«3. Medizinische Klinik und Poliklinik – Hämatologische Forschung Cks1 is a critical regulator of hematopoietic stem cell cycling, quiescence and ...»
In Addition to the control via ubiquitin ligase mediated proteosomal degradation of the CIP/KIP family of CDK inhibitors and p130, SCFSkp2 controls other proteins that may also have important roles in HSC/HPC, such as IkB [199, 200], β-catenin  and E2F1 . IkB is an inhibitory protein in the canonical NF-kB pathway, degradation of which is necessary to induce NF-kB activity . Recently, NF-kB was shown as a positive regulator of HSC quiescence  through noncanonical signaling, which does not involve IkB . This finding indicates that the control of the SCFSkp2 in the NF-kB pathway probably does not apply in HSC, which on the other hand correlates with the observed effects in this study, since lack of Cks1 resulted in increased HSC DISCUSSION quiescence (Chapters 4.2.10.-4.2.13.) and a hypothetical NF-kB inhibition would lead to the opposite effect. β-catenin, another SCFSkp2 target  is shown to promote HSC self-renewal in a contact dependent manner through the Wnt signaling pathway . However, stabilization of β-catenin in the HSC resulted in HSC exhaustion , excluding SCFSkp2 as a possible regulator of this pathway in HSC, since the opposite effect was observed in this study (Chapters 4.2.10.-4.2.13.).
Cks1 also functions in regulating CDK expression and activity at least partly independent of SCFSkp2 [143, 206], thus additional studies in this direction might reveal a complex role of Cks1 in regulating HSC homeostasis.
Figure 33 represents the hypothetical involvement of the SCFSkp2 targets, which were shown to accumulate in Cks1-/- HSC/HPC (Chapter 4.1.1.) during early hematopoiesis.
Figure 33: Hypothetical model representing the role of SCFSkp2-Cks1 in early hematopoiesis and the possible CKI involvement.
5.3. Cks1 mediates the balance between lymphoid and myeloid lineage differentiation through regulation of p27 In addition to engraftment and self-renewal studies using transplantation assays, 5-FU analysis provides information about the regenerative capacity of HSC and progenitor differentiation after ablation of mature hematopoietic subsets [171, 178-180]. A remarkable observation was made after 5-FU treatment of Cks1 respectively p27-deficient mice and mice lacking both, Cks1 and p27 (Chapter 4.3.3). While loss of Cks1 resulted in an increase of BDISKUSSION lymphocytes and decrease in T-lymphocytes and granulocyte percentage, in p27-/- BM the opposite effect was observed. The simultaneous loss of Cks1 and p27 resulted in a phenotype similar to WT, indicating that Cks1 regulation of the differentiation after chemo-ablative stress occurs probably through the downstream target p27. A study showing increased T-lymphocyte proliferation in p27-/- mice supports this finding . Also, after transplantation analysis, Cks1-/- B-lymphocytes were increased and Cks1-/- -delivered T-lymphocyte were decreased (Chapter 4.3.4.). However in this case, the opposed effect after the transplantation of p27-/- cells was sustained in the DKO group, indicating a stronger effect of the p27 loss in the DKO cells after serial transplantations.
Interestingly, serial BM transplantations of p27-/- /p57-/- induced the same phenotype in the adult hematopoietic populations as 5-FU treatment of p27-/mice, T cell and myeloid populations were increased and B cell population were decreased .This finding and the fact that the opposite effect in mice lacking Cks1 was observed after CD150+ LSK transplantations (4.2.12. and 4.2.13.), clearly indicates the Cks1 involvement in differentiation after hematopoietic stress as a regulator of p27 (Figure 33).
The HSC population itself might be heterogeneous and can be subdivided into myeloid-biased and lymphoid-biased stem cells with different properties .
Myeloid-biased stem cells, which preferentially differentiate to myeloid cells display improved engraftment and self-renewal capacities compared to lymphoid-biased stem cells . The transplantations with sorted HSC in this project (4.2.12. and 4.2.13.) reveal Cks1-/- HSC as mainly lymphoid-biased.
They were more quiescent, displayed decreased engraftment and delivered more B-lymphocytes. It is therefore possible that Cks1 affects the distribution of myeloid- and lymphoid-biased HSC, an observation that provides an interesting field for further research.
