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Integrated Stress Response (ISR) facilitates cellular adaptation to variable environmental conditions by reprogramming cellular response. Activation of ISR was reported in neurological disorders and solid tumours, but its function in hematological malignancies remains largely unknown. Previously we showed that ISR is activated in chronic myeloid leukemia (CML) CD34+ cells, and its activity correlates with disease progression and imatinib resistance. Here we demonstrate that inhibition of ISR by small molecule ISRIB, but not by PERK inhibitor GSK2656157, restores sensitivity to imatinib and eliminates CM Blast Crisis (BC) D34+ resistant cells. We found that in Patient Derived Xenograft (PDX) mouse model bearing CD34+ imatinib/dasatinib-resistant CML blasts with PTPN11 gain-of-function mutation, combination of imatinib and ISRIB decreases leukemia engraftment. Furthermore, genes related to SGK3, RAS/RAF/MAPK, JAK2 and IFNγ pathways were downregulated upon combined treatment. Remarkably, we confirmed that ISRIB and imatinib combination decreases STAT5 phosphorylation and inhibits expression of STAT5-target genes responsible for proliferation, viability and stress response. Thus, our data point to a substantial effect of imatinib and ISRIB combination, that results in transcriptomic deregulation and eradication of imatinib-resistant cells. Our findings suggest such drug combination might improve therapeutic outcome of TKI-resistant leukemia patients exhibiting constitutive STAT5 activation.

The unique bone marrow microenvironment is created by stromal cells and such physical conditions as hypoxia. Both hypoxia and interactions with stromal cells have a significant impact on the biology of leukemia cells, changing their sensitivity to antileukemic therapies. Thus, it is crucial to introduce biological systems, which enable the investigation of leukemia-stroma cross-talk and verification of novel therapies effectiveness under such bone marrow niche-mimicking conditions. Here, we have established an experimental setup based on the hypoxic co-culture of stromal cells with different cell lines derived from various leukemia patients. Flow cytometry enables simultaneous fluorescent tracking of viable cells and analysis of fundamental cellular processes, also to monitor the basal vital state of cells in the hypoxic co-culture. This is critically important, as the stromal cells deliver a big variability of signals to protect leukemia cells and provide drug resistance. Therefore, keeping stromal cells at the healthy state is crucial during experimental procedures. In the proposed studies, viability, apoptosis, proliferation, ROS production, and mitochondrial membrane potential were monitored in both cell types, which were separated on the basis of the fluorescence of a cell tracker. We have shown that the proposed hypoxic co-culture conditions do not affect basal live parameters of stromal cells, indicating the relevance of proposed model. Finally, we utilized this experimental setup to monitor the stroma-mediated protection of leukemia cells from the imatinib-induced cell death, which contributes to the leukemia progression and development of therapy resistance. Altogether, we recommend such flow cytometric strategy as an elementary screen of the vital state of stromal cells, which should be performed when using the co-culture hypoxic models. The proposed approach can also be broadly used for other studies of the leukemia-stroma cross-talk and of the part played by the leukemic microenvironment in drug screening studies.

The recently announced new methodologies to detect mRNA molecules in single cells offer opportunities for research, medicine and molecular diagnostics. The NanoFlare RNA Detection Probes are tools for characterizing RNA content (not localization) using fluorescence-based approaches in living cells. Combined with flow cytometry, NanoFlares have expanded the available possibilities of quantitative analysis of mRNA level in a single cell. Herein we present that in some cases, the specific NanoFlare probes (SmartFlares) detect different amounts of mRNA compared to qPCR. Using the previously published model, in which we studied influence of BCR-ABL oncogene on BRCA1 mRNA translation, we found that the NanoFlare-mediated measurement of mRNA was affected by the assembly of stress granules, structures which store mRNA in complexes with RNA binding proteins. With the usage of chemical compounds we confirmed that under conditions supporting assembly of stress granules, the detection of mRNAs by these probes was decreased, whereas disassembly resulted in the increased mRNAs detection. Altogether, we showed that assembly of stress granules could interfere with mRNA accessibility to the NanoFlare RNA Detection Probes, indicating that the SmartFlares could recognize only the translationally active pool of mRNA, contrary to qPCR. This can significantly influence the quality of obtained data and should be taken into consideration while planning the analysis of mRNA markers using NanoFlares.

BRCA1 tumor suppressor regulates crucial cellular processes involved in DNA damage repair and cell cycle control. We showed that expression of BCR-ABL1 correlates with decreased level of BRCA1 protein, which promoted aberrant mitoses and aneuploidy as well as altered DNA damage response. Using polysome profiling and luciferase-BRCA1 3'UTR reporter system here we demonstrate that downregulation of BRCA1 protein in CML is caused by inhibition of BRCA1 mRNA translation, but not by increased protein degradation or reduction of mRNA level and half-life. We investigated 2 mRNA-binding proteins - HuR and TIAR showing specificity to AU-Rich Element (ARE) sites in 3'UTR of mRNA. BCR-ABL1 promoted cytosolic localization of TIAR and HuR, their binding to BRCA1 mRNA and formation of the TIAR-HuR complex. HuR protein positively regulated BRCA1 mRNA stability and translation, conversely TIAR negatively regulated BRCA1 translation and was found localized predominantly in the cytosolic stress granules in CML cells. TIAR-dependent downregulation of BRCA1 protein level was a result of ER stress, which is activated in BCR-ABL1 expressing cells, as we previously shown. Silencing of TIAR in CML cells strongly elevated BRCA1 level. Altogether, we determined that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein level in BCR-ABL1 -positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in "BRCAness" CML and BCR-ABL1 -positive ALL cells.