Cyclin C Regulates the Quiescence of Human CD34+CD38- Hematopoietic Stem Cells.

Abstract The relative quiescence of adult hematopoietic stem cells (HSCs) at steady state represents an important regulatory mechanism for maintaining their self-renewal and engraftment capacity, as well as their resistance to cytotoxic insults. However, the specific mechanisms regulating the intermittent entry of HSCs into the cell cycle are not well characterized. Here we provide the evidence that cyclin C (CCNC) specifically promotes the G0/G1 transition of human CD34+CD38- HSCs, and thus can significantly affect the loss of HSC self-renewal capacity in in vitro culture. Based on the recently hypothesized specific function of CCNC in G0 exit of human fibroblasts, we have analyzed the effects of CCNC loss on the behavior of human cord blood HSCs. We achieved a highly efficient knockdown of CCNC expression (&gt;90%) using lentiviral shRNA (shCCNC) transduction of freshly isolated human cord blood CD34+ cells, allowing the in vitro assessment of early cell cycle regulation in HSCs. First, we observed a 3-fold increase in the G0 fraction of shCCNC transduced CD34+ cells compared to the empty vector control, based on the Pyronin Y and Hoechst 33342 staining 72h after infection. The depletion of CCNC did not prevent cell cycle progression beyond the G1 entry, as we observed no significant changes in the G1/S/G2-M distribution, indicating that critical CCNC activities may be restricted to the G0 checkpoint. Consistent with the reported enrichment of functional HSCs in the G0 fraction, CCNC knockdown (CCNC KD) cells showed increased activity in all surrogate in vitro assays of stem cell-ness tested: a ∼3 fold increase in CD34+ population after long term culture, a ∼2.5 fold increase in long-term culture initiating cells (LTC-ICs) and a ∼3.5 fold increase in cobblestone area forming cells (CAFCs). In contrast, CFU assays using freshly sorted shCCNC cells (and cells obtained after one-week culture in cytokines) showed only a minimal decrease in total colony number, with no difference in colony composition or morphology, indicating no significant effect on hematopoietic progenitor cell differentiation. However, we did observe a prominent effect on secondary CFUs after 2 and 3 weeks in liquid culture (i.e. using the delta assay), namely a 2-fold and 30-fold increase in shCCNC over control culture respectively, again indicating a specific function of CCNC on the more primitive cells. Consistently, CCNC KD robustly enhanced CD34 expression and secondary CFU maintenance in sorted CD34+CD38- cells (HSCs); both markers of hematopoietic cell immaturity were rapidly lost in CD34+CD38+ cells (i.e. the committed progenitor cells) with no detectable effect of shCCNC transduction. Finally, we have found that these effects of CCNC depletion are likely the result of its initial loss of function, as transient CCNC KD, using siRNA transfection of CD34+ cells, produced similar biological effects as the constitutive lentiviral shCCNC expression. Collectively, these data indicate a cell context-dependent effect of CCNC KD on the initial rate of cell cycle entry by quiescent HSCs and suggest that this approach could be used to preserve their functional capacity in culture, potentially enhancing the ex vivo expansion of HSCs, as well as their use in gene therapy protocols. Transplantation of transduced CD34+ cells into sublethally irradiated immunodeficient mice is now under way to establish the potentially beneficial effects of CCNC KD on the engraftment and repopulating capability of cultured HSCs.</jats:p

Generation of a Novel, Multi-Stage, Progressive, and Transplantable Model of Multiple Myeloma

