ALBERT

Description: Adult hematopoietic stem cells (HSCs) primarily reside in the hypoxic bone marrow microenvironment, and preferentially utilize anaerobic glycolysis to obtain energy. Cited2 is a cytokine-inducible gene, which plays various roles during mouse development. Our previous studies showed that deletion of Cited2 in adult mouse results in loss of HSC quiescence, increased apoptosis, and impaired HSC reconstitution capacity (Blood 2012, 119:2789-2798). In this study, we conditionally deleted Cited2 in Cited2fl/fl;Mx1-Cre mice and investigated the role of Cited2 in the metabolic regulation of HSCs. First, we examined mitochondrial alterations in Cited2 knockout (KO) long-term (LT-) and short-term (ST-) HSCs defined as “Flt3-CD34- LSK” and “Flt3-CD34+ LSK”, respectively. Staining with MitoTracker Green revealed that deletion of Cited2 resulted in a significant increase in mitochondrial mass in both LT- and ST-HSCs but not in the whole bone marrow cells. To explore the morphological changes of mitochondria in Cited2 KO HSCs, we sorted Flt3-LSK cells (containing LT- and ST- HSCs) and performed electron microscopy ultrastructural analysis. The mitochondria in wild type (WT) HSCs were mostly small, round or oval, and dark (Figure 1). However, Cited2 KO HSCs displayed markedly elongated and brighter mitochondria, similar to those observed in aged WT HSCs (20–24 months old mice) by others. The frequency of Cited2 KO LT-HSCs with high mitochondrial membrane potential was significantly increased (8.5% in WT versus 15.1% in KO). Furthermore, the reactive oxygen species (ROS) levels in Cited2 KO HSCs were significantly higher than those in WT controls. To further understand the metabolic changes in Cited2 KO HSCs, we measured glucose uptake using fluorescent indicator 2-NBDG. Glucose uptake was unchanged in the Cited2 KO LT- and ST- HSCs. Also, intracellular ATP content was maintained at the normal levels in Cited2 KO LT-HSCs, although slightly increased in ST-HSCs compared with WT controls. To assess the utilization of glycolysis in Cited2 KO HSCs, glycolytic flux was determined by glucose-derived 13C-lactate production using Gas Chromatography–Mass Spectrometry (GC-MS). We found that the rate of 13C-lactate production was significantly lower in both LT- and ST-HSCs lacking Cited2 than in WT controls. To further confirm this finding, we treated HSCs with antimycin A (AMA), a specific inhibitor of mitochondrial electron transport chain. We found that Cited2 KO HSCs displayed increased NADH after AMA treatment, compared with the WT control, indicating that mitochondrial respiration was increased in KO HSCs and produced more NADH. At the molecular level, deletion of Cited2 significantly reduced the expression of metabolism related genes in HSCs, such as lactate dehydrogenase (LDH) B and LDHD, pyruvate dehydrogenase kinase (Pdk) 2 and Pdk4, PYGL (phosphorylase, glycogen, liver), and GPX1 (glutathione peroxidase 1). Notably, Pdk2 and Pdk4 were recently shown to be critical controllers of glycolysis and checkpoint for cell cycle in HSCs. Consistent with reduced expression of Pdk, the phosphorylation of PDH-E1α was significantly decreased in Cited2 KO HSCs. Akt, mTOR, and FoxOs are known regulators of mitochondrial functions in HSCs. We found that Akt-mTOR signaling activity was increased in Cited2 KO HSCs, as indicated by increased phosphorylation of Akt and S6 ribosomal protein. However, in vitro treatment of LT-HSCs with mTORC1 inhibitor rapamycin did not resume decreased expression of LDHB, LDHD, Pdk2, and Pdk4, suggesting that elevated mTORC1 activity may not be the major contributor to the downregulation of glycolysis related genes. Meanwhile, we also found that in Cited2 KO LT-HSCs, phosphorylation of FoxO1 and FoxO3 was increased, both of which are known regulators of Pdk4 expression. Interestingly, in vitro treatment of LT-HSCs with PI3/Akt inhibitor LY294002, partially rescued the expression of Pdk4. Together, these findings suggest that the downregulation of Pdk4 in Cited2 KO HSCs is likely mediated by the inactivation of FoxOs caused by the elevated Akt activity. In summary, these results show that loss of Cited2 attenuates HSCs' glycolytic metabolism while simultaneously enhancing their overall mitochondrial oxidative phosphorylation, thus suggesting a critical role of Cited2 in the maintenance of adult HSC glycolytic metabolism likely through regulating LDH, Pdk, and Akt activity. Disclosures: No relevant conflicts of interest to declare.

