Ozhan Ocal

Rgs16 and Rgs8 in embryonic endocrine pancreas and mouse models of diabetes

SUMMARY Diabetes is characterized by the loss, or gradual dysfunction, of insulin-producing pancreatic β-cells. Although β-cells can replicate in younger adults, the available diabetes therapies do not specifically target β-cell regeneration. Novel approaches are needed to discover new therapeutics and to understand the contributions of endocrine progenitors and β-cell regeneration during islet expansion. Here, we show that the regulators of G protein signaling Rgs16 and Rgs8 are expressed in pancreatic progenitor and endocrine cells during development, then extinguished in adults, but reactivated in models of both type 1 and type 2 diabetes. Exendin-4, a glucagon-like peptide 1 (Glp-1)/incretin mimetic that stimulates β-cell expansion, insulin secretion and normalization of blood glucose levels in diabetics, also promoted re-expression of Rgs16::GFP within a few days in pancreatic ductal-associated cells and islet β-cells. These findings show that Rgs16::GFP and Rgs8::GFP are novel and early reporters of G protein-coupled receptor (GPCR)-stimulated β-cell expansion after therapeutic treatment and in diabetes models. Rgs16 and Rgs8 are likely to control aspects of islet progenitor cell activation, differentiation and β-cell expansion in embryos and metabolically stressed adults.

The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity.

A compound that binds to tubulin in an unusual way has superior antitumor efficacy and safety and has a distinctive impact on microtubule curvature and dynamics. Throwing a curve ball to cancer Drugs such as vinca alkaloids, which target tubulin and interfere with microtubule function in mitosis, are commonly used for the treatment of cancer. Unfortunately, they also damage microtubules in normal undividing cells including neurons, resulting in toxicity. Wieczorek et al. identified a drug called DZ-2384, which may offer a safer alternative to the vincas. The authors found that although DZ-2384 is very effective at targeting cancer cells by inhibiting mitosis, it preserves the microtubule network in non-dividing cells and in primary neurons at effective doses and is much safer in mouse models. By analyzing the structure of tubulin with different compounds, the authors determined that DZ-2384 binds at the vinca site but induces a distinctive change in the curvature of growing tubulin protofilaments, which may explain its unusual effects on microtubule dynamics and decreased toxicity. Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.

A rapid in vivo screen for pancreatic ductal adenocarcinoma therapeutics

ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States, and is projected to be second by 2025. It has the worst survival rate among all major cancers. Two pressing needs for extending life expectancy of affected individuals are the development of new approaches to identify improved therapeutics, addressed herein, and the identification of early markers. PDA advances through a complex series of intercellular and physiological interactions that drive cancer progression in response to organ stress, organ failure, malnutrition, and infiltrating immune and stromal cells. Candidate drugs identified in organ culture or cell-based screens must be validated in preclinical models such as KIC (p48Cre;LSL-KrasG12D;Cdkn2af/f) mice, a genetically engineered model of PDA in which large aggressive tumors develop by 4 weeks of age. We report a rapid, systematic and robust in vivo screen for effective drug combinations to treat Kras-dependent PDA. Kras mutations occur early in tumor progression in over 90% of human PDA cases. Protein kinase and G-protein coupled receptor (GPCR) signaling activates Kras. Regulators of G-protein signaling (RGS) proteins are coincidence detectors that can be induced by multiple inputs to feedback-regulate GPCR signaling. We crossed Rgs16::GFP bacterial artificial chromosome (BAC) transgenic mice with KIC mice and show that the Rgs16::GFP transgene is a KrasG12D-dependent marker of all stages of PDA, and increases proportionally to tumor burden in KIC mice. RNA sequencing (RNA-Seq) analysis of cultured primary PDA cells reveals characteristics of embryonic progenitors of pancreatic ducts and endocrine cells, and extraordinarily high expression of the receptor tyrosine kinase Axl, an emerging cancer drug target. In proof-of-principle drug screens, we find that weanling KIC mice with PDA treated for 2 weeks with gemcitabine (with or without Abraxane) plus inhibitors of Axl signaling (warfarin and BGB324) have fewer tumor initiation sites and reduced tumor size compared with the standard-of-care treatment. Rgs16::GFP is therefore an in vivo reporter of PDA progression and sensitivity to new chemotherapeutic drug regimens such as Axl-targeted agents. This screening strategy can potentially be applied to identify improved therapeutics for other cancers. Editors choice - Drug Discovery Collection: The study describes a rapid in vivo screen for novel strategies to treat pancreatic ductal adenocarcinoma (PDA).

