Abstract P09: Splicing and translation in chronic myeloid leukemia cells adapting to bone marrow microenvironment and chemotherapy

Abstract

Abstract Extravasation of leukemia cells implies a microenvironment change affecting the cell response to chemotherapy. Resistance is supported by cell adaptation to extracellular conditions founded on the post-transcriptional regulation of gene expression. Previous studies showed that mild activation of the cellular stress response supports therapy resistance of chronic myeloid leukemia (CML) cells [PMID: 23095523]. To better understand CML-CP cell characteristics in the bone marrow niche, we modeled human CML-bone marrow (BM) crosstalk. We employed various experimental setups in co-culture under hypoxia and spheroids, and utilized three approaches to model human BM in mice (subcutaneously formed ‘ossicles’ and organoids, and pre-seeded 3D printed scaffolds). Imaging of luciferase activity in mice or flow cytometry analysis of isolated cells showed that human primary CML-CP cells or K562 cells were resistant to imatinib and PARP1 inhibitor treatment in the presence of stromal mesenchymal cells, as observed in vitro previously [PMID: 33027668]. We also found that pre-implantation differentiation of mesenchymal stem cells to osteoblasts had an impact on the sensitivity of K562 to imatinib treatment indicating the pivotal role of the cellular composition of the stromal microenvironment. Cells isolated from in vivo (subcutaneous) organoids in imatinib-treated mice expressed markers of erythroid-like differentiation. In vitro, long-term culture in a setup modeling hypoxic bone marrow mimics imatinib treatment. We have recently shown the impact of a hypoxic BM-like setup on the regulation of translation and demonstrated differences in the nascent proteome, verified by an approach involving biorthogonal labeling of nascent peptides combined with click-it chemistry for quantitative mass spectrometry (QuaNCAT-MS) [PMID: 37123244]. Effects on co-transcriptional splicing were investigated by long-read sequencing of nascent RNA identifying genes that in K562 cells display a failure of polyA cleavage, leading to transcriptional readthrough. Many of these genes encode erythroid proteins. Analysis of selected nascent transcripts revealed crosstalk between polyA cleavage and splicing that is orchestrated by stress responses driven by the microenvironment, potentially playing a role in cell fate decisions. Interestingly, a comparison of imatinib with a PARP1 inhibitor and homoharringtonin treatments revealed that the level of transcriptional readthrough and changes in splicing are specific to the kind of therapeutic agent. Our results show that co- and post-transcriptional control (translation and splicing) are important components of the adaptation of leukemic cells to the microenvironment that modulates therapy resistance. I will discuss how gene-specific coupling between splicing and polyA cleavage in response to the microenvironment controls protein levels in leukemia. This work was supported by research grants from the National Science Centre 2018/30/M/NZ3/00274 to P.P-B., 2018/29/B/NZ3/01778 to K.P., and the National Institutes of Health R01GM140735 to K.M.N. Citation Format: Ewa Kozlowska, Remigiusz Serwa, Morgan Shine, Magda Wolczyk, Paulina Pilanc-Kudlek, Agata Kominek, Bac Viet Le, Ewa Walejewska, Joanna Idaszek, Wojciech Swieszkowski, Katarzyna Piwocka, Tomasz Skorski, Karla M Neugebauer, Paulina Podszywalow-Bartnicka. Splicing and translation in chronic myeloid leukemia cells adapting to bone marrow microenvironment and chemotherapy [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P09.