The Mir-193 Family Antagonizes Stem Cell Pathways and Is a Potent Tumor Suppressor in Childhood and Adult Acute Myeloid Leukemia
Bibliographic record
Abstract
Abstract MicroRNAs (miRNAs) are essential for maintenance and differentiation of normal hematopoietic cells and their dysregulation is strongly implicated in leukemias. In order to identify tumor suppressor miRNAs in the context of hematological malignancies, we performed two complementary miRNA expression screenings in normal hematopoiesis as well as in childhood and adult acute myeloid leukemias (AML). We reasoned that tumor suppressor miRNAs are upregulated in mature myeloid cells, as compared to normal hematopoietic stem and progenitor cells (HSPCs), and downregulated in AML. Based on this screening strategy, we identified the miR-193 family members as potent suppressors of HSPC activity and AML growth. During normal hematopoiesis mmu-miR-193a-3p is exclusively expressed in mature myeloid cells and absent in normal HSPCs. Accordingly, in a cohort of 165 pediatric AML patients hsa-miR-193b-3p was broadly repressed throughout the cytogenetically characterized subgroups. In addition, in a cohort of 43 adult AML patients, its homolog hsa-miR-193a-3p was significantly upregulated in APL cases (p=0.0025, n=7) compared to bone marrow from healthy donors (n=5). To assess the impact of the miR-193 family members on AML maintenance and development, we lentivirally expressed miR-193a/b in the MLL-rearranged cell lines ML2 and THP1, which induced monocytic differentiation in concert with calcitriol treatment, measured by CD11b/CD14 expression (p=0.024). Consistently, enforced miR-193-expression led to a significant growth disadvantage in ML2 and THP1 cells (p=<0.001 and p=0.02, respectively) as well as to reduced colony formation (p=0.008) in methylcellulose-based colony-forming unit (CFU) assays. Noteworthy, these effects were not restricted to MLL-rearranged AML cell lines only, but were also evident in six other AML cell lines representing the most common AML subgroups, such as t(8;21) and t(15;17). Beyond the growth-suppressive and differentiation-inductive effect of miR-193 in human AML cell lines, overexpression of miR-193a caused a significant decrease of proliferation in murine bone marrow cells immortalized in vitro by retroviral expression of Hoxa9 or Hoxa9 and Meis1 (p=0.019 and p=0.008, respectively). Based on these findings in AML, we further investigated the impact of the miR-193 family on normal hematopoiesis. We retrovirally expressed miR-193a in 32D cells treated with granulocyte-colony stimulating factor (G-CSF), which resulted in a strong induction of myeloid differentiation already after day 2 (p=0.006) as assessed by CD11b/Gr-1 surface marker expression. We lentivirally transduced mouse lineage negative (Lin-) HSPCs and transplanted them into irradiated isogenic recipients. Bleedings performed on weeks 4, 8 and 11, as well as the examination of the bone marrow on week 11, showed a severe competitive disadvantage of miR-193-transduced cells (week 11: 2% GFP+ miR-193- vs. 25% GFP+ miR-NSC-transduced cells). These results were further refined using highly purified ESLAM (CD45+ EPCR+ CD48− CD150+) HSCs which failed to reconstitute hematopoiesis when overexpressing miR-193a, indicated by the absence of miR-193a/GFP+ cells at week 8 post transplantation. These observations might be explained by a potent G1 cell cycle arrest in HSPCs when overexpressing miR-193a/b (4-fold decrease in the S phase population) and induction of apoptosis. Our results in normal and malignant hematopoiesis suggested that the miR-193 family acts globally through targeting relevant stem cell pathways. To validate this hypothesis we quantified the knockdown of ten predicted miR-193 target genes. qRT-PCR analysis confirmed the down regulation of KIT, KRAS, SOS2 (key components of the MAPK signaling pathway) and CCND1, a CDK regulator of G1/S phase transition. We propose a dual regulatory platform where firstly, miR-193 targets CCND1 and controls the cell cycle kinetics of stem cells. Secondly, miR-193 interferes with the KIT proto-oncogene and the RAS pathway thereby inhibiting crucial pro-proliferation and anti-apoptotic signaling cascades. Taken together, we identified the miR-193 family as a pan-tumor suppressor in childhood and adult AML. Its anti-leukemic effect is mediated by targeting the stem cell KIT/SOS2/RAS/RAF axis. 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) | 0.001 | 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".