Abstract 5182: A rapid in vivo screen for pancreatic ductal adenocarcinoma therapeutics using the tumor marker Rgs16::GFP

Pancreatic Ductal Adenocarcinoma (PDA) is the most lethal major cancer in the USA due to lack of early diagnostics and effective treatments. Activating Kras mutations (such as Kras G12D ) occur early in tumor progression and are present in about 90% of PDA. Pancreatic Intraepithelial Neoplasms (PanIN) are the most common initial neoplastic lesions and those with activating Kras alleles typically progress to carcinoma in situ and metastasize. Receptor Tyrosine Kinases (RTK) and G-protein Coupled Receptors (GPCR) can indirectly activate Kras and are therefore potential drug targets. We previously showed that a feedback inhibitor of GPCRs, Regulator of G-protein Signaling 16 (Rgs16), is expressed in pancreatic progenitors during embryonic development. Rgs16 expression continues postnatally in ducts and beta cells during isletogenesis but is absent in normal glycemic adults. On the other hand, Rgs16 expression returns to ducts and islet beta cells after chronic hyperglycemia in mouse models of Type 1 and Type 2 Diabetes mellitus. Our hypothesis is that Rgs16::GFP is induced in these pancreatic cells in response to GPCR agonists released during chronic stress. In an effort to conduct drug screens in primary duct cell culture, we investigated Rgs16::GFP expression in pancreatic neoplasia by crossing it into a mouse model of aggressive PDA, called KIC (Kras G12D ; Cdkn2a L/L ; Ptf1a::Cre). In Rgs16::GFP-KIC mice, Rgs16 was expressed at the initiation of PDA in early PanINs, as early as two weeks after birth (P15), and throughout tumor progression. Rgs16::GFP expression increased with tumor mass through one month of age (P29). We used this tumor specific characteristic of Rgs16 expression to set-up a two-week in vivo quantitative assay to test chemotherapeutic drug effectiveness. In a proof-of-principle study, the standard PDA therapeutics gemcitabine and nab-Paclitaxel reduced Rgs16::GFP expression and tumor burden compared to untreated mice at P29. Targeting Axl, an RTK highly expressed in PDA, with two different inhibitors of Axl signaling, BGB324 or warfarin, in combination with gemcitabine and nab-Paclitaxel further reduced PDA initiation and progression compared to standard chemotherapy alone. Survival studies with Rgs16::GFP-KIC mice showed that treatment with gemcitabine and warfarin extended median life span about two weeks while nearly doubling the maximum life span compared to untreated group. In summary, Rgs16::GFP-KIC mice provide an in vivo model to rapidly identify more effective PDA chemotherapeutics and treatment protocols. Citation Format: Ozhan Ocal, Victor Pashkov, Rahul K. Kollipara, James B. Lorens, Galvin H. Swift, Rolf A. Brekken, Thomas M. Wilkie. A rapid in vivo screen for pancreatic ductal adenocarcinoma therapeutics using the tumor marker Rgs16::GFP. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5182.

Abstract A188: Diazonamide DZ 2384, a potential therapeutic for pancreatic cancer, binds to tubulin with a unique impact on microtubule dynamics and tubulin curvature

