3 bin... Molecular and cellular biology\| PubMed ID: 20065032	2010
Regulation of the AGS3Â·G{alpha}i signaling complex by a seven-transmembrane span receptor. The Journal of biological chemistry\| PubMed ID: 20716524	2010
IKKÎ² regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells. PloS one\| PubMed ID: 21490922	2011
Abnormal hematopoietic phenotypes in Pim kinase triple knockout mice: Pim kinase regulates hematopoiesis. Journal of hematology & oncology\| PubMed ID: 23360755	2013
The haploinsufficient tumor suppressor, CUX1, acts as an analog transcriptional regulator that controls target genes through distal enhancers that loop to target promoters. Nucleic acids research\| PubMed ID: 28369554
Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset. Oncotarget\| PubMed ID: 28415816
Gene dosage effect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS. Blood\| PubMed ID: 29592892

Ningfei An

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