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«3. Medizinische Klinik und Poliklinik – Hämatologische Forschung Cks1 is a critical regulator of hematopoietic stem cell cycling, quiescence and ...»

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TECHNISCHE UNIVERSITÄT MÜNCHEN

3. Medizinische Klinik und Poliklinik – Hämatologische

Forschung

Cks1 is a critical regulator of hematopoietic stem cell

cycling, quiescence and homeostasis, operating

upstream of CDK inhibitors

Viktoriya Tomiatti

Vollständiger Abdruck der von der Fakultät für Medizin der Technischen

Universität München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Jürgen Ruland

Prüfer der Dissertation:

1. apl. Prof. Dr. Ulrich Keller

2. Univ.-Prof. Angelika Schnieke, Ph.D.

Die Dissertation wurde am 19. 12. 2013 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 01.04.2014 angenommen.

Für meine Eltern Pavlina und Georgi und für meinen Ehemann Sebastian Table of contents Zusammenfassung

Summary

1. Introduction

1.1. Cell cycle

1.1.1. Phases and checkpoints of the cell cycle

1.1.2. Cell cycle control: Cyclin/CDK comlexes

1.1.3. Cell cycle control: CDK inhibitors

1.2. Hematopoiesis

1.2.1. Hematopoietic stem cells

1.2.2. Regulatory signals in HSC

1.2.3. Regulation of the cell cycle in HSC

1.2.4. Chronic myeloid leukemia, a HSC disease

1.3. SCFSkp2-Cks1

1.3.1. Ubiquitination and the SCFSkp2 complex

1.3.2. SCFSkp2-Cks1 and its role in cell cycle control

1.3.3. Regulation of Cks1 expression

1.3.4. Cks1 in cancer

1.4. Aim of this thesis

2. Materials

2.1. Mice

2.2. Cell lines

2.3. Bacteria

2.4. Vectors

2.5. Antibodies

2.6. Primer

2.7. Kits

2.8. Chemicals

2.9. Equipment

2.10. Software programs

3. Methods

3.1. Genotyping

3.2. Preparation of murine tissues

3.3. Transplantation assays

3.4. 5-FU and BrdU injections

3.5. Colony forming assay

3.6. Co-cultures of bone marrow cells with a stromal cell line

3.7. Culture of bone marrow cells

3.8. Cell culture

3.9. Determination of cell number and cell vitality

3.10. Cell cycle analysis

3.11. Apoptosis assay

3.12. Retroviral transduction

3.13. Cell sorting and surface staining for flow cytometry

3.14. Intracellular staining for flow cytometry

3.16. RNA isolation

3.19. Western blotting

3.20. Statistics

4. Results

4.1. Effects of Cks1 loss on CKI levels and expression of Cks1 in HSC/HPC.............. 52  4.1.1. Cks1 loss correlates with accumulation of CKI in early hematopoietic subsets

4.1.2. High expression of Cks1 in early hematopoietic subsets

4.2. Role of Cks1 in steady state and stress hematopoiesis

4.2.1. Normal steady state adult hematopoiesis in Cks1-deficient mice

4.2.2. Decreased absolute HPC numbers and increased relative HSC numbers in Cks1-/- mice

4.2.3. Loss of Cks1 results in decreased hematopoietic colony formation................ 60  4.2.4. Decreased colony formation and HPC number after cultivation of Lin- Cks1-/cells on stromal cells

4.2.5. Cks1-/- hematopoietic cells proliferate slower in culture

4.2.6. Cks1 regulates survival of progenitor cells in vitro

4.2.7. Increased B-Lymphocytes- and decreased granulocytes frequency after cytotoxic stress in Cks1-knockout mice

4.2.8. Cks1 controls stress hematopoiesis in LSK cells: impaired regeneration after cytotoxic stress

4.2.9. Bone marrow transplantations: Cks1-/- bone marrow cells are able to reconstitute recipient mice

4.2.10. Secondary bone marrow transplantations: increased engraftment of Cks1-/cells

4.2.11. Loss of Cks1 results in increased LSK frequency and increased replating capability after cultivating in vitro

4.2.12. Transplantations of CD150+ LSK cells: loss of Cks1 leads to accumulation of LT-HSC

4.2.13. Secondary transplantations of CD150+ LSK cells: Cks1-/- HSC proliferate slower

4.3. p27 dependent and independent effects of Cks1 loss in the hematopoiesis:

analysis of double knockouts

4.3.1. Steady state hematopoiesis in p27-/- and Cks1/p27 DKO

4.3.2. Concomitant loss of Cks1 and p27 in BM cells results in better survival in vitro compared to Cks1-/- or p27-/- cells

