Gpx3 Determines Competitiveness of Normal and Leukemic Stem Cells.
Bibliographic record
Abstract
Abstract Abstract 1587 Although important efforts have been invested in the discovery of genes that regulate normal or leukemic hematopoietic stem cells (HSC) self-renewal, the number of validated candidates remains low, due largely to the unavailability of functionally pure stem cell populations. Moreover, it is often difficult to identify the normal counterpart cell from which leukemia originated, further complicating studies based on comparative gene expression. In this study, we used a series of recently characterized Hoxa9 + Meis1 acute myeloid leukemias (AML) derived from fetal liver (FL) cells (Wilhelm BT et al., submitted). These leukemias are remarkably similar in several aspects including their L-HSC frequency (between ∼1 in 100 to 350) except for one leukemia (FLA2) in which 70% of the cells show repopulation ability (i.e., L-HSC). We reasoned that comparative mRNA profiling of FLA2 to the phenotypically similar FLB1 (0.3% L-HSC) might identify genes uniquely associated with L-HSC self-renewal. We observed a 2–3-fold upregulation of Gpx3 in FLA2, which was confirmed by qRT-PCR. In accordance with this, all 14 of the tested Gpx3 promoter region CpG sequences were methylated in FLB1 and hypomethylated in FLA2 cells. The higher expression of GPx3 in FLA2 was confirmed at the protein level and reflected in elevated glutathione peroxidase activity in comparison to FLB1. Importantly, we also observed in FLA2 a relative reduction in reactive oxygen species (ROS) level (DCFDA) and a concomitant decrease in p38 MAPK activation (western blot and mass spectrometry). The correlation of Gpx3 levels with L-HSC frequency could be reflective of their functional dependence on this enzyme. FLA2 cells being difficult to manipulate ex vivo, to address this we utilized retroviruses encoding shRNAs and a GFP reporter to explore the in vivo function of FLA2 cells with downregulated Gpx3. The decrease in percentage of GFP+ donor cells when leukemia became apparent (∼19 days) from that of populations initially transplanted, was 4-fold higher following Gpx3 knockdown in comparison to shLuciferase transduction. Moreover, those shGpx3 infected FLA2 remaining at day 19 displayed a 3-fold decrease in GFP mean fluorescence intensity relative to their control counterparts. These results show that GFPhigh cells were selectively depleted, and suggest that Gpx3 is critical for the competitiveness of L-HSCs. Because redox metabolism has been implicated in HSC self-renewal, we also analyzed its expression and function in normal HSC to gain further insight into the role of GPx3 in stem cell activity. Interestingly, compared to FL-HSCs, isolated 3 and 4 week bone marrow (BM), HSCs exhibited a 39- and 6-fold decrease in Gpx3 expression, respectively. A correlation of Gpx3 levels with enhanced self-renewal was also observed in vitro as overexpression of several nuclear determinants of HSC expansion such as Hoxb4, NA10HD, Klf10 and Prdm16 promoted Gpx3 expression by 3.2 to 19.2-fold. We next infected BM cells enriched for HSCs with retroviruses carrying shRNAs to Gpx3. shRNA targeting of Gpx3 dramatically inhibited hematopoietic reconstitution. Transplantations of sublethally irradiated recipients indicated that Gpx3 knockdown significantly impaired both early and late donor-derived hematopoiesis. These results suggest that GPx3 is critical for repopulation mediated by both short and long-term repopulating cells. In reciprocal gain-of-function experiments, Lin-CD150+CD48- cells engineered to overexpress Gpx3, showed a marked competitive advantage over controls when transplanted following a 7-day ex vivo culture step. Insertional mutagenesis was ruled out as proviral integration analyses of six recipients confirmed polyclonal hematopoiesis. Moreover, some mice were in part reconstituted by the same clones, indicating that self-renewal occurred in vitro prior to transplantation. Phenotypic analysis of late-transplant hematopoietic tissues showed that Gpx3-transduced cells contributed to lymphoid and myeloid repopulation, confirming their multipotentiality. Together, these results indicate that Gpx3 enhances HSC expansion ex vivo possibly through modulation of self-renewal activity. In conclusion, a unique model of primary L-HSC was exploited to identify Gpx3 as a critical determinant for the competitiveness of L-HSCs and complementary experiments demonstrated a key role for this gene in normal HSC self-renewal. Disclosures: No relevant conflicts of interest to declare.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".