ALBERT

Description: Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM. Methods: Previous 2D models were compared to a novel 3D co-culture model (agarose matrix interlayer, 100 microwells/cm², 1.5mm in depth, permeable for oxygen+cytokines, but not for BMSCs utilizing U266, RPMI-8226, OPM-2 and primary BM patient (pt) cells, with and without HS-5 vs. M210B4 stroma support (Fig. A + Fig. B.a. for pt characteristics). Analyses covered Trypan Blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, Br J Hematol. 2013). In a next step, primary bone-derived stroma cells were acquired from bones of C57BL/6 J mice. Bones were flushed, digested and FACS sorted in order to acquire single BM and bone bystander cell subtypes (MSPCs [mesenchymal stem and progenitor cells], endothelial cells, osteoblasts, PAS [PDFGRalphaSca1] and CaRs [CXCL12-abundant reticular cells]) which were then compared to HS-5 and M210B4 with regard to growth support, cytokine secretion and protection from anti-MM substances. Results: MMCLs and pt specimens were cultured at different concentrations (10 vs. 100 cells per microwell) with and without M210B4 demonstrating a growth advantage with vs. without M210B4 (Fig. B.b). Liquid overlay technique allowed cluster formation of pt specimens leading to more reliable propagation of pt material for up to 20d of culture. Apoptotic changes were assessed by confocal microscopy of RPMI8226 co-expressing fluorescently labelled histone 2B-mCherry (red) and cytochrome c-GFP (green) as indicators of late and early apoptosis. Comparing BMSCLs with regard to their MM growth support capacities, human HS-5 proved even more beneficent than M210B4 stroma (Fig. B.b). Phenotypic analyses of pt specimens showed decreased CXCR4 expression with vs. without BMSCs suggesting a dynamic regulation of homing molecules. The model was then used as an ex-vivo platform allowing both cytotoxicity and cell cycle analyses for the combination of bortezomib (Btz) vs. Cfz with ARRY-520 (kinesin spindle protein inhibitor). Btz (10nM) and Cfz (20nM) proved significantly cytotoxic compared to the control (U266 and pt specimens, respectively) after 48h of single agent treatment Fig.B.c). Compared to Btz (10nM, B10) and Cfz (20nM, C20) as single agents, the additional combination with ARRY-520 showed stronger additive cytotoxicity for Btz (A5+B10, median: 37.5% vs. 13.1%) than for Cfz (A5+C20, 38.6% vs. 34.1%). To note, the model could also be utilized for more profound analyses as depicted for G2/M cell cycle studies in Fig.B.d. 5nM ARRY-520 (A5) led to significant accumulation of OPM-2 cells in G2/M arrest after 48h of treatment confirming prior analyses (Hernández-Garcia Blood Suppl 4710,2014; Fig. B.d). Co-culture studies with different subsets of BM and bone-derived bystander cells are currently ongoing and will be presented at the meeting (Fig. B.e). Conclusions: More complex, 3D bone-derived high-throughput in vitro models are urgently needed to better predict the potency of preclinically tested agents and to better estimate the likelihood of their later clinical adoption into phase I-II trials. With this work, we provide an innovative model which reflects the BM microenvironment as a crucial predictor for in-vivo sensitivity as shown for ARRY-5200. This ex-vivo approach helps to better incorporate MM growth support by bone and BM-derived bystander cells and thus depicts a valid tool to better characterize the role of the BM niche in myeloma. Figure 1. Figure 1. Disclosures Engelhardt: Deutsch Krebshilfe: Other: grant. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Description: 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.

Description: 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.