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Podszywalow-Bartnicka, P.

Publications and source records attributed to Podszywalow-Bartnicka, P..

4 recordsLinked to original sources

TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

Chronic myeloid leukemia (CML) is treated with Abl1 tyrosine kinase inhibitors (TKIs). Quiescent cancer cells residing in the bone marrow (BM) can survive the treatment and cause CML relapse. We previously found that a subset of alternative splicing (AS) changes detected in CML cells surviving months of therapy are initiated within hours of treatment onset. Here, we investigated how AS in CML cells is modulated by the human BM microenvironment. By incorporating humanized BM niche models in vivo, we uncovered stroma-induced transcriptome adaptation that influences transcriptional regulation, transmembrane transport, lipid metabolism, the tricarboxylic acid cycle, and respiratory electron transport. We identified RNA-binding protein TIAR (T-cell intracellular antigen-related protein) as a key mediator of CML survival under TKI imatinib treatment. Our data show TIAR-dependent coordination of RNA processing with the metabolic program induced by stromal interaction. Quantitative nascent proteome analysis revealed that TIAR silencing affects the synthesis of metabolic enzymes and proteins involved in imatinib-induced erythroid differentiation. Besides, TIAR knockdown increased lipid peroxidation in untreated cells and decreased reduction potential in cells upon imatinib treatment. Taken together, TIAR deficiency reduces CML survival, possibly by inducing ferroptosis. These findings identify TIAR-dependent RNA processing within the BM niche as a previously unrecognized mechanism of CML therapy resistance and a potential therapeutic vulnerability.

cancer biology↗

Transcriptional readthrough precedes alternative splicing programs triggered in CML cells by imatinib

Cellular stresses induce transcription readthrough, whereby RNA polymerase II elongates past a genes polyadenylation cleavage site without RNA cleavage. Readthrough has been reported in several cancer types. Here, we use long-read sequencing of nascent RNA to quantify transcriptional readthrough in chronic myelogenous leukemia (CML) cells and characterize early responses to the targeted therapeutic, imatinib. We show that the amount, length, and gene-specificity of readthrough increase within 1 hour, while gene expression and alternative splicing alterations emerge later. Strikingly, imatinib-dependent mRNA isoform changes involved "readthrough chimeras", in which exons from an upstream gene are alternatively spliced to exons in a downstream gene. Modifications in mRNA isoforms and chimera levels detected at 18 hours were also found in imatinib-resistant K562 as well as CML patient cells, suggesting a cascade of early changes in the fidelity of transcription and splicing, leading to long-term adjustment in gene expression and the development of therapy resistance. TeaserPrecision RNA sequencing was used to discover the earliest response of leukemia cells treated with targeted therapy: transcriptional readthrough.

molecular biology↗

DNA polymerase theta-mediated DNA repair is a functional dependency and therapeutic vulnerability in DNMT3A deficient leukemia cells

Myeloid malignancies carrying somatic DNMT3A mutations (DNMT3Amut) may be refractory to standard therapy. DNMT3Amut leukemia cells accumulate toxic DNA double strand breaks (DSBs) and stalled replication forks, rendering them dependent on DNA damage response (DDR). We report here that DNA polymerase theta (Pol{theta}), a key element in DSB repair by end-joining (TMEJ) and in fork restarting, is essential for survival and proliferation of DNMT3Amut leukemia cells. Pol{theta} is overexpressed in DNMT3Amut leukemia cells due to abrogation of PARP1 PARylation-dependent UBE2O E3 ligase-mediated ubiquitination and proteasomal degradation of Pol{theta}. In addition, PARP1-mediated recruitment of the SMARCAD1-MSH2/MSH3 repressive complex to DSBs was diminished in DNMT3Amut leukemia cells which facilitated loading of Pol{theta} on DNA damage and promoting TMEJ and replication fork restart. Pol{theta} inhibitors enhanced the anti-leukemic effects of standard drugs such as FLT3 kinase inhibitor quizartinib, cytarabine +/- doxorubicin, and etoposide in vitro and in mice with DNMT3Amut leukemia. Altogether, Pol{theta} is an attractive target in DNMT3Amut hematological malignancies.

cancer biology↗

High-throughput formulation of reproducible 3D cancer microenvironments for drug testing in myelogenous leukemia

Targeting cancer microenvironment is currently one of the major directions in drug development and preclinical studies in leukemia. Despite the variety of available chronic myelogenous leukemia 3D culture models, the reproducible generation of miniaturized leukemia microenvironments, suitable for high-throughput drug testing, has remained a challenge. Here, we use microfluidics to generate over ten thousand highly monodisperse leukemic-bone marrow hydrogel microbeads per minute. We employ gelatin methacrylate (GelMA) as a model extracellular matrix (ECM) and tune the concentration of the biopolymer, as well as other possible components of the ECM (fibrin, hyaluronic acid), cell concentration and the ratio of leukemic cells to bone marrow cells within the microbeads. This allows to achieve optimal cell viability and the propensity of the encapsulated cells to microtissue formation, while also warranting long-term stability of the microbeads in culture. We administer model kinase inhibitor, imatinib, at various concentrations to the microbeads and, via comparing mono-and co-culture conditions (cancer alone vs cancer-stroma), we find that the stroma-leukemia crosstalk systematically protects the encapsulated cells against the drug-induced cytotoxicity, confirming therefore that our system mimics the physiological stroma-dependent protection. We finally discuss applicability of our model to (i) studying the role of direct-or close-contact interactions between leukemia and bone marrow cells embedded in 3D ECM on the stroma-mediated protection, and (ii) high-throughput screening of anti-cancer therapeutics in personalized therapies.

cancer biology↗