Description: Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood, representing 31% of all tumors, and about 85% of children with ALL have B-cell ALL. Although the survival rate is approaching 90%, ALL remains the main cause of death from disease in children and young adults. The activity of histone deacetylases (HDAC) in childhood ALL is increased compared with that in normal peripheral blood mononuclear cells or bone marrow cells. Treatment of mice engrafted with T or B-ALL cells with HDAC inhibitor (HDACi) increases the acetylation of Histone 3 and Histone 4 and prolongs survival of these mice. Vorinostat (Suberoylamilide Hydroxamic Acid, SAHA) was the first HDACi approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Currently, several clinical trials are being conducted to evaluate its effects on other cancers, including ALL. However, some patients are resistant to HDACi therapy, and concerns regarding toxic side effects of HDACi exist due to the roles of HDACs in multiple pathways. Therefore, identification of new therapeutic targets is required which could improve the efficacy of HDACi by reducing the dose of HDACi administered without compromising the treatment benefits but alleviating the side effects of HDACi. CBP/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)–rich tail 2 (CITED2) is a cytokine-inducible gene that plays various roles during mouse development and, in particular, is essential for normal hematopoiesis. While the role of CITED2 in the pathogenesis of leukemia is currently unclear, dysregulation of CITED2 has been implicated in various types of leukemia, including ALL, in which downregulation of CITED2 is frequently observed. In this study, we tested the hypothesis that CITED2 may enhance the sensitivity of human pediatric pre-B ALL cells to HDACi SAHA. SAHA treatment of NALM-6 and 697 cells (human pediatric pre-B ALL cell lines) significantly induced apoptosis and cell cycle arrest in a dose dependent manner. The protein level of CITED2 was not affected by SAHA treatment. Although overexpression of CITED2 alone only slightly increased apoptosis, it significantly enhanced apoptosis resulted from SAHA treatment in both NALM-6 (15.1% versus 39.2%) and 697 cells (9.4% versus 14.6%) as assessed by annexin V/Propidium Iodide double staining and flow cytometry analysis (Figure 1). Accordingly, compared with control (i.e. NALM-6 cells transduced with GFP), overexpression of CITED2 also greatly reduced mitochondrial membrane potential of NALM-6 cells caused by SAHA treatment. To explore the potential mechanisms underlying enhanced apoptosis by overexpression of CITED2 in NALM-6 cells treated with SAHA, we determined the levels of pro- and anti- apoptotic proteins by Western blot and real-time quantitative PCR. We found that SAHA treatment increased the levels of pro-apoptotic molecules Bak, Puma, and Noxa, and decreased the levels of anti-apoptotic molecule Bcl-xL and apoptosis inhibitors XIAP and survivin. Importantly, overexpression of CITED2 markedly increased the protein levels of pro-apoptotic molecules Bak and Bim. Furthermore, knockdown of Bim by shRNA significantly attenuated apoptosis in Cited2 overexpressing NALM-6 cells treated with SAHA. Taken together, these results suggest that modulation of the CITED2 activity may confer its cooperative effect with SAHA in pre-B ALL cells and warrant future evaluation of such a combination in inducing apoptosis of primary ALL cells. Disclosures: No relevant conflicts of interest to declare.

Description: Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1–dependent and HIF-1–independent pathways.

