In this report we demonstrate that a single E3 ubiquitin ligase, Fbw7, is an essential regulator of HSC quiescence and self-renewal. Fbw7 is expressed in quiescent HSCs, and its expression is down-regulated upon the loss of their self-renewing capacity. These effects are cell autonomous, as Fbw7−/− LSKs are unable to compete with WT counterparts both in vitro and in vivo. The loss of LSK absolute numbers and self-renewal abilities are also reflected at the early stages of lymphopoiesis, as Mx-cre+Fbw7f/f mice have lower numbers of B cell, and almost no T cell, progenitors. To mechanistically explain these LSK phenotypes, we have performed comparative gene expression studies using Fbw7−/− stem cells and MPs. These studies demonstrated that Fbw7 deletion targets specific cell cycle regulators and suppresses the expression of several genes previously suggested to control HSC function. Thus, we have identified a novel function for the proteasome–ubiquitin ligase complex as a genetic “switch” that controls HSC cycle entry and self-renewal.
Our studies are in agreement with a recent report by Matsuoka et al. (44
). These investigators also demonstrate that Fbw7 is essential to maintain HSC quiescence and self-renewal, and suggest that c-Myc is a key Fbw7 substrate in HSCs. They also report that Fbw7−/−
animals die by either extreme pancytopenia at 12 wk after polyI-polyC injection or by T-ALL. Development of T-ALL was a later event, as disease appeared only after 16 wk after treatment. Only very few (n
= 2) of our polyI-polyC–injected Fbw7f/f
animals were allowed to reach these time points, and all developed pancytopenia. Thus, we cannot directly compare rates of T-ALL induction, as all other polyI-polyC–injected animals presented in this study were analyzed at very early time points. However, both Fbw7-targeted animals developed T-ALL with similar kinetics when crossed to the Lck-cre strain (unpublished data and reference 35
). A discrepancy between the two studies exists over the detection of apoptosis in Fbw7−/−
LSK cells. However, Matsuoka et al. (44
) reported elevated percentages of apoptosis only in HSCs purified from the marrows of severely cytopenic mice 12 wk after deletion. In the studies presented here, the analysis was performed using LSK cells purified from noncytopenic Fbw7−/−
mice 3 wk after polyI-polyC deletion. At that time point there was a significant alteration of HSC quiescence and a reduction of LSK (and LT-HSC) absolute cell numbers. In agreement with our findings, Matsuoka et al. (44
) failed to detect an increased frequency of apoptosis in HSCs purified from the BM of noncytopenic animals, even at 12 wk after deletion.
There are few other examples of stem cell function or differentiation being regulated by specific E3 ubiquitin ligases (30
). Initially, the UBR1/2 E3 ligases were shown to be essential for neurogenesis due to their effects on the differentiation and survival of neural stem cells and progenitors (45
). Also, in the neural system, the Mindbomb1 ligase regulates expression of Notch ligands and consequently controls differentiation of neural stem cells (46
). Finally, DDB1, an E3 ligase member of the cullin4A complex, is important for the viability, genomic integrity, and maintenance of neural stem cells (47
). Information on the role of any of the multiple E3 ligases on HSC function is scarce and indirect. Studies using a panel of chemical proteasome inhibitors have shown that suppression of proteasome function induced cell death in human CD34+
hematopoietic progenitors (48
). Moreover, primary CD34+
leukemic stem cells also enter apoptosis in response to treatment with the MG-132 proteasome inhibitor (49
). However, these were crude experiments that cannot address the complexity of regulation of proteasome function in HSCs by the different ubiquitin ligase complexes. Experiments similar to the ones described in this report, which include E3 ligase expression mapping and gene-targeting experiments, are thus essential for the detailed understanding of the importance of ubiquitination in HSC function.
Multiple protein substrates of Fbw7 have been identified. These include the cell cycle regulator cyclin E and transcription factors such as c-Myc and Notch (50
) that could directly or indirectly regulate stem cell function. Our protein expression analyses showed no stabilization of Notch1 in the BM of Mx-cre+
mice. In fact, we were unable to detect any Notch1-IC protein expression at all. This was not due to any technical obstacle, as Notch1 was significantly stabilized and overexpressed in the thymus of these mice. Also, we were unable to detect CD4+
cells in the Fbw7−/−
BM (not depicted), usually a reliable and rapid indicator of aberrant Notch activation (51
). However, we cannot exclude that Notch stabilization occurs in a small fraction of LSK cells, and this change in protein expression is below the sensitivity of the experimental method used. Indeed, Matsuoka et al. (44
) detected Notch1 overexpression using confocal microscopy of individual Fbw7−/−
LSK cells. In agreement with these results, our RT-PCR analysis of Fbw7−/−
LSK cells has revealed a persistent overexpression of Hes-1 and Deltex-1, both well-characterized targets of the Notch pathway ().