Microtubules are critical for cell proliferation, cellular invasion, migration and trafficking. As such, anti-mitotic tubulin binding agents continue to be a cornerstone of adjuvant chemotherapies across many different tumor indications. A major challenge in the development of new anti-tubulin agents is to overcome toxicities associated with targeting microtubule dynamics while maintaining a high degree of anti-cancer potency. Diazonamide A is a natural product isolated from Diazona angulata, which has previously been shown to block cell division at mitosis but with an unusual safety profile compared to other anti-mitotics. DZ 2384 is a novel and more potent synthetic analog of diazonamide A. In an unbiased functional genomics, biochemical and high resolution structure approach to determine its cellular target, we found that DZ 2384 binds in the vinca domain of tubulin but imparts distinct effects on microtubule dynamics compared to vinorelbine that targets the same site. DZ 2384 and vinorelbine both inhibit the growth rate of microtubules; however, DZ 2384 also increases the rescue frequency while vinorelbine decreases the growth length of microtubules increasing the time spent in a paused or attenuated state. These dynamic characteristics are consistent with the observations that the microtubule network is preserved in interphase cells and in primary cortical neurons treated with DZ 2384 compared to vinorelbine. X-ray crystallography and electron microscopy studies demonstrate that DZ 2384 causes a straightening of curved protofilaments, an effect that has not been observed for other vinca-domain binders so far, and which may account for the observed differences in microtubule dynamics and toxicity of this class of compounds. DZ 2384 has potent anti-tumor activity in xenograft models of pancreatic and colon cancer and a higher therapeutic window than vinorelbine considering body weight, blood chemistry, hematology and bone marrow. DZ 2384 was also tested in a KrasG12D-driven genetically engineered murine model of pancreatic ductal adenocarcinoma that carries a Rgs16::GFP reporter transgene to enable tumor burden quantitation. In this model, DZ 2384 demonstrates strong antitumor activity in combination with gemcitabine; comparable with or better than that of gemcitabine + Abraxane on developing pancreatic tumors. DZ 2384 also reduces new tumor formation in this model. Taken together, DZ 2384 represents a novel class of microtubule-targeting agents that operates with a distinct mechanistic impact on microtubule dynamics and structure. We propose DZ 2384 as a promising new agent for the treatment of pancreatic ductal adenocarcinoma. Citation Format: Michal Wieczorek, Joseph Tcherkezian, Cynthia Bernier, Ozhan Ocal, Sami Chabaan, Yanneve Rolland, Claude Godbout, Mark Hancock, Cecilia Rocha, Natacha Olieric, Andrea E. Prota, Michel O. Steinmetz, Thomas M. Wilkie, Rolf A. Brekken, Hui Ding, Patrick Harran, Gordon C. Shore, Gary Brouhard, Anne Roulston. Diazonamide DZ 2384, a potential therapeutic for pancreatic cancer, binds to tubulin with a unique impact on microtubule dynamics and tubulin curvature. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A188.

Abstract LB-130: Reporter genes for a rapid in vivo screen of PDA therapeutics are required for energy homeostasis in pancreatic cancer-associated malnutrition

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths. Limited progress in developing effective therapy for PDA is partially due to the lack of a robust in vivo screen for effective drug combinations. Kras mutations (e.g. KrasG12D) are found in over 90% of human PDA and occur early in tumor progression. G Protein Coupled Receptor (GPCR) and protein kinase signaling can initiate Ras activation. Regulators of G-protein Signaling (RGS) proteins are coincidence detectors of Ras activation that feedback regulate, by virtue of their GTPase Activating Protein (GAP) activity, the intensity and duration of Gi- and Gq-coupled GPCR signaling. RGS-resistant mutations in Gq have been associated with PDA. We show a Rgs16::GFP transgene is a KrasG12D-dependent marker of all stages of neoplasia in the LSL-KrasG12D; Cdkn2af/f; p48Cre (KIC) mice. GFP is proportional to and coincident with tumor burden. Although KrasG12D is expressed in embryonic pancreas progenitor cells and in all mature acinar cells, Rgs16::GFP expression in tumors first emerges in ductal PanINs as early as 12 days post birth. The receptor tyrosine kinase Axl is highly expressed in PDA progenitor cells. The Gas6 ligand evokes Axl signaling in epithelial progenitor cells and contributes to activation of KrasG12D, PDA initiation and progression. In a proof-of-principle for drug screens, we determined that warfarin, which blocks maturation of Gas6, an Axl agonist, combined with the standard of care Gemcitabine and Abraxane (GA), significantly reduced PDA progression. In humans, partial pancreatic deficiency often precedes pancreatic cancer. Pancreatic insufficiency develops by 5 weeks in KC (LSL-KrasG12D;p48Cre) mice that express KrasG12D in all pancreas cells. KrasG12D, in the context of wild type Cdkn2a, causes dedifferentiation of acinar cells and a drastic reduction in digestive enzymes secreted by the pancreas. KC mice become malnourished but can survive over one year before succumbing to PDA. We find Intraductal Papillary Mucinous Neoplasm (IPMN) in KC mice express Rgs16::GFP by 2 weeks of age. We crossed the Rgs8-16 double knockout into KC (KC-R) mice to test if Rgs8-16 are tumor suppressor genes. Most KC-R mice die before 4 months of age because they can not maintain energy homeostasis - Rgs8-16 are required in liver to conserve energy utilization in malnourished mice. The effects of Rgs8-16 deficiency on exocrine pancreas function, acinar-to-ductal metaplasia (ADM), apoptosis and tumor progression in KC-R mice are under investigation. As a reporter gene, Rgs16::GFP faithfully tracks PDA progression and sensitivity to new drug regimens that inhibit KrasG12D mediated oncogenesis. Supported by NCI CA161624. Citation Format: Ozhan Ocal, Yalda Zolghadri, Galvin H. Swift, Rolf A. Brekken, Thomas M. Wilkie. Reporter genes for a rapid in vivo screen of PDA therapeutics are required for energy homeostasis in pancreatic cancer-associated malnutrition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-130. doi:10.1158/1538-7445.AM2015-LB-130

