Enriched MicroRNA-126 Bioactivity Marks the Primitive Compartment In AML and Regulates LSC Numbers
Bibliographic record
Abstract
Abstract Abstract 94 Previous work has shown miRNAs to be dysregulated in acute myeloid leukemia (AML), however, there is little known regarding miRNA expression and function in leukemia stem cells (LSC). In order to elucidate the role of miRNA in LSC, we performed miRNA profiling on fractionated subpopulations of primary AML patient samples. Supervised analysis guided by the in vivo SCID leukemia initiating capacity (SL-IC) of each sub-population generated a unique miRNA signature associated with LSC enriched fractions. An in vitro antagomir-based functional miRNA knockdown screen identified miR-126, our top array candidate, for further study. After RT-PCR validation, the biological activity of miR-126 was confirmed at single cell resolution by using a novel bidirectional lentivirus miRNA reporter system in the 8227 cell line in vitro and within primary AML patient samples xenografted into immune-deficient NSG mice. These data suggest that primitive AML cells may express high levels of bioactive miR-126 relative to more “differentiated” blast populations. To test the hypothesis that AML stem cells are marked by high miR-126 bioactivity, we FACS sorted miR-126 genetic reporter vector transduced primary AML patient samples and transplanted these populations into immune-compromised secondary mouse recipients. The results of these proof-of-concept experiments demonstrates our ability to prospectively isolate LSC enriched fractions in all 4 AML patient samples tested using only a single biomarker, miR-126. Finally, to understand the functional relevance of miR-126 expression within primitive AML cells, stable enforced expression and knockdown of miR-126 was achieved using lentiviral vectors. Enforced expression of miR-126 in CD34+CD38- 8227 cells resulted in reduced AML blast colony formation, an increase/maintenance of CD34+ cells and a decrease in differentiation marker positive (CD14, CD15) AML blasts. Similarly, enforced miR-126 expression in 4 primary AML xenografts resulted in a several fold increase of CD34+CD117+ lentivirus marked leukemia cells after 12 weeks. In addition, the miR-126 cells showed reduced differentiation marker expression (CD14, CD15) with no significant differences in AML graft size. To determine if the expanded population had SL-IC activity or was a downstream leukemic progenitor, limiting dilution assays were performed by transplantation of FACS sorted lentivirus marked cells into secondary recipient mice for 12 weeks. A 13 fold increase in LSC activity was observed with miR-126 forced expression compared to control vector expressing cells. These data suggest that high levels of miR-126 bioactivity may support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Conversely, in vitro knockdown of miR-126 in CD34+CD38- 8227 cells increased AML blast colony formation, while no phenotype was observed in xenotransplanted primary AML, with secondary LDA transplant experiments ongoing. Target prediction algorithms and previously described target genes were used to ascertain the principal signaling pathway(s) under direct control of miR-126 in primitive AML cells. In summary, these experiments demonstrate that miR-126 is more abundantly expressed and biologically active within the leukemia stem/progenitor cell compartment of the AML functional hierarchy and serves to regulate AML stem cell numbers. Disclosures: No relevant conflicts of interest to declare.
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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 | 0.001 |
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".