Abstract Abstract 327 Multiple myeloma is characterized by the progressive expansion of monoclonal plasma cells in the bone marrow, which leads to the production of serum and/or urine monoclonal proteins and systemic complications including lytic bone lesions, renal abnormalities hypercalcemia, and infections. Although the treatment of multiple myeloma has vastly improved, multiple myeloma remains a generally incurable disease. Transgenic mouse models have been generated that develop plasma cell accumulations or myeloma, however these models are quite imperfect in mimicking the human disease. Quite serendipitously, we have generated a multi-stage, progressive, and transplantable mouse model of multiple myeloma, crossing a genetically modified mouse with aberrant class switch recombination with another modified mouse that has elevated DNA damage response signaling. We have reported that cells expressing the hypermorphic Rad50s allele show constitutive ATM activation, leading to cancer predisposition and aggressive hematopoietic failure in Rad50s/s mice. While deficiency of the transcription factor Mef/Elf4, which regulates the quiescence of hematopoietic stem/progenitor cells, can mitigate hematopoietic failure observed in Rad50s/s mice, we found that 70% of Mef−/−Rad50s/s mice more than 200 days old died from multiple myeloma, plasmacytoma, or plasma cell leukemia, confirmed by pathology, immunohistochemistry, flowcytometry (CD138/B220 profiles), and PCR analysis for VDJ recombination. Prior to the onset of the plasma cell neoplasms, the Mef−/−Rad50s/s mice show abnormal plasma cell accumulation in the peripheral blood and bone marrow, which worsens with age. As the mice age, they also develop progressive increases in g-globulin levels and decreases in serum albumin levels. Monoclonal protein peaks were frequently observed in the serum of mice older than 200 days, and in step with the progressive nature of these manifestations, anemia and lower bone mineral density becomes apparent as the mice further age. Overall, the median survival of the Mef−/−Rad50s/s mice is approximately 470 days. The plasma cell neoplasms derived from Mef−/−Rad50s/s mice can be transplanted into recipient mice and the onset of the transplanted disease is markedly accelerated, to approximately 4 weeks post transplantation. Thus, the transplanted neoplastic Mef−/−Rad50s/s plasma cells appear to be more aggressive than the original ones. Taken together, our findings suggest that the Mef−/−Rad50s/s animal model can recapitulate the spectrum and pace of human plasma cell neoplasms, including the progression from monoclonal gammopathy to multiple myeloma. Class switch recombination is facilitated in Mef−/−Rad50s/s B cells in vitro, compared with control, Mef−/−, and Rad50s/s B cells, thus the plasma cell neoplasms found in Mef−/−Rad50s/s mice may result from Rad50s-driven oncogenesis. This novel Mef−/−Rad50s/s myeloma animal model should be useful for the drug screening of novel anti-myeloma compounds, as well as defining the pathogenesis of multiple myeloma/plasma cell neoplasms. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Cyclin C Regulates the Quiescence of Human CD34+CD38- Hematopoietic Stem Cells

Abstract The relative quiescence of adult hematopoietic stem cells (HSCs) at steady state represents an important regulatory mechanism for maintaining their self-renewal and engraftment capacity, as well as their resistance to cytotoxic insults. However, the specific mechanisms regulating the intermittent entry of HSCs into the cell cycle are not well characterized. Here we provide the evidence that cyclin C (CCNC) specifically promotes the G0/G1 transition of human CD34+CD38- HSCs, and thus can significantly affect the loss of HSC self-renewal capacity in in vitro culture. Based on the recently hypothesized specific function of CCNC in G0 exit of human fibroblasts, we have analyzed the effects of CCNC loss on the behavior of human cord blood HSCs. We achieved a highly efficient knockdown of CCNC expression (>90%) using lentiviral shRNA (shCCNC) transduction of freshly isolated human cord blood CD34+ cells, allowing the in vitro assessment of early cell cycle regulation in HSCs. First, we observed a 3-fold increase in the G0 fraction of shCCNC transduced CD34+ cells compared to the empty vector control, based on the Pyronin Y and Hoechst 33342 staining 72h after infection. The depletion of CCNC did not prevent cell cycle progression beyond the G1 entry, as we observed no significant changes in the G1/S/G2-M distribution, indicating that critical CCNC activities may be restricted to the G0 checkpoint. Consistent with the reported enrichment of functional HSCs in the G0 fraction, CCNC knockdown (CCNC KD) cells showed increased activity in all surrogate in vitro assays of stem cell-ness tested: a ∼3 fold increase in CD34+ population after long term culture, a ∼2.5 fold increase in long-term culture initiating cells (LTC-ICs) and a ∼3.5 fold increase in cobblestone area forming cells (CAFCs). In contrast, CFU assays using freshly sorted shCCNC cells (and cells obtained after one-week culture in cytokines) showed only a minimal decrease in total colony number, with no difference in colony composition or morphology, indicating no significant effect on hematopoietic progenitor cell differentiation. However, we did observe a prominent effect on secondary CFUs after 2 and 3 weeks in liquid culture (i.e. using the delta assay), namely a 2-fold and 30-fold increase in shCCNC over control culture respectively, again indicating a specific function of CCNC on the more primitive cells. Consistently, CCNC KD robustly enhanced CD34 expression and secondary CFU maintenance in sorted CD34+CD38- cells (HSCs); both markers of hematopoietic cell immaturity were rapidly lost in CD34+CD38+ cells (i.e. the committed progenitor cells) with no detectable effect of shCCNC transduction. Finally, we have found that these effects of CCNC depletion are likely the result of its initial loss of function, as transient CCNC KD, using siRNA transfection of CD34+ cells, produced similar biological effects as the constitutive lentiviral shCCNC expression. Collectively, these data indicate a cell context-dependent effect of CCNC KD on the initial rate of cell cycle entry by quiescent HSCs and suggest that this approach could be used to preserve their functional capacity in culture, potentially enhancing the ex vivo expansion of HSCs, as well as their use in gene therapy protocols. Transplantation of transduced CD34+ cells into sublethally irradiated immunodeficient mice is now under way to establish the potentially beneficial effects of CCNC KD on the engraftment and repopulating capability of cultured HSCs

