Jessica Wagner

TLRs Regulate the Gatekeeping Functions of the Intestinal Follicle-Associated Epithelium

Initiation of adaptive mucosal immunity occurs in organized mucosal lymphoid tissues such as Peyer’s patches of the small intestine. Mucosal lymphoid follicles are covered by a specialized follicle-associated epithelium (FAE) that contains M cells, which mediate uptake and transepithelial transport of luminal Ags. FAE cells also produce chemokines that attract Ag-presenting dendritic cells (DCs). TLRs link innate and adaptive immunity, but their possible role in regulating FAE functions is unknown. We show that TLR2 is expressed in both FAE and villus epithelium, but TLR2 activation by peptidoglycan or Pam3Cys injected into the intestinal lumen of mice resulted in receptor redistribution in the FAE only. TLR2 activation enhanced transepithelial transport of microparticles by M cells in a dose-dependent manner. Furthermore, TLR2 activation induced the matrix metalloproteinase-dependent migration of subepithelial DCs into the FAE, but not into villus epithelium of wild-type and TLR4-deficient mice. These responses were not observed in TLR2-deficient mice. Thus, the FAE of Peyer’s patches responds to TLR2 ligands in a manner that is distinct from the villus epithelium. Intraluminal LPS, a TLR4 ligand, also enhanced microparticle uptake by the FAE and induced DC migration into the FAE, suggesting that other TLRs may modulate FAE functions. We conclude that TLR-mediated signals regulate the gatekeeping functions of the FAE to promote Ag capture by DCs in organized mucosal lymphoid tissues.

Role of p97 AAA-ATPase in the Retrotranslocation of the Cholera Toxin A1 Chain, a Non-ubiquitinated Substrate*

The enzymatic A1 chain of cholera toxin retrotranslocates across the endoplasmic reticulum membrane into the cytosol, where it induces toxicity. Almost all other retrotranslocation substrates are modified by the attachment of polyubiquitin chains and moved into the cytosol by the ubiquitin-interacting p97 ATPase complex. The cholera toxin A1 chain, however, can induce toxicity in the absence of ubiquitination, and the motive force that drives retrotranslocation is not known. Here, we use adenovirus expressing dominant-negative mutants of p97 to test whether p97 is required for toxin action. We find that cholera toxin still functions with only a small decrease in potency in cells that cannot retrotranslocate other substrates at all. These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function.

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro- translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endo-somal sorting of the toxin-GM1 complex.

Ganglioside GM1-mediated Transcytosis of Cholera Toxin Bypasses the Retrograde Pathway and Depends on the Structure of the Ceramide Domain

Background: Mechanisms for intracellular lipid sorting remain poorly understood. Results: Polarized epithelial cells sort ganglioside GM1, the receptor for cholera toxin, into distinct retrograde and transcytotic pathways, provided that GM1 contains ceramide domains with short or unsaturated fatty acid chains. Conclusion: Sphingolipid sorting depends on ceramide structure, implicating a mechanism for lipid sorting by lipid shape. Significance: The results identify a lipid-sorting pathway across epithelial barriers with clinical applications. Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.

Analysis of Adhesion Molecules Involved in Leukocyte Homing into the Basolateral Pockets of Mouse Peyer's Patch M Cells

The basolateral membranes of intestinal M cells are invaginated to form large intraepithelial “pockets” that are populated by specific sub-sets of mucosal leukocytes, including CD4+ T cells, memory and naïve B cells, and occasional dendritic cells. The adhesion molecules involved in leukocyte trafficking and/or retention within this unique immunological niche are unknown. In this study, we used immunofluorescence microscopy and a battery of monoclonal antibodies to identify the adhesion molecules expressed by leukocytes situated within the intracellular pockets of mouse Peyers patch (PP) M cells. M cell associated leukocytes (MAL) consistently stained positive for integrin α4β7, and integrin LFA-1 (CD11a/CD18), but were rarely positive for L-selectin (CD62L) or the mucosal integrin αEβ7. However, neither the α4β7 ligands MadCAM-1 or VCAM-1, nor the LFA-1 ligand ICAM-1, were detected on M cell basolateral membranes. To determine whether integrins α4β7 or LFA-1 play a functional role leukocyte homing to M cell pockets, we examined M cells in mice deficient in integrin β7 or CD11a/CD18. Although PP from CD18-/- or integrin β7-/- mice were reduced in number and size as compared to age-matched controls, we identified M cells in both strains of mice. However, mice lacking CD18 (but not integrin β7) had significantly fewer leukocytes within M cell pockets as compared to control animals, suggesting LFA-1 (but not α4β7) may contribute, in part, to leukocyte trafficking into and/or retention within this unique immunological niche.

