Abstract: JAK2V617F+ myeloproliferative neoplasms (MPNs) frequently progress into leukemias, but the factors driving this process are not understood. Here, we find excess Hedgehog (HH) ligand secretion and loss of PTCH2 in myeloproliferative disease, which drives canonical and noncanonical HH-signaling. Interestingly, Ptch2−/− mice mimic dual pathway activation and develop a MPN-phenotype with leukocytosis (neutrophils and monocytes), strong progenitor and LKS mobilization, splenomegaly, anemia, and loss of lymphoid lineages. HSCs exhibit increased cell cycling with improved stress hematopoiesis after 5-FU treatment, and this results in HSC exhaustion over time. Cytopenias, LKS loss, and mobilization are all caused by loss of Ptch2 in the niche, whereas hematopoietic loss of Ptch2 drives leukocytosis and promotes LKS maintenance and replating capacity in vitro. Ptch2−/− niche cells show hyperactive noncanonical HH signaling, resulting in reduced production of essential HSC regulators (Scf, Cxcl12, and Jag1) and depletion of osteoblasts. Interestingly, Ptch2 loss in either the niche or in hematopoietic cells dramatically accelerated human JAK2V617F-driven pathogenesis, causing transformation of nonlethal chronic MPNs into aggressive lethal leukemias with & gt;30% blasts in the peripheral blood. Our findings suggest HH ligand inhibitors as possible drug candidates that act on hematopoiesis and the niche to prevent transformation of MPNs into leukemias.

Abstract: MPNs often transform into secondary AML associated with poor prognosis and high mortality due to limited therapeutic options. Here we show that aberrant Hedgehog ligand secretion in MPN and CML not only induces constitutive Smoothened-dependent canonical HH signaling causing GLI1 transcription, but also causes PTCH1-dependent non-canonical HH signaling leading to constitutive ERK activation. Our investigations show, that depletion of the Ptch2 receptor in vitro and in vivo recapitulates overactivation of both pathways, while depletion of Ptch1 only reflects constitutive Gli1 activation pinpointing the important role of Ptch1 for Erk activation (Figure 1). Ptch2-/- mice develop a pronounced hematopoietic phenotype with leukocytosis driven by an increase in neutrophils, anemia, thrombocytopenia, loss of T cells combined with a strong increase of cKit+ progenitor and stem cells in the peripheral blood and increased extramedullary hematopoiesis causing splenomegaly reflecting a MPN phenotype. HSCs (Lin- cKit+ Sca1+) residing within the BM (bone marrow) showed enhanced cycling properties causing exhaustion and loss of LKS cells over time (Figure 2), but improved stress hematopoiesis after 5-FU treatment. Niche change experiments show that cytopenias and loss of LKS cells are caused by overactivated HH signaling within the niche cells, causing depletion of osteoblasts and alterations of essential niche factors like Cxcl12, Angiopoietin, Stem cell factor, Thrombopoietin or Jagged1 (Figure 2). In contrast, the hematopoietic Ptch2-/- is responsible for leukocytosis and even promotes HSC expansion and replating capacity in vitro. Interestingly, depletion of Ptch2 in the niche or within hematopoietic cells dramatically altered JAK2V617F-driven pathogenesis causing transformation of a non-lethal chronic myeloproliferative disease into an aggressive AML-like disease with up to 30% blasts in the peripheral blood (Figure 3). In contrast, the BCR-ABL-driven leukemia was exclusively accelerated by the cell intrinsic Ptch2-/-, but not by cell extrinsic HH activation. In conclusion, combined constitutive canonical and non-canonical HH activation induced by depletion of Ptch2 causes a MPN phenotype driven by cell intrinsic, but mainly cell extrinsic mechanisms and accelerates myeloproliferative diseases caused by JAK2V617F and BCR-ABL into acute leukemias. Therefore HH ligand-blocking antibodies or a combination of ERK and SMO inhibitors might be a potential treatment option to prevent transformation of MPNs. Figure 1. Diagram showing canonical and non-canonical HH signaling in Wt, Ptch1-/- and Ptch2-/- cells. Depletion of Ptch1 causes canonical pathway activation driving Gli1 transcription but lacks activation of Erk. In contrast, depletion of Ptch2 induces canonical Gli transcription and also directs HH ligands towards Ptch1 increasing non-canonical Erk phosphorylation. B BM cells of Wt, Ptch1+/- and Ptch2-/- mice were checked for Gli1 and Ptch1 