Generation of a Novel, Multi-Stage, Progressive, and Transplantable Model of Multiple Myeloma

Abstract Abstract 327 Multiple myeloma is characterized by the progressive expansion of monoclonal plasma cells in the bone marrow, which leads to the production of serum and/or urine monoclonal proteins and systemic complications including lytic bone lesions, renal abnormalities hypercalcemia, and infections. Although the treatment of multiple myeloma has vastly improved, multiple myeloma remains a generally incurable disease. Transgenic mouse models have been generated that develop plasma cell accumulations or myeloma, however these models are quite imperfect in mimicking the human disease. Quite serendipitously, we have generated a multi-stage, progressive, and transplantable mouse model of multiple myeloma, crossing a genetically modified mouse with aberrant class switch recombination with another modified mouse that has elevated DNA damage response signaling. We have reported that cells expressing the hypermorphic Rad50s allele show constitutive ATM activation, leading to cancer predisposition and aggressive hematopoietic failure in Rad50s/s mice. While deficiency of the transcription factor Mef/Elf4, which regulates the quiescence of hematopoietic stem/progenitor cells, can mitigate hematopoietic failure observed in Rad50s/s mice, we found that 70% of Mef−/−Rad50s/s mice more than 200 days old died from multiple myeloma, plasmacytoma, or plasma cell leukemia, confirmed by pathology, immunohistochemistry, flowcytometry (CD138/B220 profiles), and PCR analysis for VDJ recombination. Prior to the onset of the plasma cell neoplasms, the Mef−/−Rad50s/s mice show abnormal plasma cell accumulation in the peripheral blood and bone marrow, which worsens with age. As the mice age, they also develop progressive increases in g-globulin levels and decreases in serum albumin levels. Monoclonal protein peaks were frequently observed in the serum of mice older than 200 days, and in step with the progressive nature of these manifestations, anemia and lower bone mineral density becomes apparent as the mice further age. Overall, the median survival of the Mef−/−Rad50s/s mice is approximately 470 days. The plasma cell neoplasms derived from Mef−/−Rad50s/s mice can be transplanted into recipient mice and the onset of the transplanted disease is markedly accelerated, to approximately 4 weeks post transplantation. Thus, the transplanted neoplastic Mef−/−Rad50s/s plasma cells appear to be more aggressive than the original ones. Taken together, our findings suggest that the Mef−/−Rad50s/s animal model can recapitulate the spectrum and pace of human plasma cell neoplasms, including the progression from monoclonal gammopathy to multiple myeloma. Class switch recombination is facilitated in Mef−/−Rad50s/s B cells in vitro, compared with control, Mef−/−, and Rad50s/s B cells, thus the plasma cell neoplasms found in Mef−/−Rad50s/s mice may result from Rad50s-driven oncogenesis. This novel Mef−/−Rad50s/s myeloma animal model should be useful for the drug screening of novel anti-myeloma compounds, as well as defining the pathogenesis of multiple myeloma/plasma cell neoplasms. Disclosures: No relevant conflicts of interest to declare