Description: Abstract 912 Hematopoietic stem cells (HSCs) are thought to be localized in hypoxic microenvironment of the bone marrow (BM) and can remain quiescent or differentiate into multiple blood cell lineages. A number of factors have been found to regulate HSC quiescence in either cell-intrinsic or cell-extrinsic manner. Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2), a member of a newly identified transcriptional modulator, is a cytokine inducible gene and plays various roles during mouse development. In particular, Cited2 is essential for fetal liver hematopoiesis. In this study, we used conditional knockout strategy to delete Cited2 in order to further investigate its function in adult hematopoiesis. Sequential injection of poly(I)-poly(C) (pI-pC) efficiently deleted the Cited2 gene in Cited2fl/fl;Mx1-Cre mice. In this mouse model (Cited2−/− mice), the white blood cell (WBC) count, BM cellularity and Lin−Sca-1+c-Kit+ (LSK) cell number were within the normal range. However, the long-term HSC (LT-HSC; defined as Flt3−CD34−LSK or CD48−CD150+LSK) frequency was significantly decreased. Cited2−/− mice also exhibited increased apoptosis in both LSK and CD34−LSK cells. In addition, Cited2 deficiency led to loss of HSC quiescence evidenced by cell cycle analysis and BrdU incorporation assay. HSC reconstitution capacity was significantly impaired assessed by 5-fluorouracil (5-FU) treatment and transplantation experiments. Transcriptional profiling revealed that multiple HSC quiescence and hypoxia related genes were affected, including p57, Hes1 and Egr1. Recent studies have shown that both HIF-1α-deficient and HIF-1α-stabilized HSCs result in impaired hematopoietic reconstitution, suggesting that precise regulation of HIF-1α level is essential for maintaining HSC quiescence and transplantation activity. Cited2 has been shown to be a negative regulator for HIF-1α through competitive binding to CBP/p300 with higher affinity. In addition, we previously showed that HIF-1α haploinsufficiency (HIF-1α+/−) partially rescues the heart defects in Cited2−/− embryos and HIF-1α deletion (HIF-1α−/−) rescues aberrant vasculature in Cited2−/− embryonic lens. These findings prompted us to explore whether defects of Cited2−/− HSC in adult mice are mediated by dysregulated HIF-1 activity by generating Cited2fl/flHIF-1αfl/fl;Mx1-Cre mice. Additional deletion of HIF-1α partially rescued impaired HSC quiescence and reconstitution capacity caused by Cited2 deficiency. Cited2−/− HIF-1α−/− HSCs displayed comparable apoptosis to Cited2−/− counterparts. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to a normal level. Taken together, these results suggest that Cited2 regulates HSC quiescence through HIF-1 dependent and independent pathways. Disclosures: No relevant conflicts of interest to declare.

Description: Tyrosine kinase inhibitors (TKIs), VEGF/VEGF receptor inhibitors (VEGFIs) and immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced cancers including non-small-cell lung cancer (NSCLC). This study aims to evaluate the utility of plasma cell-free DNA (cfDNA) as a prognostic biomarker and efficacy predictor of chemotherapy (CT) with or without these precision therapies in NSCLC patients. Peripheral cfDNA levels in 154 NSCLC patients were quantified before and after the first target cycle of chemotherapy. The correlations of cfDNA with tumor burden, clinical characteristics, progression-free survival (PFS)/disease-free survival (DFS), objective response ratio (ORR), and therapy regimens were analyzed respectively. Baseline cfDNA, but not post-chemotherapeutic cfDNA, positively correlates with tumor burden. Notably, cfDNA kinetics (cfDNA Ratio, the ratio of post-chemotherapeutic cfDNA to baseline cfDNA) well distinguished responsive individuals (CR/PR) from the non-responsive (PD/SD). Additionally, cfDNA Ratio was found negatively correlated with PFS in lung adenocarcinoma (LUAD), but not lung squamous-cell carcinoma (LUSC) which may be due to a limited number of LUSC patients in this cohort. LUAD patients with low cfDNA Ratio have prolonged PFS and improved ORR, compared to those with high cfDNA Ratio. When stratified by therapy regimen, the predictive value of cfDNA Ratio is significant in patients with chemotherapy plus VEGFIs, while more patients need be included to validate the value of cfDNA Ratio in other regimens. Thus, the kinetics of plasma cfDNA during chemotherapy may function as a prognostic biomarker and efficacy predictor for NSCLC patients.