ALBERT

Description: Article The cyclin-dependent kinase inhibitors p18 and p27 confer advantage to the propagation of haematopoietic stem cells (HSCs). In this manuscript, the authors demonstrate that p18 is a potent negative regulator of HSC self-renewal, and identify novel small molecules putatively inhibiting p18 that promote HSC growth in culture and mouse transplant assays. Nature Communications doi: 10.1038/ncomms7328 Authors: Yingdai Gao, Peng Yang, Hongmei Shen, Hui Yu, Xianmin Song, Liyan Zhang, Peng Zhang, Haizi Cheng, Zhaojun Xie, Sha Hao, Fang Dong, Shihui Ma, Qing Ji, Patrick Bartlow, Yahui Ding, Lirong Wang, Haibin Liu, Yanxin Li, Hui Cheng, Weimin Miao, Weiping Yuan, Youzhong Yuan, Tao Cheng, Xiang-Qun Xie

Description: Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

Description: Homing, lodgment, survival and proliferation are critical early determinants for the later outcomes of hematopoietic stem cell (HSC) or bone marrow transplantation (BMT). The irradiated bone marrow microenvironment may also pose an exhausting effect to the repopulating potential of donor HSCs, but the mechanisms for the effect are largely unknown. To determine whether these early events contribute to the exhausting effect, we have examined the kinetics of transplanted HSCs in 10 Gy lethally irradiated (IR) mice in comparison with transplanted HSCs in non-irradiated (NR) mice. 18 hours after transplantation, we found that the absolute number of homed Lin-Sca-1+ cells was not significantly different between IR and NR recipients. To examine the cell proliferative rate, CFSE staining together with flow cytometry was used to track the cell divisions of transplanted cells in the recipient marrow. While there were no detectable cell divisions in NR hosts, we detected 3 cell divisions in the Lin-Sca-1+ cell population 48 hours after BMT, thereby excluding the possibility that proliferation of hematopoietic cells was constrained in IR hosts. Regarding the expression of HSC associated markers, despite the similar expression of Sca-1 expression in both NR and IR recipients, the c-Kit was significantly downregulated to a nearly absent level in IR recipients, but it was not altered in NR recipients 18 hours post transplantation. The downregulation appeared to be transient since c-Kit was readily detectable after short-term engraftment. To functionally correlate c-Kit downregulation with long-term engraftment and self-renewal potential of transplanted HSCs, we sorted the homogeneous c-Kit+ cells (CD45.2+) and injected them into NR or IR recipients (CD45.1) at 5x106 cells/mouse. As expected, c-Kit became absent in IR hosts but not in NR hosts 18 hours after transplantation. We then harvested the homed cells and performed a competitive repopulation experiment involving the use of different congenic mice as secondary recipients at the dose of 1.3x 104 CD45.2 cells mixed with 1x105 competitive cells per mouse (n=4). Relative to the competitor cells (CD45.2/CD45.1 F1) in a same recipient, engraftment of the cells from IR recipients was lower than from NR recipients at each monthly time point (6 months). Moreover, the relative engraftment to competitor cells from IR recipients gradually declined to a minimal ratio of 0.03 while the engraftment from NR recipients sustained at a ratio of 0.3 after long-term engraftment. Finally, to further assess the self-renewal of the repopulated cells in the secondary recipients, 2 x 105 sorted CD45.2+ cells together with an equal number of competitor cells were re-transplanted into tertiary recipients. None of the mice (0/3) transplanted with cells originating from IR hosts were engrafted, but all mice (3/3) transplanted with the cells originating from NR recipients were engrafted as assessed at 6 months after tertiary transplantation. Given the previous studies by others showing that c-Kit signaling is involved in HSC lodgment and mobilization, we propose here that c-Kit downregulation in IR hosts impairs the lodging process of donor HSCs in the “niches” and as a consequence, the quality of the transplanted HSCs may be compromised. Therefore, further defining the molecular mechanisms for c-Kit downmodulation may guide us to develop novel approaches aimed to enhance the efficacy of HSC transplantation.

