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Role of MPL expression in determining heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells

ABSTRACT: Using a CML mouse model, we identified differences in gene expression between leukemic compared with non-leukemic LTHSC, including increased expression of the thrombopoietin (THPO) receptor MPL. LTHSC expressing high levels of MPL showed enhanced JAK/STAT signaling and proliferation in response to THPO in vitro, and increased leukemogenic capacity in vivo compared to LTHSC with low MPL expression. Although both G0 and S-phase subpopulations were increased in MPL expressing LTHSC, LSC capacity was restricted to quiescent cells. Inhibition of MPL expression in CML LTHSC resulted in reduced THPO-induced JAK/STAT signaling and leukemogenic potential. Similar observations were made with LTHSC from CML patients. MPL expressing LTHSC demonstrated reduced sensitivity to BCR-ABL TKI treatment but demonstrated increased sensitivity to JAK inhibitors. Our studies identify MPL expression levels as a key determinant of heterogeneous leukemia-initiating capacity and drug sensitivity of CML LTHSC, and suggest that MPL-expressing CML stem cells are critical targets for therapy.

ORGANISM(S): Mus musculus

PROVIDER: GSE76123 | GEO | 2016/06/01

SECONDARY ACCESSION(S): PRJNA306362

REPOSITORIES: GEO

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Project description:Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Gene expression analysis was performerd of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling. BM cells were flushed from the femurs and tibias of C57Bl/6 donor mice. Lineage-marker negative (lin-) cells were isolated by magnetic cell sorting using lineage-specific antibodies (GR1, CD11b, CD45R/B220, CD3e, TER-119). Prior to viral transduction, lin- BM cells were prestimulated for 24-48h in StemSpan, containing 10 ng/ml murine SCF, 20 ng/ml murine Thpo, 10 ng/ml recombinant human FGF-1, 20 ng/ml murine IGF2, 1% penicillin/streptomycin, 2 mM glutamine. Lin- cells were transduced twice on two following days with an MOI of 10 with retroviral vectors on Retronectin coated and preloaded wells (10 μg/cm2). On day four after isolation, 5x10^5 cells/mouse were intravenously injected into lethally irradiated C57Bl/6 mice (10 Gy). Eight weeks after transplantation Lin-Sca1+cKit+ (LSK) cells were isolated from dnMpl and control transplanted mice by fluorescent activated cell sorting. For each sample, BM of 2-5 mice were pooled. RNA was isolated using RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany). RNA quality was assessed using the Agilent 2100 Bioanalyzer.

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Role of MPL expression in determining heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells

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