4.3.3. Cks1 controls the regeneration of mature lineages after cytotoxic stress through p27 regulation

4.3.4. Serial transplantations with p27-/- and Cks1/p27 DKO BM

4.4. The role of Cks1 in a HSC disease

4.4.1. Cks1 transcript levels are regulated by BCR-ABL acitivity

4.4.2. BCR-ABL expression in Cks1-/- BM cells results in decreased colony forming capability

5. Discussion

5.1. Cks1 controls the HSC/HPC pool at steady state in vivo and protects HPC from apoptosis in vitro

5.2. Downstream targets of the SCFSkp2-Cks1 complex are strongly involved in selfrenewal and quiescence balance in HSC





5.3. Cks1 mediates the balance between lymphoid and myeloid lineage differentiation through regulation of p27

5.4. E3 ubiquitin ligases are critical factors in the control of HSC fate

5.5. Potential regulators upstream of Cks1 in HSC

5.6. Cks1 is an essential regulator in cancer cells

5.7. Conclusion

6. List of tables and figures

7. Abbreviations

8. References

9. Publications

–  –  –

Zusammenfassung Der Prozess der Blutbildung, Hämatopoese beinhaltet eine hieratische Abfolge hematopoetischer Stammzellen (HSC), Progenitorzellen (HPC) und adulter Blutzellen. Zellzyklusregulation spielt eine Schlüsselrolle während des stationären Ruhezustandes von HSC, der Induktion der Blutproduktion und der Differenzierung. Sie ist ein wichtiger Mechanismus, der unkontrollierter Zellproliferation und Tumorentstehung entgegenwirkt. Cyclin/Cyclin dependent kinase (Cyclin/CDK) Komplexe und die CDK Inhibitoren (CKI) sind wichtige Elemente, die die Übergänge der Zellzyklusphasen kontrollieren. Regulation der CKI Level geschieht vornehmlich auf post-translationaler Ebene, durch Kontrolle der Proteinstabilität. Dieser Mechanismus wurde am gründlichsten für den CKI p27 untersucht, dessen Abbau durch den E3 Ubiquitinligase Komplex SCFSkp2 veranlasst wird. Die effiziente Ubiquitinylierung von phosphoryliertem p27 durch den SCFSkp2 benötigt zusätzlich das Protein Cks1, welches ebenfalls für die SCFSkp2-vermittelte post translationale Regulation der CKI p21, p57 und des Mitglieds der Rb-Familie, p130, benötigt wird.

Aufgrund der hohen Bedeutung der CKI für die Hämatopoese und des SCFSkp2vermittelten Abbaus von CKI für den Zellzyklusablauf, wurde im Rahmen dieses Projektes die hämatopoetische Rolle des limitierenden Faktors des SCFSkp2Komplexes, Cks1 untersucht. Um p27-abhängige Effekte des Cks1-Verlustes zu bestimmen, wurden zusätzlich Experimente mit Doppel-Knockouts für Cks1 und p27 durchgeführt.

Die Ergebnisse zeigen, dass die Expression von Cks1 in den frühen, „long-term“ HSC (LT-HSC) erhöht war. Der Verlust von Cks1 in HSC/HPC resultierte in erhöhten Proteinleveln von p21, p27, p57 und p130. Diese Akkumulation war in den frühesten HSC Populationen, den CD150+ LSK am stärksten ausgeprägt.

Während Cks1-/- Mäuse eine normale adulte Hämatopoese aufwiesen, wurde im Knochenmark ein Abfall der Vorläufer-Populationen und eine Akkumulation der LT-HSC beobachtet. Dies deutete auf einen verzögerten Austritt der Cks1defizienten HSC aus dem Ruhezustand hin. Diese Annahme wurde durch 5-FU induzierte Stresshämatopoese Experimente und Transplantationsversuche bewiesen. Der Verlust von Cks1 führte zu einer verzögerten Stress-Antwort nach 5-FU Behandlung und zu einer erhöhten LT-HSC Dichte nach Transplantation.

ZUSAMMENFASSUNG

Beide Beobachtungen wurden von einem Abfall der BrdU-Inkorporation in den Cks1-/- LT-HSC begleitet, was auf einen verlangsamten Zellzyklus hindeutet. In vitro, führte der Verlust von Cks1 zu einer Sensibilisierung der HPC gegenüber Apoptose und einem verringerten Koloniewachstum. Andererseits konnten die Cks1-/- LSK nach Replatierungen länger bestehen und mehr Kolonien in den späteren Passagen bilden, was im Einklang mit den Transplantationsversuche war. Dies bestätigt die Annahme, dass Cks1 den Ruhezustand der HSC kontrolliert.

Da Cks1-/-p27-/- Mäuse keinen Wildtyp ähnlichen Phänotyp aufwiesen oder p27-/Zellen keinen reziproken Phänotyp gegenüber Cks1-/- Zellen zeigten, war keiner der oben erwähnten Effekte des Cks1-Verlustes ausschliesslich auf p27 Akkumulation zurückzuführen. Durch Cks1 bedingte Regulation des p27 Proteinlevels wurde die Verteilung der adulten hämatopoetischen Populationen nach 5-FU induziertem Stress gesteuert. Eine 5-FU vermittelte Ablation der reifen, sich teilenden Zellen und die dadurch bedingte Ausdifferenzierung der HSC, resultierte in einem Anstieg der Cks1-/- B-Lymphocyten und einem Abfall der Cks1-/- Granulocyten. Der entgegengesetzte Effekt wurde in p27-/- Mäusen beobachtet, während die Doppel-Knockouts einen Wildtyp-ähnlichen Phänotyp aufwiesen.