Dose intensification of TRAIL-inducing ONC201 inhibits metastasis and promotes intratumoral NK cell recruitment

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax–/– tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti–PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

Abstract 3245: Preclinical evaluation of the imipridone family of small molecules, including analogues of clinical-stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212

We previously identified a novel, potent anti-cancer small molecule ONC201, which upregulates the integrated stress response (ISR) through ATF4/CHOP/DR5 and acts as a dual inactivator of Akt and ERK, leading to TRAIL gene activation. After completing a first-in-human phase I clinical trial that revealed exceptional safety, therapeutic pharmacokinetic (PK) profile and tumor engagement, ONC201 is under investigation in several advanced cancer Phase I/II trials. Given the unique imipridone core chemical structure of ONC201, we synthesized a family of analogues in an effort to identify additional chemical family members with distinct therapeutic properties. Based on in vitro potency improvements in human cancer cell lines and therapeutic window approximations with normal human fibroblasts, select analogues were investigated in animals for toxicity, maximum tolerated dose (MTD), and antitumor efficacy. ONC212 is one of the most promising new imipridones that was further evaluated to establish the PK profile, oral bioavailability, and efficacy in tumor types that are less sensitive to ONC201. Compared to ONC201, we noted distinct and more rapid kinetics of activity as well as improved potency in multiple human cancer cell lines in vitro. ONC212 has a broad therapeutic window, an acceptable PK profile, and is orally well-tolerated in mice. With no evidence of toxicity at efficacious doses in both colon and triple negative breast cancer, we have begun further evaluation of antitumor efficacy studies in ONC201-resistant tumor types. Efficacy studies with ONC212 are ongoing in melanoma models that are sensitive to ONC212 but less sensitive to ONC201 in vitro. Preliminary data indicates potent tumor growth reduction by ONC212 in vivo in ONC201-resistant melanoma xenografts. With a wide safety margin, potent antitumor activity in ONC201-insenstive tumors, and drug-like characteristics, ONC212 is being further developed as a drug candidate from the new imipridone class of compounds that complements the spectrum of activity of ONC201. Citation Format: Jessica Wagner, C. Leah Kline, Gary Olson, Bhaskara Nallaganchu, Richard Pottorf, Varun Prabhu, Martin Stogniew, Joshua Allen, Wafik El-Deiry. Preclinical evaluation of the imipridone family of small molecules, including analogues of clinical-stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3245. doi:10.1158/1538-7445.AM2017-3245

Abstract 1102: Anti-tumor effects of imipridone ONC201 in combination with anti-angiogenic agents significantly impact on colorectal cancer growthin vivo

Small molecule imipridone ONC201 is an investigational anti-tumor agent with a wide therapeutic index and broad-spectrum efficacy in vivo. ONC201 upregulates intratumoral TRAIL expression and the integrated stress response pathway. A Phase I clinical trial using ONC201 as therapy in advanced cancer patients has been completed and the drug has progressed into several Phase II trials in multiple cancer types. Colorectal cancer (CRC) remains one of the leading causes of cancer worldwide and metastatic disease continues to have a poor prognosis. Clinical trials in CRC and other tumor types have demonstrated that therapeutics targeting the vascular endothelial growth factor (VEGF) pathway, such as bevacizumab, are effective in combination with certain chemotherapeutic agents. Bevacizumab is a humanized monoclonal antibody that targets VEGF and is an FDA-approved treatment for advanced CRC patients, in addition to other tumor types. We are investigating the potential combination of VEGF inhibitors such as bevacizumab and ONC201 in both CRC xenograft and patient-derived xenograft (PDX) studies. Our results demonstrate significant tumor regression and occasional tumor ablation in human xenografts with the combination of ONC201 with bevacizumab, and in syngeneic MC-38 colorectal cancer xenografts using a murine VEGF-A inhibitor. Non-invasive angiogenesis imaging demonstrated the impact of this combination on decreasing tumor growth and tumor metastasis. With the use of both a murine VEGF inhibitor in syngeneic models, and bevacuzimab in human cell line-derived xenografts, we have demonstrated that combining anti-angiogenic therapies with ONC201 could enhance antitumor efficacy. Thus, ONC201 in combination with anti-angiogenic therapies such as bevacizumab represents a promising combinatorial approach that may be exploited in the clinic for the treatment of CRC. Citation Format: Jessica Wagner, Wafik El-Deiry. Anti-tumor effects of imipridone ONC201 in combination with anti-angiogenic agents significantly impact on colorectal cancer growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1102. doi:10.1158/1538-7445.AM2017-1102

Abstract 1067: Anti-cancer efficacy of imipridones in pancreatic cancer: single agent ONC212 or combination of ONC201 with IGF1-R inhibition