expression via qPCR (transcript levels are shown relative to Hprt or Gapdh) and for Erk phosphorylation via flow cytometry (n≥3 each genotype, t-test). C Western Blot of 293T cells transfected with siRNA against Ptch1 or Ptch2 and blotted with antibodies against Ptch1, Ptch2, pErk and total Erk. Figure 1. Diagram showing canonical and non-canonical HH signaling in Wt, Ptch1-/- and Ptch2-/- cells. Depletion of Ptch1 causes canonical pathway activation driving Gli1 transcription but lacks activation of Erk. In contrast, depletion of Ptch2 induces canonical Gli transcription and also directs HH ligands towards Ptch1 increasing non-canonical Erk phosphorylation. B BM cells of Wt, Ptch1+/- and Ptch2-/- mice were checked for Gli1 and Ptch1 expression via qPCR (transcript levels are shown relative to Hprt or Gapdh) and for Erk phosphorylation via flow cytometry (n≥3 each genotype, t-test). C Western Blot of 293T cells transfected with siRNA against Ptch1 or Ptch2 and blotted with antibodies against Ptch1, Ptch2, pErk and total Erk. Figure 2. Cell cycle analysis of HSCs (Lin- cKit+ Sca1+) cells using Ki67 and FxCycle Violet Stain (n = 3). B Percentage of HSCs within the BM assessed by flow cytometry (n ≥ 6 each genotype) at 3 and 12 months of age. C BM niche cells from Wt and Ptch2-/- mice were purified via FACS. RNA samples were analyzed via Affymetrix GeneChip Mouse Gene ST 2.0 and evaluated via KEGG and oncogenic signature gene sets. + indicates significantly increased canonical or non-canonical HH-signaling pathway within specified Ptch2-/- subpopulations. D-F qPCR for Scf (D), Cxcl12 (E) and Thpo (F) in sorted OBCs, MSPCs, Endo and CaR cells purified from Wt and Ptch2-/- mice. Transcript levels are shown relative to Gapdh. Figure 2. Cell cycle analysis of HSCs (Lin- cKit+ Sca1+) cells using Ki67 and FxCycle Violet Stain (n = 3). B Percentage of HSCs within the BM assessed by flow cytometry (n ≥ 6 each genotype) at 3 and 12 months of age. C BM niche cells from Wt and Ptch2-/- mice were purified via FACS. RNA samples were analyzed via Affymetrix GeneChip Mouse Gene ST 2.0 and evaluated via KEGG and oncogenic signature gene sets. + indicates significantly increased canonical or non-canonical HH-signaling pathway within specified Ptch2-/- subpopulations. D-F qPCR for Scf (D), Cxcl12 (E) and Thpo (F) in sorted OBCs, MSPCs, Endo and CaR cells purified from Wt and Ptch2-/- mice. Transcript levels are shown relative to Gapdh. Figure 3. Kaplan-Meier survival of WT or Ptch2-/- C57BL/6 mice transplanted with JAK2V617F-GFP or empty-GFP transduced WT or Ptch2-/- C57BL/6 BM cells (n≥5 each group). B PB samples were analyzed for cKit+ CD11b+ myeloid blasts by flow cytometry. Bethesda criteria published 2002 claim 20% blasts as critical value between MPN and AML. C Representative image of the pronounced splenomegaly within the Ptch2-/- group. Figure 3. Kaplan-Meier survival of WT or Ptch2-/- C57BL/6 mice transplanted with JAK2V617F-GFP or empty-GFP transduced WT or Ptch2-/- C57BL/6 BM cells (n≥5 each group). B PB samples were analyzed for cKit+ CD11b+ myeloid blasts by flow cytometry. Bethesda criteria published 2002 claim 20% blasts as critical value between MPN and AML. C Representative image of the pronounced splenomegaly within the Ptch2-/- group. Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction The CXCL12/CXCR4 axis was shown to be a major regulator for the interaction of hematopoietic stem cells (HSCs) with the niche and interruption of this pathway mobilizes HSCs from the bone marrow (BM).Therefore CXCR4 antagonists like AMD3100 (Plerixafor) are used in the clinic for the collection of HSCs from patients who fail to mobilize HSCs in response to G-CSF.In our previous studies, we could show that the serine/threonine kinase PIM1 is regulating the surface expression of the CXCR4 receptor on HSCs. In addition, PIM1-deficient HSCs fail to home to a wild type (WT) BM niche. Based on these results, we aimed to improve HSC mobilization by combining CXCR4 and PIM inhibition. Methods In order to study the mobilization efficiency in the murine model, mice were treated with AMD3100 alone or in combination with LGB321, a novel pan-PIM inhibitor. The mice were sacrificed at various timepoints and peripheral blood (PB) was isolated. The percentage of HSCs was then determined by flow cytometry. The mechanism of HSC mobilization was studied in isolated HSC and stroma populations by analyzing mRNA levels and