Modeling pharmacological inhibition of mast cell degranulation as a therapy for insulinoma

Abstract

Myc, a pleiotropic transcription factor that is deregulated and/or overexpressed in most human cancers, instructs multiple extracellular programs that are required to sustain the complex microenvironment needed for tumor maintenance, including remodeling of tumor stroma, angiogenesis, and inflammation. We previously showed in a model of pancreatic β-cell tumorigenesis that acute Myc activation in vivo triggers rapid recruitment of mast cells to the tumor site and that this is absolutely required for angiogenesis and macroscopic tumor expansion. Moreover, systemic inhibition of mast cell degranulation with sodium cromoglycate induced death of tumor and endothelial cells in established tumors. Hence, mast cells are required both to establish and to maintain the tumors. Whereas this intimates that selective inhibition of mast cell function could be therapeutically efficacious, cromoglycate is not a practical drug for systemic delivery in humans, and no other systemic inhibitor of mast cell degranulation has hitherto been available. PCI-32765 is a novel inhibitor of Bruton tyrosine kinase (Btk) that blocks mast cell degranulation and is currently in clinical trial as a therapy for B-cell non-Hodgkin lymphoma. Here, we show that systemic treatment of insulinoma-bearing mice with PCI-32765 efficiently inhibits Btk, blocks mast cell degranulation, and triggers collapse of tumor vasculature and tumor regression. These data reinforce the notion that mast cell function is required for maintenance of certain tumor types and indicate that the Btk inhibitor PCI-32765 may be useful in treating such diseases.

Figures

Figure 1

PCI-32765 inhibits mast cell degranulation. In vitro quantitation of mast cell degranulation by hexaminidase release assay. Rat RBL-2H3 mast cells were incubated with PCI-32765 and degranulation then triggered by IgE receptor cross-linking. The extent of degranulation was quantitated by assaying release of hexosaminidase. Numbers represent means of duplicate measurements.

Figure 2

Short-term (1 week) treatment with PCI-32765 induces death of both tumor and adjacent endothelial cells. (A) Hematoxylin and eosin (H&E) staining of pancreata harvested from Ins-MycERTAM;RIP7-Bcl-xL mice treated with PCI-32765 only (left panel), with tamoxifen only (middle panel), or with both tamoxifen and PCI-32765 (right panel). (B) TUNEL staining of sections described above. TUNEL-positive apoptotic cells are indicated by black circles. (C) Meca32 (red) and TUNEL (green) double staining showing the presence of dying endothelial cells in tumors treated with PCI-32765. Hoechst nuclear counterstaining (blue) is shown in the left panels. i, ii, iii, and iv indicate higher magnification of TUNEL-positive apoptotic endothelial cells.

Figure 3

PCI-32765 inhibits the proliferation of tumor cells. Ki67 (proliferative marker) staining of sections of pancreata harvested from Ins-MycERTAM;RIP7-Bcl-xL mice treated with PCI-32765 only (left panel), with tamoxifen only (middle panel), or both (right panel). Lower inserts show higher-magnification images of Ki67-positive cells. Quantification of Ki67-positive cells is also provided for of each genotype and treatment condition.

Figure 4

PCI-32765 does not compromise glucose tolerance. Mice previously subjected to 24-hour fasting were challenged by i.p. injection of a bolus of glucose (10 µl/g of 1 M stock solution), and their blood glucose levels were measured every 20 minutes during the ensuing 2 hours. Continuous lines represent mice pretreated for 3 days with PCI-32765, whereas broken lines depict vehicle-treated controls.

Figure 5

Oral administration of PCI-32765 achieves complete Btk occupancy and systemically blockades mast cell degranulation. (A) Btk occupancy assay on splenocyte lysates derived from Ins-MycERTAM;RIP7-Bcl-xL mice treated with tamoxifen and either PCI-32765 or vehicle control. The black arrow indicates band relative to Btk. A schematic of the assay is shown on the left. (B) Toluidine blue staining of pancreatic stroma of Ins-MycERTAM;RIP7-Bcl-XL mice treated with tamoxifen only (left panels) or with both tamoxifen and PCI-32765 (right panels). The smaller panels on both sides show higher magnification of single mast cells.

Figure 6

Long-term treatment with PCI-32765 causes tumor regression. Tumor bearing mice (already treated with tamoxifen for 2 weeks) were treated with PCI-32765 or vehicle control in combination with a daily tamoxifen injection for 4 weeks. (A) H&E staining of sections of pancreata harvested from Ins-MycERTAM;RIP7-Bcl-xL mice treated with PCI-32765 only (i), with tamoxifen only (6 weeks in ii and 2 weeks in iii) or both (iv). (B) Meca32 staining (red) and Hoechst nuclear counterstaining (blue) of the same sections as described in A.

Figure 7

Human insulinomas exhibit significant mast cells infiltration. Biopsies from human tumors were analyzed by H&E (A) and immunohistochemistry using chromogranin A antibody (B), insulin antibody (C), and c-Kit antibody (D) to identify mast cells. Inserts (i) and (ii) show high-magnification images of degranulating mast cells.