Regulation of Hematopoietic Stem Cell Quiescence - A Novel Role for p53

Abstract Although the p53 tumor suppressor can elicit cell-cycle arrest or apoptosis in hematopoietic cells upon DNA damage, its function during steady-state hematopoiesis is largely unknown. We demonstrated that the Ets transcription factor MEF/ELF4 regulates both HSC proliferation/self-renewal and quiescence, as Mef null mice exhibit greater numbers of hematopoietic stem cells and the Mef null HSCs are more quiescent than normal. As such, the hematopoietic compartment of Mef null mice shows significant resistance to chemotherapy and radiation (Lacorazza et al., Cancer Cell, 2006). In this study, we have investigated the mechanisms utilized by MEF/ELF4 to regulate the quiescence and self-renewal of hematopoietic stem cells, identifying p53 as a key regulator. We have recently found that Mef null mouse embryonic fibroblasts (mefs) accumulate p53 and undergo premature senescence; MEF appears to surpress the expression of p53 by directly upregulating Mdm2 (G. Sashida et al., submitted). We hypothesized that p53 may play a role in the enhanced stem cell quiescence or the increased HSC frequency seen in Mef null mice. To examine this, we generated p53−/− Mef −/− mice and compared HSC biology in the double knock out mice (p53−/− Mef −/−) vs p53 null mice, Mef null mice and wt mice. Loss of p53 decreased the fraction of Pyronin Ylow Lin-Sca-1+ cells, suggesting fewer quiescent HSCs, and staining of CD34-LSK cells for the proliferation marker Ki67 also showed enhanced HSC proliferation in the absence of p53 (with fewer quiescent cells present). These data suggest that p53 promotes quiescence in HSCs, and in the absence of p53, HSCs more readily enter the cell cycle. When we analyzed the DKO (p53−/− Mef −/−) mice, we observed that the percentage of G0 HSCs returned to normal, indicating that p53 is essential for maintaining the enhanced quiescence of MEF null HSCs. p21 is a major target gene of p53 in cells, and has been shown to play an important role in maintaining HSC quiescence. As expeceted, we found elevated levels of p21 mRNA in MEF null LSK cells and reasoned that p21 may account for their enhanced quiescence. We generated p21 −/− Mef −/− mice, which are viable, born at normal mendelian frequency and appear grossly normal. However, cell cycle analysis of HSCs obtained from p21 −/− Mef −/− mice showed that the enhanced quiescence in MEF null HSCs did not depend on p21, indicating that other p53 target genes play an important role in maintaining stem cell quiescence. We therefore utilized transcript profiling (Microarray studies and quantitative PCR analysis) to identify potential p53-regulated genes that may be differentialy expressed in LSK cells isolated from wild type, p53−/−, Mef −/−, and p53−/− Mef −/− mice. By ChiP and luciferase reporter assays, we show for the first time that Gfi-1 and Necdin are direct transcriptional targets of p53 in HSCs and both Gfi-1 and Necdin regulate the enhanced quiescence exhibited in MEF null HSCs. Taken together, our work defines a novel role for p53 in the maintenance of HSC quiescence. Furthermore, HSC quiescence and self-renewal appear to be mediated by different p53 target genes during steady state hematopoiesis

Regulation of Hematopoietic Stem Cell Quiescence - A Novel Role for p53.