surface expression of CXCR4, its ligand CXCL-12 and other factors, which are crucial for the interaction of HSCs and stromal cells. Results We found that CXCR4 inhibition using AMD3100 leads to a compensatory upregulation of CXCR4 surface expression on total BM cells as well as HSCs. This effect can be reverted by deficiency or inhibition of PIM1 (Figure 1A). As a consequence, HSC mobilization using AMD3100 is strongly enhanced and prolonged in Pim1-deficient mice compared to WT animals (Figure 1B). Likewise, treatment of WT animals with AMD3100 in combination with LGB321 leads to increased and prolonged HSC mobilization compared to animals treated only with AMD3100 (Figure 1C). Besides the downregulation of CXCR4 on HSCs, we found that the Cxcl-12 expression as well as CXCR4 surface expression in CXCL12-abundant reticular (CAR) cells is dramatically decreased in Pim1-deficient mice, which even further increases the mobilization capacity of Pim1-deficient mice. Conclusion Our findings indicate, that PIM1 inhibition counteracts the compensatory upregulation of the CXCR4 receptor on HSCs after Plerixafor treatment and decreases CXCL12 levels within the bone marrow niche. Therefore, targeting PIM kinases in combination with CXCR4 inhibition could improve the collection of stem cells in patients at risk for poor mobilization. Figure 1. PIM1-deficiency or -inhibition enhances HSC mobilization in combination with AMD3100-treatment. (A) Statistical analysis of CXCR4 surface expression on Pim1-/- or WT LSK cells after AMD3100-treatment for 24h. PIM1-deficiency reverts the increased CXCR4 surface expression after AMD3100-treatment. (B) Time course of total LSK cells per ml PB of AMD3100-treated mice. Pim1-/- mice show significantly higher LSK counts. (C) Statistical analysis of total LSK cells per ml of LGB321+AMD3100 treated mice compared to AMD3100-only treated WT animals. PIM-inhibition significantly increases mobilization efficiency of AMD3100. Figure 1. PIM1-deficiency or -inhibition enhances HSC mobilization in combination with AMD3100-treatment. (A) Statistical analysis of CXCR4 surface expression on Pim1-/- or WT LSK cells after AMD3100-treatment for 24h. PIM1-deficiency reverts the increased CXCR4 surface expression after AMD3100-treatment. (B) Time course of total LSK cells per ml PB of AMD3100-treated mice. Pim1-/- mice show significantly higher LSK counts. (C) Statistical analysis of total LSK cells per ml of LGB321+AMD3100 treated mice compared to AMD3100-only treated WT animals. PIM-inhibition significantly increases mobilization efficiency of AMD3100. Disclosures No relevant conflicts of interest to declare.

Abstract: Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by impaired stem cell function resulting in pancytopenia. Diagnosis of IBMFS presents a major challenge due to limited diagnostic tests and overlapping phenotypes. For that reason, novel and clinical relevant biomarkers and possible targets are urgently needed. Our study defines NIPA as an IBMFS gene, which is significantly downregulated in a distinct subset of MDS-type refractory cytopenia of childhood patients. Mechanistically, NIPA binds FANCD2 and regulates its nuclear abundance. The stabilization of both non- and monoubiquitinated FANCD2 identifies NIPA as an essential player in the Fanconi Anemia (FA) pathway. NIPA thereby prevents MMC hypersensitivity visualized by increased numbers of chromosome radials and reduced cell survival after induction of interstrand crosslinks. To provide proof of principle, re-expression of FANCD2 in Nipa deficient cells restores MMC sensitivity. In a knockout mouse model, Nipa deficiency leads to major cell intrinsic long-term repopulation defects of hematopoietic stem cells (HSCs), with impaired self-renewal in serial transplantations and a bias towards myeloid differentiation. Unresolved DNA damage in Nipa deficient HSCs causes increased sensitivity to cell death and leads to progressive, age-related loss of the HSC pool. Induction of replication stress triggers the phenotypic reduction and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the mice thereby mimicing Fanconi Anemia. Taken together, our study adds NIPA to the short list of FA-associated proteins being essential for a functional DNA repair/FA/BRCA axis and thereby emphasizing its impact as potential diagnostic marker and/or possible target in bone marrow failure syndromes. Disclosures Niemeyer: Celgene: Consultancy.