Paul G. Rack

Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade

Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)], including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action, and their phenotypes reveal that Gli processing, Gli activation, and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo, and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers.

Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (KrasG12D), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

The Imidazopyridine Derivative JK184 Reveals Dual Roles for Microtubules in Hedgehog Signaling

Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

Arhgap36-dependent activation of Gli transcription factors

Significance The Hedgehog (Hh)/Gli signaling pathway is a key regulator of embryonic patterning and tissue homeostasis, and its inappropriate activation can lead to several human cancers, including basal cell carcinoma, medulloblastoma, and meningioma. To better understand the mechanisms that control Hh pathway state, we have conducted a genome-scale cDNA overexpression screen for signaling proteins that promote Gli-dependent transcription. Our studies reveal Arhgap36 to be a potent Gli activator, yielding the first functional insights, to our knowledge, for this Rho GAP family member. We also find that ARHGAP36 is overexpressed in a subset of medulloblastomas, suggesting that this Gli-activating protein plays an important role in tumorigenesis. Hedgehog (Hh) pathway activation and Gli-dependent transcription play critical roles in embryonic patterning, tissue homeostasis, and tumorigenesis. By conducting a genome-scale cDNA overexpression screen, we have identified the Rho GAP family member Arhgap36 as a positive regulator of the Hh pathway in vitro and in vivo. Arhgap36 acts in a Smoothened (Smo)-independent manner to inhibit Gli repressor formation and to promote the activation of full-length Gli proteins. Arhgap36 concurrently induces the accumulation of Gli proteins in the primary cilium, and its ability to induce Gli-dependent transcription requires kinesin family member 3a and intraflagellar transport protein 88, proteins that are essential for ciliogenesis. Arhgap36 also functionally and biochemically interacts with Suppressor of Fused. Transcriptional profiling further reveals that Arhgap36 is overexpressed in murine medulloblastomas that acquire resistance to chemical Smo inhibitors and that ARHGAP36 isoforms capable of Gli activation are up-regulated in a subset of human medulloblastomas. Our findings reveal a new mechanism of Gli transcription factor activation and implicate ARHGAP36 dysregulation in the onset and/or progression of GLI-dependent cancers.

In vivo imaging of Hedgehog pathway activation with a nuclear fluorescent reporter.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue regeneration, and its dysregulation can lead to birth defects and tumorigenesis. Understanding how this signaling mechanism contributes to these processes would benefit from an ability to visualize Hedgehog pathway activity in live organisms, in real time, and with single-cell resolution. We report here the generation of transgenic zebrafish lines that express nuclear-localized mCherry fluorescent protein in a Gli transcription factor-dependent manner. As demonstrated by chemical and genetic perturbations, these lines faithfully report Hedgehog pathway state in individual cells and with high detection sensitivity. They will be valuable tools for studying dynamic Gli-dependent processes in vertebrates and for identifying new chemical and genetic regulators of the Hh pathway.

Abstract LB-34: 3-Dimensional air-liquid interface organoid culture of primary human tumor biopsies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The application of primary organoid cultures to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. We have previously described a single air-liquid interface culture method that can be used to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach, and pancreas (Ootani et al., Nature Medicine 15(6):701-6 (2009); Li et al., in press). We have recently modified this methodology to allow the robust long-term 3-dimensional culture of a wide variety of primary neoplasms from human biopsy samples. These primary tumor organoids accurately recapitulate the architecture and histopathology of the tumors from which they were derived, including epithelial and mesenchymal compartments. These tumor organoids can be further passaged into 96-well format to enable a variety of genetic and pharmacologic manipulations with the overall goal of enabling mechanistic studies of tumorigenesis and obtaining clinically actionable information regarding chemosensitivity and resistance to therapy. Citation Format: James T. Neal, Michael Cantrell, Paul Rack, Calvin J. Kuo. 3-Dimensional air-liquid interface organoid culture of primary human tumor biopsies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-34. doi:10.1158/1538-7445.AM2014-LB-34