LAMTOR2 Regulates Stem Cell Maintenance and Acts in Lineage Choice By Modulating Cytokine Receptor-Mediated Signaling
Authors: Kotlarz Daniel , Brandes Gudrun , Adrian Schwarzer , Silke Glage , Guntram Büsche , Dietmar Pfeifer , Huber Lukas A , Klein Christoph 
Published: November 2019

Abstract
Background
We have previously reported a novel primary immunodeficiency syndrome caused by a mutation in the 3’ untranslated region of the gene p14 (also known as late endosomal/lysosomal adaptor, MAPK and MTOR activator 2; LAMTOR2) resulting in impaired endosomal biogenesis (Bohn et al, Nature Medicine 2007). The p14-deficient patients are characterized by congenital neutropenia associated with growth failure, partial albinism, as well as B cell and cytotoxic T cell deficiency. Here, we studied the role of the endosomal adaptor protein p14 in hematopoieses using a Cre/loxP-based conditional knockout mouse model.
Methods
We have generated a conditional Mx1Cre-p14flox/flox (p14-/-) mouse model, in which postnatal deletion of p14 can be induced by intraperitoneal injection of polyinosinic-polycytidylic acid (pIpC, 5 x 250 µg, every other day). The hematopoietic system of p14-/- mice was analyzed by tissue sections, flow cytometry, biochemical assays, and electron microscopy studies. Hematopoietic stem cells were analyzed by competitive transplantation studies and in vitro colony-forming unit assays.
Results
p14-/- mice die 3 - 6 weeks after treatment with pIpC. Disruption of p14 was associated with anemia, thrombocytopenia, and moderate leukocytosis in the peripheral blood. Pathological examination of p14-/- mice revealed a splenomegaly and myeloproliferative infiltrations in the bone marrow and spleen. Flow cytometric analysis of p14-deficient nucleated bone marrow cells showed a massive expansion of immature myelomonocytic cells (Gr-1lowCD11b+, pre-M), whereas the granulocytic (Gr-1highCD11b+), B cell (CD19+B220+), and erythroid (TER-119+) lineages were severely reduced. Similarly, FACS analysis revealed a remarkable accumulation of the pre-M population in the spleen, whereas the erythroid lineage was increased at various developmental stages as determined by expression of TER-119 and CD71, indicating enhanced extramedullary hematopoiesis. Immunophenotypic characterization of the hematopoietic stem cell (HSC) compartment showed a significant depletion of long-term HSCs (lin-Sca-1+c-kit+CD150+CD48-) accompanied by an increase in the proportion of short-term HSCs (lin-Sca-1+c-kit+CD150+CD48+) and multipotent progenitors (lin-Sca-1+c-kit+CD150-CD48+). BrdU incorporation assays documented enhanced cycling of lin-Sca-1+c-kit+ cells (LSK) 4 days after pIpC injection. Transcriptome analysis detected a strong enrichment of myeloid-specific and a loss of “stemness” gene sets within the p14-deficient LSK gene signature. Bone marrow transplantation studies showed that the p14-deficient bone marrow cells are less competent in repopulating lethally irradiated mice and that the phenotype of myeloid hyperplasia in p14-/- mice is transplantable and caused by cell-intrinsic effects. As a potential pathomechanism for the myeloproliferative phenotype in p14-/- mice we documented an altered cytokine receptor-mediated signaling in p14-deficient hematopoietic progenitor cells (HPC). Whereas p14-deficient HPC exhibited an increased response to GM-CSF and IL-3 with respect to ERK1/2 phosphorylation and colony formation, G-CSF signaling was substantially impaired corroborating with the decreased frequency of granulocytic Gr-1highCD11b+ cells in p14-/-mice.
Conclusion
Our data suggest that conditional deletion of p14 in mice causes a myeloproliferative disorder associated with increased sensitivity of HPC to GM-CSF receptor-mediated signaling.

Disclosures
No relevant conflicts of interest to declare.