Abstract Although the p53 tumor suppressor can elicit cell-cycle arrest or apoptosis in hematopoietic cells upon DNA damage, its function during steady-state hematopoiesis is largely unknown. We demonstrated that the Ets transcription factor MEF/ELF4 regulates both HSC proliferation/self-renewal and quiescence, as Mef null mice exhibit greater numbers of hematopoietic stem cells and the Mef null HSCs are more quiescent than normal. As such, the hematopoietic compartment of Mef null mice shows significant resistance to chemotherapy and radiation (Lacorazza et al., Cancer Cell, 2006). In this study, we have investigated the mechanisms utilized by MEF/ELF4 to regulate the quiescence and self-renewal of hematopoietic stem cells, identifying p53 as a key regulator. We have recently found that Mef null mouse embryonic fibroblasts (mefs) accumulate p53 and undergo premature senescence; MEF appears to surpress the expression of p53 by directly upregulating Mdm2 (G. Sashida et al., submitted). We hypothesized that p53 may play a role in the enhanced stem cell quiescence or the increased HSC frequency seen in Mef null mice. To examine this, we generated p53−/− Mef −/− mice and compared HSC biology in the double knock out mice (p53−/− Mef −/−) vs p53 null mice, Mef null mice and wt mice. Loss of p53 decreased the fraction of Pyronin Ylow Lin-Sca-1+ cells, suggesting fewer quiescent HSCs, and staining of CD34-LSK cells for the proliferation marker Ki67 also showed enhanced HSC proliferation in the absence of p53 (with fewer quiescent cells present). These data suggest that p53 promotes quiescence in HSCs, and in the absence of p53, HSCs more readily enter the cell cycle. When we analyzed the DKO (p53−/− Mef −/−) mice, we observed that the percentage of G0 HSCs returned to normal, indicating that p53 is essential for maintaining the enhanced quiescence of MEF null HSCs. p21 is a major target gene of p53 in cells, and has been shown to play an important role in maintaining HSC quiescence. As expeceted, we found elevated levels of p21 mRNA in MEF null LSK cells and reasoned that p21 may account for their enhanced quiescence. We generated p21 −/− Mef −/− mice, which are viable, born at normal mendelian frequency and appear grossly normal. However, cell cycle analysis of HSCs obtained from p21 −/− Mef −/− mice showed that the enhanced quiescence in MEF null HSCs did not depend on p21, indicating that other p53 target genes play an important role in maintaining stem cell quiescence. We therefore utilized transcript profiling (Microarray studies and quantitative PCR analysis) to identify potential p53-regulated genes that may be differentialy expressed in LSK cells isolated from wild type, p53−/−, Mef −/−, and p53−/− Mef −/− mice. By ChiP and luciferase reporter assays, we show for the first time that Gfi-1 and Necdin are direct transcriptional targets of p53 in HSCs and both Gfi-1 and Necdin regulate the enhanced quiescence exhibited in MEF null HSCs. Taken together, our work defines a novel role for p53 in the maintenance of HSC quiescence. Furthermore, HSC quiescence and self-renewal appear to be mediated by different p53 target genes during steady state hematopoiesis.</jats:p

Necdin Regulates Hematopoietic Stem Cell Quiescence and Sensitivity to Genotoxic Stress

Abstract Abstract 379 Necdin, a member of MAGE (melanoma antigen) family proteins, is a growth suppressing protein that was first identified in post mitotic neurons. The gene encoding necdin is one of several deleted in individuals with Prader-Willi syndrome, a neurobehavioural disorder associated with an increased risk of myeloid leukemia. It is reported that necdin interacts with p53 and represses p53-mediated apoptosis in neurons, but its role in hematopoiesis is largely unknown. Recently, we defined a critical role of p53 in regulating hematopoietic stem cell quiescence, and identified necdin as a target gene of p53, that is highly expressed in LT-HSCs (Liu Y et al., Cell Stem Cell, 2009). To define the role of necdin in hematopoiesis, we have analyzed the hematopoietic compartment of necdin-null mice. As necdin-null mice die perinatally, we first investigated fetal hematopoiesis and found no alteration in the frequency of fetal liver HSCs, defined as Lin-Sca1+Mac1+CD48-CD150+ within the fetal liver cells. Although necdin-null fetal liver HSCs increase serial replating capability in methylcellulose and maintain stemness in long-term stromal based cultures better than wild type HSCs, necdin-null fetal liver HSCs repopulate lethally irradiated recipient mice similar to wild type HSCs, in primary, secondary, and tertiary serial bone marrow transplantation assays. In addition, necdin-null HSCs show almost comparable repopulating ability as wild type HSCs, after secondary competitive bone marrow transplantation assays. These imply that necdin is dispensable for HSC self renewal. On the other hand, BM-derived necdin-null HSCs show decreased quiescence 4 months after transplantation, and increased proliferation as indicated by in vivo BrdU incorporation assays. Furthermore, recipient mice repopulated with necdin-null HSCs show enhanced sensitivity both to weekly 5-FU administration and to total body irradiation, as manifested by increased mortality. This suggests that the decreased quiescence of necdin-null HSCs leads to their depletion under conditions of genotoxic stress. Gene expression profiling studies have identified several deregulated signaling pathways in the necdin-null HSCs. Expression of several p53 target genes is altered in irradiated necdin-null HSCs, which may account for their enhanced radiosensitivity. We are now investigating these necdin target genes to clarify how necdin functions to critically regulate HSC quiescence. We are also determining whether targeting necdin could be a therapeutic approach to eliminate quiescent leukemia stem cells, using a murine CML model. Disclosures: No relevant conflicts of interest to declare