5.4. E3 ubiquitin ligases are critical factors in the control of HSC fate The observations made in Cks1-deficient mice show overlap with those reported for the Skp2-/- mice [197, 198]. These studies revealed that Skp2deficient mice possess decreased marrow cellularity and increased frequencies of LT-HSC, which seem to have an increased ability to self-renew in vivo. Both Skp2 and Cks1 function in the same SCF complex . The similarity in the DISCUSSION functional modulations of hematopoiesis in Cks1-/- animals compared to Skp2 deficiency demonstrates that the function of Skp2 in hematopoiesis is uniquely dependent on Cks1 and that the SCFSkp2 ubiquitin ligase is critically important in the control of HSC fate. The present study additionally reveals that the accumulation of SCFSkp2-Cks1 targets is most prominent in the earliest phenotypic hematopoietic population (CD150+ LSK) (Chapter 4.1.1.). These results suggest that the E3 ligase is specifically active in the earliest HSC. Since deficiency of either Skp2 or Cks1 reduces cell cycle entry and maintains a larger population of self-renewing cells, rapid up-regulation of SCFSkp2-Cks1 might be at the center for the recruitment of quiescent cells into active cell cycle which is required to initiate a regenerative response.
Deletion of E3 ubiquitin ligases such as Asb2 , c-Cbl , Itch  and VHL  results in similar phenotype as in Cks1-/- or Skp2-/- mice, where HSC engraftment or self-renewal is increased. This is remarkable, because these ligases target different molecules in HSC than SCFSkp2-Cks1. For instance, c-Cbl acts as negative regulator of HSC self-renewal probably through the TPO/cMpl signaling pathway, which is known to be crucial for the maintenance of HSC [210, 213] and Itch seems to play a similar role to c-Cbl, but via the Notch signalling pathway . VHL is the E3 ligase involved in hypoxia-inducible factor-1alpha (HIF-1a) protein degradation  and HIF-1a is needed in HSC to maintain their quiescence .
On the other hand, deficiency of other E3 ubiquitin ligases such as Fancl  and Fbxw7  result in contrary effects to Skp2 and Cks1 deficiency, as their deletion causes stem cell exhaustion. For instance, Fancl ubiquitinates βcatenin, however the ubiquitin chain, which Fancl adds to β-catenin, possesses an atypical expansion with nonproteolytic functions, increasing β-catenin activity and inducing expansion of multilineage progenitors . A recent study shows the PI3K/Akt pathway, which is involved in self-renewal and survival of HSC, to induce Fancl levels  supporting the idea that this E3 ligase plays a contrary role than the SCFSkp2 complex. One of the Fbxw7 ligase targets is c-Myc, which overexpression leads to HSC exhausting due to accelerated differentiation . And loss of Fbxw7 in BM resulted in c-Myc accumulation  explaining the similarity to the c-Myc overexpression phenotype in Fbxw7 knockouts.
DISKUSSION Taken together, these studies demonstrate that E3 ligases play a central role in the control of HSC behaviour and are strongly involved in the fine-tuning of the complex HSC homeostasis.
5.5. Potential regulators upstream of Cks1 in HSC SCF ubiquitin ligase complexes use a family of F-box proteins as substrate adaptors to mediate the degradation of a large number of regulatory proteins involved in diverse processes. The coordinate expression of such substrate adaptors is therefore of utmost importance [196, 218]. In the case of SCFSkp2, Cks1 adds an additional level of substrate specificity [172, 218], implying that the regulation of Cks1 transcript and protein levels should be tight.
The transcription factor c-Myc, a key protein for acquisition and maintenance of stem cell properties [217, 219] has also been identified as a positive transcriptional regulator of Cks1 . Conditional loss of c-Myc in the BM resulted in accumulation of HSC, which were diminished in their ability to differentiate . Since lack of Cks1 also resulted in elevated HSC quiescence (Chapters 4.2.10.-4.2.13.), it is possible, that c-Myc is one of the positive regulators of Cks1 activity in HSC.
While Myc regulation of Cks1 is indirect , another transcription factor, NFY, binds directly to the Cks1 promoter to regulate its expression during the cell cycle . Deficiency for Cks1 resulted in higher sensitivity of HPC against apoptosis (Chapter 4.2.6.), slower cycling (Chapters 4.2.5., 4.2.8. and 4.2.13.) and increased persistence of CD150+ HSC (Chapters 4.2.10-4.2.13). NF-Ya deletion is shown to create an accumulation of HSC in G2/M, prompts apoptosis and causes hematopoietic failure , a phenotype that could involve reduced levels of Cks1.
One known regulator of HSC quiescence is TGF-β . TGF-β signaling limits the activation of Akt, induced by SCF or TPO stimulation and promotes HSC survival but not proliferation, thus maintaining quiescence . Besides, TGF-β induces p57 expression and prevents cyclin D1 sequestration [66, 102], supporting the finding, that p57 is required for the quiescence in HSC .
