Interaction of cancer cells with their microenvironment generated by stromal cells is essential for tumor cell survival and influences the localization of tumor growth. Here we demonstrate that hedgehog ligands secreted by bone-marrow, nodal and splenic stromal cells function as survival factors for malignant lymphoma and plasmacytoma cells derived from transgenic Emu-Myc mice or isolated from humans with these malignancies. Hedgehog pathway inhibition in lymphomas induced apoptosis through downregulation of Bcl2, but was independent of p53 or Bmi1 expression. Blockage of hedgehog signaling in vivo inhibited expansion of mouse lymphoma cells in a syngeneic mouse model and reduced tumor mass in mice with fully developed disease. Our data indicate that stromally induced hedgehog signaling may provide an important survival signal for B- and plasma-cell malignancies in vitro and in vivo. Disruption of this interaction by hedgehog pathway inhibition could provide a new strategy in lymphoma and multiple myeloma therapy.

The hedgehog pathway (Hh) is one of several key developmental pathways whose deregulation is known to induce tumor formations in mice and humans including medulloblastoma or basal cell carcinoma. Certain human solid tumors (prostate cancer, pancreatic cancer) have been identified which require sustained Hh-Gli signaling for proliferation. Here, we show for the first time a crucial role of Hedgehog signalling in hematopoietic malignancies. In our experiments, we demonstrate that the Hh pathway is essential for survival of more than 70% of primary lymphomas extracted from transgenic Eμ-Myc mice or Cdkn2a−/− mice (INK4Arf−/−). 34 Myc-lymphomas (10 B-cell lymphomas, 12 plasmablastomas, 10 plasmocytomas, 2 mixed lymphomas) and 10 Cdkn2a−/− lymphomas were isolated from bone marrow, spleen or lymph nodes from diseased mice and expanded on bone marrow stroma cells from Cdkn2a−/− mice. Inhibition of Hh signaling by Cyclopamine (2–5 μM), an alkaloid which inhibits smoothened activation, induced apoptosis in 80% of B-cell lymphomas, 82% of plasmablastomas and 60% of multiple myelomas without affecting stroma proliferation. Protein or transcript levels of Hh pathway downstream targets, such as Gli1, Ptch or Bcl2 but not BMI1 were downregulated after 24h treatment with Cyclopamine. Overexpression of Bcl2 or Gli3 could inhibit apoptosis induction by Cyclopamine. High levels of Ihh protein could be detected in our stroma cell line as well as in bone marrow stroma from various mouse strains. Stimulation of lymphoma cells with recombinant Shh or Ihh protein could overcome apoptosis induction induced by removal of the stromal layer, indicating Ihh as a mediator between stroma and lymphoma cells. Injection of luciferazed lymphoma cells into C57Bl6 mice induced lymphoma formation within 2–6 weeks, as shown by Xenogen luciferase imaging. Subcutaneous treatment of lymphoma injected mice with Cyclopamine could inhibit lymphoma formation and even reduce lymphoma mass in mice with already fully developed lymphomas, indicating stroma-lymphoma interaction through hedgehog signaling as an important survival mechanism for lymphoma cells also in vivo. Our data demonstrates that proliferation and survival of a majority of B-cell lymphomas and plasmacytomas is dependent on intact Hh signaling in vitro and in vivo, suggesting disruption of this pathway as a novel approach in lymphoma therapy.