P1310: A ROBUST APPROACH FOR THE GENERATION OF FUNCTIONAL HEMATOPOIETIC PROGENITOR CELL LINES TO MODEL LEUKEMIC TRANSFORMATION

Abstract

Background: Preclinical research aims at developing robust methods to maintain, expand and manipulate haematopoietic stem/progenitor cells (HSPC) ex vivo, as these cells are infrequent and their immortalization is hardly achieved. The transcription factor, LIM homeobox protein 2 (LHX2), has a critical role in maintaining self-renewal and has been reported to facilitate ex vivo expansion of immature HSPCs. Aims: We aimed to establish a robust and low-cost murine HSPC in vitro model system that allows maintenance and expansion of functional LSK (lin-, Sca-1+, c-Kit+) cells, and guarantees long-term multipotency. Methods: We generated HPCLSK cell lines from high-purity sorted murine LSK cells by Lhx2 overexpression. SCF/IL-6 dependent HPCLSK cells are kept on agarose-coated plates to prevent adherence-induced myeloid differentiation. HPCLSK cells can be genetically modified e.g. by retroviral transduction, which serves as a source to generate LSCs harboring hematopoietic stem cell oncogenes including BCR/ABLp210+, MLL-AF9 or FLT3-ITD/NRASG12D. Immunophenotyping, transcriptome analysis and functionality tests of HPCLSK and LSC lines were carried out. Results: HPCLSK cells preserve LSK markers despite continuous proliferation. HPCLSK cells efficiently home to the BM, blood, spleen, and thymus and are able to differentiate into myeloid and lymphoid lineages in vitro and in vivo. Numbers of HPCLSK–derived myeloid and lymphoid progenitors in the BM and differentiated blood cells are comparable to BM-injected mice. Transcriptome analysis of HPCLSK lines showed a ST-HSC/MPP1 signature, which corresponds to the earliest proliferating HSPCs. Injection of transformed HPCLSK-LSC (BCR/ABLp210, MLL-AF9, FLT3-ITD/NRASG12D) lines in immunocompromised mice induced myeloid leukemia. All diseased animals displayed elevated WBC counts, blast-like cells in the blood and suffered from splenomegaly. To test the applicability of the model system for drug sensitivity tests we employed Imatinib mesylate (IM). As in patients, HPCLSK-BCR/ABLp210 cells show partial IM resistance and leads to the enrichment of undifferentiated LSK cells. Summary/Conclusion: We created a robust method of culturing functional HSPCs, which are immortalized and can be expanded indefinitely. HPCLSK lines can be rapidly generated from transgenic mouse models and are a useful system for functional assays that require high cell numbers. By oncogenic transformation we created leukemia triggering LSCs. These findings support the relevance of HPCLSK cell system that represents a unique tool to compare LSCs to non-transformed HPCLSK cells in vitro and in vivo, which can be adapted for high-scale preclinical compound screening.