Medizinische Universität Graz - Research portal
Selected Publication:
Berg, J.
The role of micro-RNAs in the development of chronic myelomonocytic leukemia
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medical University of Graz; 2021. pp. 127
[OPEN ACCESS]
FullText
- Authors Med Uni Graz:
- Advisor:
- Höfler Gerald
- Sill Heinz
- Zebisch Armin
- Altmetrics:
- Abstract:
- Chronic myelomonocytic leukemia (CMML) is an aggressive neoplastic disease originating from malignant hematopoietic stem cells (HSCs). CMML leukemogenesis is often driven by genetic mutations occurring in HSCs, frequently affecting genes of the epigenetic machinery or signaling mutations, that hyperactive the RAS-MAPK/ERK pathway. Therapeutic options for CMML are limited, especially in unfit patients, where the use of hypomethylating agents (HMAs) shows only temporary efficacy. Deregulated micro RNAs (miRNAs, miRs) are potential players in CMML pathogenesis making them promising targets for future therapeutic intervention. In the first part of this thesis, we focused on miRNAs involved in CMML leukemogenesis. Initially, we confirmed results from a microarray miRNA expression screen performed in my master thesis. We additionally analyzed Lin-/Sca1+/c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) from a Kras G12D-induced CMML mouse model and demonstrated the consistent downregulation of miR-125a. We additionally corroborated these findings in 36 primary CMML specimen employing qPCR analysis. Functional in-vitro assays in myeloid cell lines with stable overexpression of miR-125a showed anti-leukemic effects. Next, we explored the molecular mechanisms leading to the decrease of miR-125a levels using bisulfite sequencing. This revealed the hypermethylation of the upstream/promoter region of miR-125a. Interestingly, the HMA azacitidine (Aza) could reverse hypermethylation and significantly increase miR-125a levels in myeloid cell lines. Further, qPCR analysis of serially obtained primary CMML specimen demonstrated higher miR-125a levels after HMA treatment, an effect specifically seen in patients with clinical response. To investigate the potential role of miR-125a in mediating Aza efficacy, we used miRNA inhibitors and CRISPR/Cas9-mediated knockout to prevent HMA-induced miR-125a upregulation in myeloid cell lines. Most importantly, the silencing of miR-125a reduced the cytotoxic efficacy of Aza in these cells, indicating that miR-125a mediates some of the anti-leukemic effects of HMAs. The second part of this thesis is based on the next generation sequencing (NGS) analysis of our CMML patient cohort. We observed a potential co-occurrence of RAS (NRAS and KRAS) and EZH2 mutations, which we aimed to investigate further. Previous clinical studies that tested targeted therapy against RAS-driven myeloid neoplasms (MNs) using MEK inhibitors have so far delivered disappointing results. We hypothesized, that ineffectiveness of this treatment approach is caused by other co-occurring genetic aberrations. Based on our preliminary results we investigated the co-existence of mutations in RAS modulators (RASmut defined as: KRAS, NRAS, CBL, NF1, PTPN11 mutations) and inactivation of the histone methyltransferase EZH2 (EZH2inact defined as mutations and/or chromosomal aberrations). In a data mining approach, we found EZH2inact significantly enriched in 450 primary CMML and acute myeloid leukemia (AML) patients with RASmut, which also correlated with shorter overall survival. Additional in-vitro assays could demonstrate that pharmacologic and shRNA-mediated inhibition of EZH2 in RASmut myeloid cell lines amplified oncogenic MAPK/ERK signaling. Interestingly, the co-occurrence of RASmut/EZH2inact also increased the sensitivity to MEK inhibitors in these cells. Finally, we explored the underlying mechanisms of RASmut/EZH2inact-mediated MAPK/ERK hyperactivation. RNA-sequencing analysis could show the upregulation of genes that activate the RAS-MAPK/ERK pathway. Taken together, RASmut and EZH2inact often co-exist in MN and synergistically amplify RAS-MAPK/ERK signaling. In turn, the increased dependency on the hyperactivation of RAS-signaling sensitize RASmut/EZH2inact cells to MEK inhibition, thus giving a first rational for novel therapy approaches in selected MN cases.