Description: Our recent study demonstrated that the cyclin-dependent kinase inhibitor (CKI) p18Ink4c (p18), also an INK4 family protein acting at early G1-phase, exerts its inhibitory role during the self-renewing division of murine hematopoietic stem cells (HSC) in vivo (Nature Cell Biology 2004). Down-modulating p18 may permit enhanced stem cell expansion in vitro, a hypothesis that is now being testing in our laboratory. To provide the proof-of-the concept, we first took advantage of the murine system by testing the in vivo reconstituting ability of cells that had been cultured under the Dexter culture condition for 19 weeks. 2–20x105 cells with non-adherent and adherent populations were transplanted into lethally irradiated hosts. 3 of 7 mice revealed long-term engraftment in the p18−/− transplanted group (0.5–33% engraftment levels) while there was no engraftment in the p18+/+ group (n=7). Moreover, a substantial level (38.6% on average) of long-term engraftments (7 months) in multilineage was achieved in secondary recipients transplanted with the p18−/− cells (n=3), demonstrating the self-renewal potential of the expanded HSCs after the extended period of long-term culture. These data strongly indicate that p18 absence is able to substantially mitigate the differentiating effect of the ex vivo culture conditions on HSCs and therefore offer a strong rationale for targeting p18 in human HSC expansion. P18 mRNA was detected by RT PCR in human CD34+ cells with a higher expression level in the more primitive subset: CD34+CD38−. To explore the possibility of targeting p18 for expanding human HSCs, we have employed the RNA interference (RNAi) technology in CD34+ cord blood cells. We screened a pool of small interfering RNA (siRNA) oligos and three of them were able to effectively reduce p18 expression by 60–80% in 48 hours as assessed by both RNA and protein analyses in human cells. Further, we tested both transient and permanent delivery methods for introducing the RNAi effect in the CD34+CD38− cells. To demonstrate whether the RNAi method would be sufficient to impact the outcome of cell division after a single or limited cell cycle(s), we chose the nucleofector technology and were able to achieve 48.30±11.66% of transduction efficiency with good viability (50.63±9.38%, n=3) in human CD34+ cells. After a single electroporation pulse, we were able to increase by 2-fold the CD34+CD38− cells associated with the same magnitude of increased colony forming activity under culture condition supplemented with SCF, TPO and Flt3. To observe the long-term effect of p18 downregulation in human HSCs, we constructed a p18 short hairpin (shRNA)-expressing lentiviral vector that was engineered to have the mouse U6 promoter upstream of a CMV-EGFP expression cassette. A transduction efficiency of 30–60% was achieved after overnight infection of the human CD34+ cells with the p18 shRNA or with control lentiviral vectors pseudotyped with the VSV-g envelope. 72–96 hours after the transduction, human p18 protein can be knocked down by the p18 siRNA lentivector at near 100% in the HeLa cell line as determined on the western blot, and at more than 50% in human primary CD34+ cells as determined by real time RT PCR. We are currently undertaking further study aimed at assessing the repopulating ability of the transduced human HSCs with lentivirus-mediated p18 shRNA in NOD/SCID mice. Together, these findings suggest that down-modulating p18 might be a feasible approach for manipulating human HSCs ex vivo.

Description: Abstract 917 Post-transcriptional regulation such as RNA editing in hematopoiesis and lymphopoiesis is poorly understood. ADAR1 (adenosine deaminase acting on RNA-1) is a RNA editing enzyme essential for embryonic development. Disruption of the ADAR1 gene was shown to cause defective embryonic hematopoiesis (Wang Q et al, Science 2000). Moreover, we have recently obtained direct evidence for the preferential effect of ADAR1 deletion on adult hematopoietic progenitor cells as opposed to the more primitive cells via a RNA-editing dependent mechanism by different conditional gene deletion strategies (Xufeng R et al PNAS 2009, in press). To further determine the role of ADAR1 in T cell development, we generated a mouse model in which ADAR1 was deleted specifically in T lymphocytes by interbreeding ADAR1lox/lox mice with Lck-Cre transgenic mice. In our current study, we report that ADAR1 is essential for T cell differentiation at the late progenitor stage in the thymus, coincident with T cell receptor-α/β expression. In ADAR1lox/loxLck-Cre mice, mature T cells decreased dramatically in peripheral blood, spleen and lymph nodes in comparison to littermate controls. In the thymus, the production of CD4+/CD8+ double positive cells was severely impaired and massive cell death was observed in pre-T cell populations. Within the pro-T cells, ADAR1 deletion resulted in a significant decrease of late progenitor cells but not early progenitor subsets. In both pro-T and pre-T cell stages, defective T cell development preferentially occurred in the beta chain positive cells, but was not apparent in gamma/delta T cells. Our data demonstrated an indispensable role of ADAR1 in early T cell differentiation that correlated with T cell receptor beta chain expression, thereby indicating that RNA editing by ADAR1 is an essential event in T cell development. Disclosures: No relevant conflicts of interest to declare.

