Retroviral Ectopic Expression of a Signaling-Defective Thrombopoietin Receptor (Mpl) Induces a Systemic Loss of Hematopoietic Stem Cells in Mice,

Abstract 4175

MPL signaling, induced by the binding of Thrombopoietin (THPO), regulates megakaryopoiesis and platelet development, and is essential for hematopoietic stem cell (HSC) maintenance. Murine lineage negative, Sca-1 and c-kit positive bone marrow (BM) cells (LSK cells) and human CD34 positive cells expressing MPL have long term BM reconstitution capacities. In human patients, deficiency of MPL due to inactivating mutations in the MPL gene causes severe thrombocytopenia and progressive aplastic anemia, which is lethal if not treated by BM transplantation (BMT). This rare genetic disorder is termed congenital amegakaryocytic thrombocytopenia (CAMT).

With the final aim of developing gene therapy to treat CAMT, we explored the effects of ectopic Mpl expression in wild-type C57Bl/6 mice. The enforced overexpression of Mpl induced a rapid expansion of all three hematological cell lineages, resembling chronic myeloproliferative disease (CMPD) 2 months after BMT (n=25 mice). Surprisingly, mice subsequently developed life threatening pancytopenia with normal Thpo levels despite low platelet counts (167+/−52 × 10³/μl). The BM showed reduced LSK cell numbers (0.01+/−0.01 % versus controls 0.05+/−0.015%, p=0.03). Non-transduced, co-transplanted BM cells could not rescue the disturbed hematopoiesis. Based on these observations, we hypothesized that the ectopic overexpression of Mpl in hematopoietic cells induces a dominant-negative effect, dysregulating the Thpo-Mpl balance.

To test our hypothesis, we ectopically expressed a signaling-defective truncated dominant-negative Mpl (dnMpl) receptor, lacking the complete intracellular domain, in a BMT model in wild-type C57Bl/6 mice (n=23). Lineage negative cells were transduced with gammaretroviral vectors expressing dnMpl from the SFFV promoter in the LTRs and transplanted into lethally irradiated mice. We observed long term engraftment with 57.6+/−1.9% dnMpl positive mononuclear cells in the peripheral blood (controls 76.7+/−7.8%) after 19 weeks and severe thrombocytopenia (174 +/− 32 x10³/μl) in the dnMpl expressing animals (controls 951+/−233×10³/μl). After 23 weeks, we detected 15–34% dnMpl positive BM cells (controls 15–99%). To address the question of whether the BM of dnMpl treated mice contained fewer HSCs compared to control transplanted mice, we analyzed the LSK cell number and saw a ∼4.6-fold reduction (0.02+/− 0.005% versus control mice 0.07+/−0.012%; p=0.0016). As Thpo/Mpl signaling is known to induce HSC quiescence, we speculated that the loss of these signals induces HSC cycling. Cell cycle analysis of the LSK cells of dnMpl treated mice showed a transition into G1/S/G2, indicating a loss of quiescent HSCs (G0 3.5 +/−1.6% vs controls 29.8 +/−10.5%). This effect was even more pronounced in CD34 negative LSK cells (5.4+/−4.0% vs. 44.5+/−8.5%). Secondary transplantation of dnMpl BM showed severely reduced repopulation capacity with 5 out of 6 secondary recipients dying due to graft failure.

In selected animals we transplanted another batch of BM after 16 weeks without preconditioning of the mice. Long term repopulation of the second graft was seen in 71–79% of dnMpl treated mice (controls 0.2–1.1%) and BM cells further engrafted in secondary recipients.

To address changes in HSC gene expression, we sorted LSK cells from dnMpl BM (pool of 4–5 dnMpl mice for each sample) and also separated dnMpl positive and negative cells. Our controls were LSK cells from control transplanted mice (one per sample). RNA was analyzed on an Affymetrix Mouse Genome 430 2.0 array in triplicates. Unsupervised clustering and principle component analysis revealed that dnMpl positive and negative samples clustered, suggesting a systemic effect on HSCs. We tested more than 4000 gene sets from the MySigDB Database (Broad Institute, Cambridge, MA) for enrichment in either of the phenotypes and found a profound downregulation of gene sets containing “stemness” genes in the LSK cells of dnMpl treated mice, irrespective of cellular dnMpl expression.

In summary, we show that ectopic expression of dnMpl in a subset of BM cells disturbs Thpo/Mpl-signaling inducing thrombocytopenia and a systemic loss of HSCs. Besides underlining the strict requirement for regulated Mpl expression in gene therapy for CAMT, these data create new hypotheses for the pathogenesis of aplastic anemia and the development of non-cytotoxic conditioning regimens in BMT.