Pancreatic cancer is a highly chemo-resistant tumor type known to aggressively metastasize at an early stage with an overall five-year survival rate of ~6%. In this study we tested the efficacy of ONC201 and ONC212 in a panel of pancreatic cancer cell lines and patient-derived models in vitro and in vivo. ONC201 is the founding member of the imipridone class of small molecules with anti-proliferative and pro-apoptotic effects in various tumor types. Kline et al. and Allen et al. previously showed that ONC201 stimulates the integrated stress response by up-regulating ATF4, CHOP and DR5 followed by induction of apoptosis by up-regulating TRAIL. While the spectrum of ONC201 efficacy is broad among tumor types, pancreatic cancer cells are relatively insensitive. ONC201 exhibits low micromolar GI50 values in 3 out of 7 pancreatic cancer cell lines and 2 out of 9 patient-derived cells that were most sensitive among the pancreatic cell lines and samples tested. ONC201 induces the integrated stress response in both ONC201-sensitive and –insensitive pancreatic cancer cell lines. Interestingly, ONC201-treated resistant cells arrest in G1 and do not go through apoptosis. In order to address insensitivity to ONC201 in pancreatic cancer we explored two approaches. The first approach included treating with the ONC201 analogue ONC212, an imipridone that is currently under pre-clinical development. In vitro and in vivo studies consistently show a higher potency of ONC212 as compared to ONC201 in pancreatic cancer, especially in ONC201-insensitive models. We are currently investigating the mechanism of action of ONC212 in comparison to ONC201. The second approach to address ONC201-insensitive pancreatic cancer was combination treatment with the IGF1-R inhibitor AG1024. Western blot analysis of receptor tyrosine kinase expression levels in the panel of pancreatic cancer cell lines revealed a strong correlation between resistance to ONC201 and high expression of IGF1-R. Therefore we hypothesized that IGF1/IGF1-R might play a role in the resistance of pancreatic cancer cells to ONC201. Indeed, treatment of resistant pancreatic cancer cells with the IGF1-R inhibitor AG1024 sensitized the cells to ONC201. We are currently further exploring the involvement of IGF1-R in the ONC201 resistance mechanism. Furthermore, in-vivo studies are ongoing to validate the efficacy of the combination treatment. In summary, although pancreatic cancer is known to be refractory to many drugs, this study introduces two different approaches with imipridone small molecules ONC212 and ONC201 that show promising therapeutic potential for pancreatic cancer. Citation Format: Avital Lev, Amriti R. Lulla, Jessica Wagner, David T. Dicker, Wafik S. El-Deiry. Anti-cancer efficacy of imipridones in pancreatic cancer: single agent ONC212 or combination of ONC201 with IGF1-R inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1067. doi:10.1158/1538-7445.AM2017-1067

Abstract 3213: Antagonism of D2-like dopamine receptors plays a role in Onc201’s anticancer effects

ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that causes early activation of the integrated stress response and inactivates both Akt and ERK. Its promising safety profile and broad spectrum efficacy in vitro has been confirmed in Phase I/II trials in several advanced malignancies. Biochemical and reporter assays have shown that ONC201 is a selective and competitive antagonist of the D2-like receptors, specifically, dopamine receptor D2 (DRD2) and dopamine receptor D3 (DRD3) with a KD value of ~3 µM. The theme that dopamine and dopamine receptors are important in cancer has emerged in the literature. We hypothesize that ONC201’s interaction with DRD2 is critical for ONC201’s anticancer effects. Co-treating HCT116 and RKO colorectal cancer cells with ONC201 and dopamine or the selective D2-like receptor agonist sumanirole partially abrogated ONC201-induced ATF4/CHOP expression and apoptosis. Knocking down DRD2 expression using siRNA negated ONC201’s effects on viable cell count. Overexpressing DRD2 in a cancer cell line that has very low levels of DRD2, increased ONC201-induced PARP cleavage. Quantitative RT-PCR analyses showed that cells that have acquired resistance to ONC201 did not express detectable mRNA levels of the D2-like receptors. To further determine the anti-tumor potential of targeting the D2-like receptor, we treated different cancer cell lines with other D2-like receptor antagonists. Similar to ONC201, the D2-selective antagonist L-741,626 decreased cell viability and induced apoptosis in a number of cancer cell lines. In contrast to ONC201, however, L-741,626 has a poor therapeutic index. Our findings show that the ability of ONC201 to inhibit D2-like receptors contributes to ONC201’s antiproliferative and pro-apoptotic activity. Ongoing work is aimed at elucidating the mechanisms by which antagonism of D2-like receptors can promote apoptotic cell death, especially with regard to ATF4/CHOP/DR5 and Akt/ERK/Foxo3a/TRAIL, which have been shown to be stimulated in ONC201-treated and -sensitive tumor cells. Citation Format: Christina Leah B. Kline, Amriti Lulla, Jessica Wagner, David Dicker, Marie Baumeister, Sophie Oster, Wafik El-Deiry. Antagonism of D2-like dopamine receptors plays a role in Onc201’s anticancer effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2017-3213