Description: NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.

Description: Molecular paradigms underlying the death of hematopoietic stem cells (HSCs) induced by ionizing radiation are poorly defined. We have examined the role of Puma (p53 up-regulated mediator of apoptosis) in apoptosis of HSCs after radiation injury. In the absence of Puma, HSCs were highly resistant to γ-radiation in a cell autonomous manner. As a result, Puma-null mice or the wild-type mice reconstituted with Puma-null bone marrow cells were strikingly able to survive for a long term after high-dose γ-radiation that normally would pose 100% lethality on wild-type animals. Interestingly, there was no increase of malignancy in the exposed animals. Such profound beneficial effects of Puma deficiency were likely associated with better maintained quiescence and more efficient DNA repair in the stem cells. This study demonstrates that Puma is a unique mediator in radiation-induced death of HSCs. Puma may be a potential target for developing an effective treatment aimed to protect HSCs from lethal radiation.

Description: Ultimate success of hematopoietic stem cell transplantation (HSCT) depends not only on donor HSCs themselves but also on the host environment. Total body irradiation is a component in various host conditioning regimens for HSCT. It is known that ionizing radiation exerts “bystander effects” on nontargeted cells and that HSCs transplanted into irradiated recipients undergo proliferative exhaustion. However, whether irradiated recipients pose a proliferation-independent bystander effect on transplanted HSCs is unclear. In this study, we found that irradiated mouse recipients significantly impaired the long-term repopulating ability of transplanted mouse HSCs shortly (∼ 17 hours) after exposure to irradiated hosts and before the cells began to divide. There was an increase of acute cell death associated with accelerated proliferation of the bystander hematopoietic cells. This effect was marked by dramatic down-regulation of c-Kit, apparently because of elevated reactive oxygen species. Administration of an antioxidant chemical, N-acetylcysteine, or ectopically overexpressing a reactive oxygen species scavenging enzyme, catalase, improved the function of transplanted HSCs in irradiated hosts. Together, this study provides evidence for an acute negative, yet proliferation-independent, bystander effect of irradiated recipients on transplanted HSCs, thereby having implications for HSCT in both experimental and clinical scenarios in which total body irradiation is involved.

Description: Transplantation-associated stress can compromise the hematopoietic potential of hematopoietic stem cells (HSCs). As a consequence, HSCs may undergo “exhaustion” in serial transplant recipients, for which the cellular and molecular bases are not well understood. Hematopoietic exhaustion appears to be accelerated in the absence of p21Cip1/Waf1 (p21), a cyclin-dependent kinase inhibitor (CKI) in irradiated hosts. Our recent study demonstrated that unlike loss of p21, deletion of p18INK4C (p18), a distinct CKI, results in improved long-term engraftment, largely because of increased self-renewing divisions of HSCs in vivo. We show here that HSCs deficient in p18 sustained their competitiveness to wild-type HSCs from unmanipulated young mice, and retained multilineage differentiation potential after multiple rounds of serial bone marrow transfer over a period of more than 3 years. Further, p18 absence significantly decelerated hematopoietic exhaustion caused by p21 deficiency. Such an effect was shown to occur at the stem cell level, likely by a counteracting mechanism against the cellular senescence outcome. Our current study provides new insights into the distinct impacts of these cell-cycle regulators on HSC exhaustion and possibly HSC aging as well under proliferative stress, thereby offering potential pharmacologic targets for sustaining the durability of stressed HSCs in transplantation or elderly patients.