Interestingly, TGF-β stimulation leads to transcriptional down-regulation of Cks1 [147, 221]. If this mechanism applies to HSC, then Cks1 loss would mimic
constitutive TGF-β stimulation, thus explaining the increased quiescence in Cks1-/- HSC (Chapters 4.2.10.-4.2.13.).
Another recently identified regulator of the SCFSkp2 complex is FoxO3a , a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway [223, 224]. FoxO3a negatively regulates SCFSkp2, inhibiting the E3 ligase activity and promoting p27 stability . Since FoxO3a is known to be essential for maintenance of HSC quiescence and HSC pool  and lack of FoxO3a results in decreased expression of the promoters of quiescence p57 and p27 [73, 101], the transcription factor is also a possible SCFSkp2-Cks1 regulator in early haematopoiesis.
The potential regulators of Cks1 in early hematopoiesis represent an interesting subject of further investigation. A hypothetical model of the upstream regulators of Cks1 in early hematopoiesis, based on published studies is represented in Fig. 34.
Figure 34: Hypothetical model representing possible regulators of Cks1 in early hematopoiesis.
5.6. Cks1 is an essential regulator in cancer cells The balance between HSC quiescence and activation is not only important for regeneration but also impacts the accumulation of oncogenic mutations. Indeed, it has been shown that prolonged activation of HSC can lead to malignant transformation . Since the level of Cks1 is shown to be a critical determinant in the balance between HSC quiescence and activation (Chapter 4.2), one would predict that decreased Cks1 would favor quiescence, and DISKUSSION increased Cks1 expression promotes HSC activation. Thus, it would be likely that Cks1 levels were increased in stem cell cancers. In favor of this hypothesis, it was shown (Chapter 4.4.1.), that Cks1 transcription levels were increased in patients with CML, a classical hematopoietic stem cell disorder , and that treatment of the disease results in decrease of the Cks1 transcription levels.
Furthermore, BCR-ABL expression in Cks1-/- LSK resulted in decreased colony forming capability of these cells (Chapter 4.4.2.). In addition, in various other malignancies elevated levels of Cks1 transcript and protein are associated with high proliferation and poor prognosis [218, 226, 227], indicating that oncogeneinduced Cks1 overexpression contributes to aggressive disease and treatment resistance. Indeed, Myc overexpression or activation that leads to induction of Cks1 expression  results in suppression of p27 levels and resistance to the Abl inhibitor imatinib in CML . Further data that link SCFSkp2 to fate decisions in cancer-initiating cells come from successful pharmacological inactivation of Skp2 as a novel therapeutic means . Since not every Skp2mediated substrate recognition requires the presence of Cks1 , it is currently not clear whether for some cancer types the isolate inhibition of the Cks1-Skp2 physical interaction could be a more specific and/or less toxic approach as compared to ubiquitous SCFSkp2 inhibition. In this context it also has to be noted that loss of Cks1, but not loss of Skp2, results in a significant delay in tumorigenesis in Myc lymphoma model [148, 230], and that Cks1 has cell cycle regulatory functions independent of p27 suppression (this study and ).
In the present study, Cks1 is identified as an essential regulator of HPC/HSC fate operating upstream of CDK. p27 is shown to be an essential downstream target of the SCFSkp2-Cks1 complex in the regulation of lineage distribution at stress hematopoiesis. Cks1 loss prominently affects CD150+ LSK by inhibiting exit from quiescence and cell cycling and protecting the more mature HPC from apoptosis. Accumulation of SCFSkp2-Cks1 targets, the CKI p21, p27, p57 and the Rb family member p130 in absence of Cks1 in the earliest phenotypic hematopoietic population (CD150+ LSK) suggest that the E3 ligase SCFSkp2 is specifically active in these early cells, uniquely dependent on Cks1 and critically DISCUSSION important in the control of HSC fate. Referring to current studies, it is most likely that all Cks1 CKI targets and p130 are involved in the HSC control.
In CML, Cks1 is overexpressed and Cks1 loss results in impaired oncogeneinduced growth factor independence. Therefore Cks1 is proposed to be a central intermediate for normal and oncogene-induced cell cycle regulation in hematopoiesis and a therapeutic target in hematologic malignancies.
LIST OF TABLES AND FIGURES
6. List of tables and figures Figure 1: Regulators of the cell cycle
Figure 2: Hematopoietical tree in mice
Figure 3: SCFSkp2 – induced p27 degradation
Table1: Antibodies used for surface staining in flow cytometry
Table 2: Antibodies used for intracellular staining in flow cytometry
Table 3: Antibodies used for western blotting
Figure 4: Genotyping of (A) Cks1-/- and (B) p27-/- mice.
Figure 5: Loss of Cks1 results in accumulation of SCFSkp2 substrates.