Konference: 2015 57th ASH Annual Meeting - účast ČR
Kategorie: Nádory dětského a adolescentního věku
Téma: 603. Oncogenes and Tumor Suppressors: Poster I
Číslo abstraktu: 1244
Autoři: Kathrin Krowiorz; Razan Jammal; Stephan Emmrich; Arefeh Rouhi, Ph.D.; MD Michael Heuser; Dr. Lars Bullinger; Prof. Dr. Konstanze Döhner; Lai Courteney; Dr. Tobias Maetzig , Ph.D.; Vera Martins; Medhanie Assmelash Mulaw, BSc, MSc, PhD; Dr.rer.nat Dirk Heckl, Ph.D.; MD Christian Michel Zwaan, PhD; prof. MUDr. Jan Starý, DrSc.; MD Andre Baruchel, PhD; MD Valerie de Haas, Ph.D.; MD Dirk Reinhardt, PhD; Maarten W.J. Fornerod, Ph.D.; Prof. Dr. med. Hartmut Döhner (Doehner); Dr. Jens Ruschmann, Ph.D.; MD Marry M. van den Heuvel-Eibrink, PhD; MD R. Keith Humphries, Ph.D.; MD Florian Kuchenbauer, Ph.D.; MD Jan-Henning Klusmann
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.
Datum přednesení příspěvku: 5. 12. 2015