Im letzten Teil des Projektes wurde die Rolle von Cks1 in der hämatopoetischen Stammzellerkrankung CML untersucht. Es konnte gezeigt werden, dass Cks1 Expression durch die Aktivität der konstitutiv aktiven Kinase BCR-ABL hochreguliert wird, während Behandlung mit dem ABL Inhibitor Imatinib zu einer Unterdrückung der Cks1-Level führte. Durch In vitro Analysen wurde gezeigt, dass Cks1 die BCR-ABL induzierte klonogene Aktivität beeinflusst.

Die Daten in dieser Studie zeigen, dass Cks1 ein kritischer Regulator der Homöostase in HSC/HPC ist. Diese Regulation geschieht wahrscheinlich durch die Kontrolle der CKI-Proteinlevel als Teil des SCFSkp2 Komplexes. Cks1 ist möglicherweise ein wichtiges Zwischenglied der Oncogen-induzierten Zellzyklusregulation in hämatologischen Erkrankungen und somit ein therapeutischer Ansatzpunkt.

SUMMARY Summary Hematopoiesis, the process of blood production, involves a hierarchy of hematopoietic stem cells (HSC), progenitor cells (HPC) and mature blood cells.

Regulation of the cell cycle plays a key role during steady state and challenged blood production and also serves as a mechanism towards uncontrolled cell proliferation that might lead to blood malignancies. Cyclin/Cyclin-dependent kinase (Cyclin/CDK) complexes and their inhibitors (CKI) are the major directing elements in cell cycle transition.

The regulation of the CKI occurs mainly at the post-translational level through control of protein stability. This mechanism has been best elucidated for the CKI p27, the degradation of which is controlled by the E3 ubiquitin ligase complex SCFSkp2. For efficient ubiquitylation of phosphorylated p27, SCFSkp2 requires the presence of a small protein, the Cyclin-dependent kinase subunit 1 (Cks1). In addition to the regulation of p27 protein levels, SCFSkp2 has further been associated with the post-translational regulation of other CKI including p21, p57 and the Rb family member p130.

Considering the importance of SCFSkp2-mediated degradation of CKI for the proper cell cycle progression of HSC/HPC, the hematopoietic role of Cks1, the ratelimiting component of the SCFSkp2, was studied during this project. To reveal which effects of Cks1 loss in hematopoiesis were p27 dependent, additional experiments with double knockouts for Cks1 and p27 were performed.

Cks1 expression was increased in the early, long-term HSC (LT-HSC) and loss of Cks1 resulted in up-regulated p21, p27, p57 and p130 levels in HSC/HPC. This accumulation was most pronounced in one of the earliest HSC subsets, the CD150+ LSK. While Cks1-/- mice exhibited normal adult hematopoiesis, a significant decline in the progenitor populations and an accumulation in the LTHSC fractions were observed, indicating, that Cks1-/- HSC could be decelerated in the exit of the quiescence mode. This assumption was proven through 5-FU induced stress hematopoiesis experiments and transplantation assays. Loss of Cks1 led to a decelerated hematopoietic stress response after 5-FU treatment and increased LT-HSC frequency upon transplantation. Both observations were accompanied by decreased BrdU integration in Cks1-/- HSC indicating slower cycling of these cells. In vitro, loss of Cks1 led to sensitization towards apoptosis SUMMARY in HPC and decreased colony forming capability. On the other hand, consistent with the transplantation analysis, after serial replates Cks1-/- LSK persisted longer and formed more colonies in the later replates, confirming the suggestion, that Cks1 regulates HSC dormancy.

None of the observed effects in the Cks1-/- cells seemed to be dependent on sole p27 accumulation, since either experiments with double knockouts did not resemble the wild type phenotype or p27 knockout did not oppose the Cks1-/phenotype. Only the lineage distribution upon hematopoietic stress seemed to be controlled through Cks1 in a p27 dependent manner. Ablating the adult hematopoiesis and inducing HSC differentiation with 5-FU resulted in an increase of Cks1-/- B-lymphocytes and a decrease in Cks1-/- granulocytes. The opposite effect was observed in p27-/- mice, whereas double knockouts displayed a phenotype similar to wild type.

In the last part of this study, the role of Cks1 in the HSC disease chronic myeloid leukemia (CML) was investigated. Cks1 was shown to be up-regulated by the constitutively active tyrosine kinase BCR-ABL in CML and suppressed upon treatment with the ABL inhibitor imatinib. In vitro analysis showed that Cks1 acted as a mediator of BCR-ABL induced clonogenic activity.

Taken together, the data in this study establish Cks1 as a crucial regulator of HSC/HPC homeostasis, acting upstream of CKI. Cks1 is proposed to be a therapeutic target as a central intermediate for oncogene-induced cell cycle regulation in hematopietic malignancies.

INTRODUCTION

1. Introduction



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