Aberrant Hedgehog Signaling Represents a Novel Therapeutic Target in B Cell Lymphomas.

Aberrant self-renewal is a hallmark of cancer and is central to the initiation, maintenance and relapse of clinical disease. The cellular processes responsible for self-renewal have not been delineated in most human cancers, but it is likely that conserved pathways required for the regulation of normal stem cells are involved. Notably several highly conserved signaling pathways that regulate stem cell fate decisions during embryonic development, such as Notch, Hedgehog (Hh) and Wingless (Wnt), are inappropriately activated in a wide variety of human cancers. We recently demonstrated that the Hh signaling pathway is required for the maintenance of cancer stem cells in the plasma cell malignancy, multiple myeloma. Since myeloma stem cells phenotypically resemble normal B cells, we hypothesized that aberrant Hh signaling is a feature of other B cell malignancies. We studied established human cell lines derived from patients with classical Hodgkin lymphoma (L428, KM-H2), diffuse large B cell NHL (HT, Pfeiffer, RL, and Hs 602), and mantle cell NHL (Granta 519, Jeko-1, Rec-1) and found that expression of the Hh signaling pathway components PATCHED (PTCH), SMOOTHENED (SMO), and GLI1, 2 or 3 by RT-PCR was markedly elevated compared to normal B cells in the majority of cell lines from each subtype of lymphoma. In order to examine the functional role of Hh signaling on human lymphomas, cells were treated with recombinant human sonic Hh ligand (SHh) or the naturally occurring inhibitor of SMO, cyclopamine, followed by evaluation of clonogenic growth in methylcellulose. Resulting colony formation was significantly increased in response to activating SHh ligand, whereas treatment with cyclopamine significantly inhibited clonogenic recovery. Similarly, the inhibition of pathway signaling by neutralizing anti-Hh antibodies limited colony formation suggesting that ligand binding by PTCH was required for pathway activation similar to other non-Gorlins tumors such as small cell lung cancer, pancreatic carcinoma, and metastatic prostate cancer that lack mutation of pathway components. Furthermore, we examined the activity of a novel semi-synthetic cyclopamine analogue, IPI-609, and found that it also limited clonogenic lymphoma growth. The effects of IPI-609 were highly specific as the clonogenic recovery of cell lines lacking expression of Hh pathway components was not affected by treatment. Our previous studies in multiple myeloma have suggested that cancer stem cells can be identified by their relatively higher activity of the intracellular enzyme retinaldehyde dehydrogenase (ALDH) similar to normal hematopoietic and neural stem cells. We found that high ALDH activity could also identify rare cell populations with greater clonogenic growth potential compared to ALDH^low/neg cells in the majority of lines and treatment with cyclopamine or IPI-609 significantly reduced the relative proportion of ALDH^high cells. Therefore, the Hh signaling pathway may represent a novel therapeutic target in human lymphomas. Moreover, novel Hh inhibitors, such as IPI-609, may inhibit highly clonogenic lymphoma cancer stem cells responsible for disease relapse.