Necdin Regulates Hematopoietic Stem Cell Quiescence and Sensitivity to Genotoxic Stress.

Abstract Abstract 379 Necdin, a member of MAGE (melanoma antigen) family proteins, is a growth suppressing protein that was first identified in post mitotic neurons. The gene encoding necdin is one of several deleted in individuals with Prader-Willi syndrome, a neurobehavioural disorder associated with an increased risk of myeloid leukemia. It is reported that necdin interacts with p53 and represses p53-mediated apoptosis in neurons, but its role in hematopoiesis is largely unknown. Recently, we defined a critical role of p53 in regulating hematopoietic stem cell quiescence, and identified necdin as a target gene of p53, that is highly expressed in LT-HSCs (Liu Y et al., Cell Stem Cell, 2009). To define the role of necdin in hematopoiesis, we have analyzed the hematopoietic compartment of necdin-null mice. As necdin-null mice die perinatally, we first investigated fetal hematopoiesis and found no alteration in the frequency of fetal liver HSCs, defined as Lin-Sca1+Mac1+CD48-CD150+ within the fetal liver cells. Although necdin-null fetal liver HSCs increase serial replating capability in methylcellulose and maintain stemness in long-term stromal based cultures better than wild type HSCs, necdin-null fetal liver HSCs repopulate lethally irradiated recipient mice similar to wild type HSCs, in primary, secondary, and tertiary serial bone marrow transplantation assays. In addition, necdin-null HSCs show almost comparable repopulating ability as wild type HSCs, after secondary competitive bone marrow transplantation assays. These imply that necdin is dispensable for HSC self renewal. On the other hand, BM-derived necdin-null HSCs show decreased quiescence 4 months after transplantation, and increased proliferation as indicated by in vivo BrdU incorporation assays. Furthermore, recipient mice repopulated with necdin-null HSCs show enhanced sensitivity both to weekly 5-FU administration and to total body irradiation, as manifested by increased mortality. This suggests that the decreased quiescence of necdin-null HSCs leads to their depletion under conditions of genotoxic stress. Gene expression profiling studies have identified several deregulated signaling pathways in the necdin-null HSCs. Expression of several p53 target genes is altered in irradiated necdin-null HSCs, which may account for their enhanced radiosensitivity. We are now investigating these necdin target genes to clarify how necdin functions to critically regulate HSC quiescence. We are also determining whether targeting necdin could be a therapeutic approach to eliminate quiescent leukemia stem cells, using a murine CML model. Disclosures: No relevant conflicts of interest to declare. </jats:sec

The ETS Protein MEF Plays a Critical Role in Perforin Gene Expression and the Development of Natural Killer and NK-T Cells

AbstractWe utilized gene targeting by homologous recombination to define the role that MEF, a transcriptional activating member of the ETS family of transcription factors, plays in lymphopoiesis. MEF−/− mice have a profound reduction in the number of NK-T and NK cells. Purified MEF−/− NK cells cannot lyse tumor cell targets and secrete only minimal amounts of IFNγ. Perforin protein expression is severely impaired in MEF-deficient NK cells, likely accounting for the lack of tumor cell cytotoxicity. Promoter studies and chromatin immunoprecipitation analyses demonstrate that MEF and not ETS-1 directly regulates transcription of the perforin gene in NK cells. Our results uncover a specific role of MEF in the development and function of NK cells and in innate immunity