Similar studies excluded a role for stabilization of another Fbw7 substrate, cyclin E. Indeed, we were unable to detect any significant cyclin E stabilization in response to Fbw7 deletion in either total BM cells or purified stem cell and progenitor populations. These findings are consistent with genetic experiments reported by Loeb et al. (51
). These investigators have generated “knock-in” mice that express a mutated cyclin E, cyclin ET393A
. T393 falls within a canonical Fbw7 recognition degron, and its phosphorylation allows Fbw7 to bind and ubiquitinate cyclin E. Mutated cyclin E was stabilized and expressed at levels several-fold higher in several tissues, including lymphocytes and thymocytes. Despite this overexpression, cyclin ET393A
–homozygous mice showed no phenotypic abnormalities in all tissues examined, including the immune system. They also displayed a normal life span with no significant predisposition to autoimmune disease or tumor induction. Together with our protein expression analyses, these elegant genetic studies argue against a role of cyclin E stabilization in HSC function in response to Fbw7 deletion.
On the other hand, our studies suggested that c-Myc could be an attractive target, especially because it was previously shown to be involved in HSC differentiation (11
). Indeed, both total and phosphorylated c-Myc protein was consistently up-regulated in Fbw7−/−
BM stem cells and progenitors. These observations were consistent with the confocal microscopy studies reported by Matsuoka et al. (44
), strongly suggesting that c-Myc is indeed an important Fbw7 substrate. Putative targets of c-Myc, including p57kip2
, E2F2, and Ccnd1, were also up-regulated in Fbw7−/−
LSK cells. In agreement with this hypothesis, Wilson et al. (11
) have performed experiments in which they have overexpressed c-Myc specifically in LSK cells. Despite proper homing and inhibition of apoptosis, c-Myc–overexpressing HSCs failed to self-renew, a phenotype similar to the one reported here (11
). We should add here that although c-Myc appears to represent an important Fbw7 target, it is possible that there are other yet-unidentified Fbw7 substrates that are important for the hematopoietic phenotypes reported here.
Our presented studies identify Fbw7 as a novel essential regulator of HSC quiescence and self-renewal. Initially, deletion of Fbw7, which was normally expressed at high levels in noncycling HSCs, affects the expression of key regulators of cell cycle entry, including the D-type cyclin Ccnd1, the cell cycle inhibitor p57kip2, and the downstream transcriptional effector E2F2. The combined action of these regulators could induce exit from quiescence and cell cycle entry, as the BrdU incorporation experiments convincingly demonstrated that upon Fbw7 deletion >80% of LT-HSCs have entered cell cycle. It is tempting to hypothesize that aberrant cell cycle entry forces Fbw7−/− HSCs to exit the quiescent niche and lose their self-renewing ability. Once more, our expression studies provide mechanistic support to this hypothesis. Fbw7 deletion dramatically suppresses the expression of several genes (Mpl, Pbx3, p57, Meis1, Evi1, Eya1/2, Angpt, Thy1, and Ndn) that define a partial “LT-HSC” transcriptional signature (Table S1 and unpublished data).
The identification of Fbw7 as a key regulator of HSC quiescence and self-renewal may have very significant biological repercussions. Initially, Fbw7 function could be important for self-renewal of cancer stem cells with important implications in the therapeutic targeting of their maintenance. In agreement with this notion, Fbw7 deletion suppresses the expression of several genes involved in hematopoietic transformation (Ccnd1
, and Meis1
). It is intriguing to mention here that Fbw7 can also function as a tumor suppressor when deleted in T cells using the Lck-cre+
model (reference 35
and unpublished data), suggesting that Fbw7 function in oncogenesis could differ between uncommitted stem cells, committed progenitors, and mature lymphocytes. For all these reasons, the identification of regulators of Fbw7 activity, including specific deubiquitinating enzymes or substrate-specific priming kinases, could be valuable for both the development of HSC transplantation protocols and the suppression of transformation.