Biological impact and clinical relevance of long non-coding RNAs and post-transcriptional alterations in acute myeloid leukemia

Vollständiger Abstract

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Acute myeloid leukemia (AML) represents the most common acute leukemia in adults, marked by the abnormal expansion of myeloid progenitor cells that fail to properly differentiate. These immature cells accumulate in the bone marrow thereby distrupting normal hematopoiesis. Although advances in AML research have led to improvements in patient outcomes, the overall prognosis remains poor, particularly for relapsed and refractory cases. A deeper understanding of the molecular biology of AML is therefore urgently needed.Therefore, in this thesis, we aim to clarify how long non-coding RNAs (lncRNAs) contribute to AML pathogenesis, considering their recognized involvement in various biological functions and their altered expression patterns across many cancers, including AML. Additionally, we examine post-transcriptional regulatory mechanisms, a layer of gene regulation that has received comparatively limited attention in AML research.In Study I, using transcriptomic analyses of 7 AML patients and 5 normal bone marrow CD34+ samples, we identified 136 de novo lncRNAs that were differentially expressed in AML blasts. Among these, we characterized a novel transcript, which we named myeloid and AML-associated intergenic lncRNA (MALNC). MALNC was overexpressed in AML, particularly in patients harboring PML-RARA fusion or NPM1/IDH2R140 co-mutations, and higher expression was associated with better prognosis, independent of other established risk factors. Furthermore, MALNC knockout reduced AML cell growth and colony formation. In parallel, MALNC- depleted cells showed increased all-trans retinoic acid (ATRA)-induced differentiation and increased sensitivity to arsenic trioxide (ATO). To further characterize MALNC, transcriptomic analyses were performed in MALNC knockout clones both at baseline and following ATRA treatment, revealing modulation of genes involved in retinoic acid signaling. In addition, chromatin- binding experiments showed that MALNC interacted with genes related to the retinoic acid and Rho GTPase pathways.In Study II we employed high-throughput CRISPR-interference (CRISPRi) screens to knock down 7,996 lncRNAs and investigate their roles in AML proliferation, differentiation, and response to venetoclax. The screens identified 58 lncRNAs involved in proliferation, 4 lncRNAs affecting differentiation, and 23 lncRNAs associated with venetoclax response. Among these, AC009299.3 emerged as a candidate linked to venetoclax resistance, with patient data further showing a correlation between its expression and poor prognosis under standard chemotherapy treatment. In the proliferation screen, MIR17HG, CATG00000106133.1, and CATG00000056792.1 were identified as candidate lncRNAs promoting leukemic cell growth. Notably, CATG00000106133.1 was enriched in de novo and cytogenetically normal AML, strongly associated with NPM1 and IDH2_R140 mutations, and exhibited high expression in specific hematopoietic lineages. Functional validation through complete knockout of CATG00000106133.1, followed by transcriptomic profiling, revealed differential expression of genes involved in cytokine signaling and immune response pathways, providing insights into its role in AML biology.In study III, we investigated genes involved in post-transcriptional mechanisms in AML. Using CRISPR knockout screens from the DepMap database, the exon junction complex helicase eIF4A3, a regulator of RNA polymerase I- and II- dependent processes previously linked to cancer progression, emerged as a top essential gene across 18 AML cell lines. Transcriptomic analyses revealed higher eIF4A3 expression in AML cell lines and patient samples compared to normal controls, with gene ontology analysis highlighting RNA metabolism and translation as key affected pathways. In addition, functional experiments showed that chemical inhibition or siRNA-mediated silencing of eIF4A3 induced AML cell death through impaired ribosome biogenesis and p53 activation, as well as through p53- independent mechanisms.In summary, across these three studies, we identified novel lncRNAs and post- transcriptional regulators that expand our understanding of AML biology and drug response, pointing to their possible use as biomarkers or therapeutic targets to improve patient care.List of scientific papersI. Elisabetta Cozzi*, Anne Neddermeyer*, Xiangfu Zhong, Angelica Gamboa Cedeno, Dimitris C. Kanellis, Albin Österroos, My Björklund, Nona Struyf, Kasper Karlsson, Ying Qu, Alma Månsson, Tatjana Pandzic, Sofia Bengtzen, Christer Nilsson, Roland Fiskesund, Panagiotis Baliakas, Tom Erkers, Jiri Bartek, Olli-Pekka Kallioniemi, Hong Qian, Andreas Lennartsson, Sören Lehmann. MALNC: a new muntant NPM1/IDH2R140 and PML-RARA-associated lncRNA with impact on AML cell proliferation, maturation and drug response. Cancer Gene Therapy. (2025). https://doi.org/10.1038/s41417-025-00954-0II. Elisabetta Cozzi, Anne Neddermeyer, Sophia Miliara, Xiangfu Zhong, Tyler Weirick, Chung Chao Hon, Andreas Lennartsson, Sören Lehmann. Identification of long non-coding RNAs involved in leukemogenesis and venetoclax response in acute myeloid leukemia through functional CRISPR-dCas9 interference screens. [Manuscript]III. Sophia Miliara, Elisabetta Cozzi, Xiangfu Zhong, Isaac Chan, Karl Ekwall, Sören Lehmann, Andreas Lennartsson, Jiri Bartek, Dimitris C. Kanellis. The exon-junction complex helicase eIF4A3 holds therapeutic potential in acute myeloid leukemia. Leukemia. 2024 Mar;38(3):663-666. https://doi.org/10.1038/s41375-023-02098-2*Equal contribution