Thrombopoietin maintains cell numbers of hematopoietic stem and progenitor cells with megakaryopoietic potential.

Thrombopoietin (THPO) has long been known to influence megakaryopoiesis and hematopoietic stem and progenitor cells (HSPCs), though the exact mechanisms through which it acts are unknown. Here we show that MPL expression correlates with megakaryopoietic potential of HSPCs and identify a population of quiescent progenitor cells that show limited dependence on THPO signalling. We show that THPO is primarily responsible for maintenance of hematopoietic cells with megakaryocytic (Mk) differentiation potential and their subsequent Mk differentiation and maturation. The loss of Mks in THPO knockout (KO) mouse models results in a reduction of the Mk derived chemokine platelet factor 4 (CXCL4/PF4) in the bone marrow and administration of recombinant CXCL4/PF4 rescues the loss of progenitor cell quiescence observed in these mice. CXCL4/PF4 treatment does not rescue reduced HSPC numbers suggesting that thrombopoietin directly maintains HSPC numbers.

Modification of the bone marrow MSC population in a xenograft model of early multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of abnormal plasma cells. MM dysregulates the homeostasis of the bone niche cells like osteoclasts and osteoblasts, responsible for the bone maintenance leading to bone loss and hypercalcemia, as well as the normal immune cells leading to immunodeficiency and anemia. Osteoblasts are part of the cell population differentiating from mesenchymal stem cells (MSC). MSC also gives rise to other cell types such as adipocytes and chondrocytes. It has been observed that adipocytes support MM growth by increasing its survival and chemo-resistance. As adipocytes originate from MSC, the understanding of early modifications in the MSC population during the disease progression is of paramount importance and may help for early diagnosis of MM. Herein, we have evaluated the modification of the MSC population in the bone niche in an in vivo model of MM. Our results showed that before an observable engraftment of MM in the bone niche, the proportion of MSC population is significantly decreased, while a significant increase in adipocyte related genes such as PPARγ and CEBPα expression appears, with no difference in osteogenic differentiation. These results suggest that the bone niche is switching to a "fatty" marrow which would create an adequate microenvironment for MM. This led us to screen for and identify modulated adipokines in the sera of this in vivo MM-mice model. Such changes could reflect early signs of MM and potentially be exploited as detection biomarkers of the disease.

Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation.

During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine thrombopoietin (Thpo) both regulates platelet production and maintains HSC potential, whether Thpo controls megakaryocyte (Mk)-lineage differentiation of HSCs is unclear. Here, we show that Thpo rapidly upregulates mitochondrial activity in HSCs, an activity accompanied by differentiation to an Mk lineage. Moreover, in unperturbed hematopoiesis, HSCs with high mitochondrial activity exhibit Mk-lineage differentiation in vitro and myeloid lineage-biased reconstitution in vivo. Furthermore, Thpo skewed HSCs to express the tetraspanin CD9, a pattern correlated with mitochondrial activity. Mitochondria-active HSCs are resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo-regulated mitochondrial activity associated with mitochondrial translocation of STAT3 phosphorylated at serine 727. Overall, we report an important role for Thpo in regulating rapid Mk-lineage commitment. Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage.

The arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target.

Cancer cells, including in chronic myeloid leukemia (CML), depend on the hypoxic response to persist in hosts and evade therapy. Accordingly, there is significant interest in drugging cancer-specific hypoxic responses. However, a major challenge in leukemia is identifying differential and druggable hypoxic responses between leukemic and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme of the urea cycle, is overexpressed in CML but not normal progenitors. ARG2 is a target of the hypoxia inducible factors (HIF1-α and HIF2-α), and is required for the generation of polyamines which are required for cell growth. We therefore explored if the clinically-tested arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) would be effective against leukemic cells under hypoxic conditions. Remarkably, nor-NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxia but not normoxia. Co-treatment with nor-NOHA overcame hypoxia-mediated resistance towards BCR-ABL1 kinase inhibitors. While nor-NOHA itself is promising in targeting the leukemia hypoxic response, we unexpectedly found that its anti-leukemic activity was independent of ARG2 inhibition. Genetic ablation of ARG2 using CRISPR/Cas9 had no effect on the viability of leukemic cells and their sensitivity towards nor-NOHA. This discrepancy was further evidenced by the distinct effects of ARG2 knockouts and nor-NOHA on cellular respiration. In conclusion, we show that nor-NOHA has significant but off-target anti-leukemic activity among ARG2-expressing hypoxic cells. Since nor-NOHA has been employed in clinical trials, and is widely used in studies on endothelial dysfunction, immunosuppression and metabolism, the diverse biological effects of nor-NOHA must be cautiously evaluated before attributing its activity to ARG inhibition.