Abstract B19: Rapid in vivo screen of pancreatic ductal adenocarcinoma therapeutics

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer related deaths. Limited progress has been made in developing effective therapy for PDA. Compounding this challenge has been the lack of a systematic and robust in vivo screen for effective drug combinations. Kras mutations (e.g. KrasG12D) are found in over 90% of human PDA and occur early in tumor progression. Protein kinase and G-Protein Coupled Receptor (GPCR) signaling can initiate Ras activation. Regulators of G-protein Signaling (RGS) proteins are coincidence detectors that can be induced by and integrate multiple inputs to feedback regulate GPCR signaling. We previously described Rgs16 expression during embryonic and postnatal pancreas development in pancreatic progenitors, endocrine cells and ductal-associated cells. Here, we show that the Rgs16::GFP transgene is a KrasG12D dependent marker of all stages of neoplasia in the p48Cre; LSL-KrasG12D; Cdkn2af/f PDA mice. Although KrasG12D is expressed in embryonic pancreas progenitor cells and in all mature acinar cells, Rgs16::GFP expression in tumors first emerges in ductal pancreatic intraepithelial neoplasms as early as 12 days post birth. The distribution and intensity of expression is proportional to and coincident with tumor burden. Rgs16::GFP expression in acinar cells of distal lobes initiates only after tumor occlusion of proximal ducts. The gene expression signature of primary PDA cells in culture obtained via RNA-seq is closely related to embryonic progenitors of pancreatic ducts and endocrine cells. PDA cells in culture are highly sensitive to the chemotherapic drug gemcitabine mediated cell death. In a proof-of-principle for drug screens, we find PDA weanling mice treated with gemcitabine and Nab-paclitaxel for two weeks have significantly lower Rgs16::GFP expression and reduced tumor size and occurrence. Rgs16::GFP is a novel in vivo reporter of PDA progression and sensitivity to new drug regimens that inhibit KrasG12D mediated oncogenesis. Supported by NCI-CA161624 Citation Format: Ozhan Ocal, Blake Heath, Lee B. Rivera, Victor Pashkov, Galvin H. Swift, Rolf A. Brekken, Tom M. Wilkie. Rapid in vivo screen of pancreatic ductal adenocarcinoma therapeutics. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B19. doi: 10.1158/1557-3125.RASONC14-B19

Abstract A98: Rgs16: HTS biomarker for pancreas development, diabetes, and PDAC.

Rgs16::GFP is a novel and early reporter of GPCR signaling during beta-cell expansion and pancreatic ductal adenocarcinoma (PDAC). Novel approaches are needed to understand the contributions of endocrine progenitors and b-cell regeneration during islet expansion, and to discover new therapeutics for diabetes that will not promote PDAC. G-protein signaling is an excellent target for therapeutics but is not well understood in pancreas development and disease. Regulators of G-protein signaling (RGS) proteins can serve as biomarkers for active G protein coupled receptor (GPCR) signaling. Rgs16 is expressed in pancreatic progenitor and endocrine cells during development, then extinguish in normal glycemic weanlings (DMM 3:567). Rgs16 is reactivated in models of diabetes and PDAC. RNA-Seq was used to identify 63 GPCRs (28 orphans) expressed in PDAC cells, whose expression profile is closely related to embryonic duct and endocrine progenitors. PDAC cells have been validated for 384-well high throughput screens (HTS). A putative GPCR-ligand stimulated Rgs16::GFP expression in 90% of PDAC cells, and in islets in a regenerative model of type 1 diabetes. Mouse models of diabetes and PDAC will be used to validate small molecules identified in HTS. Rgs16 may control islet progenitor cell activation, differentiation and migration during ductal and islet expansion in embryos and metabolically stressed adults. Funding: NIH (GM61395) and Welch Foundation (I-1382) to TMW Citation Format: Thomas M. Wilkie, Ozhan Ocal, Lee B. Rivera, Rolf A. Brekken. Rgs16: HTS biomarker for pancreas development, diabetes, and PDAC. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A98.