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.

SUMMARYDiabetes is characterized by the loss, or gradual dysfunction, of insulin-producing pancreatic -cells. Although -cells can replicate in younger adults, the available diabetes therapies do not specifically target -cell regeneration. Novel approaches are needed to discover new therapeutics and to understand the contributions of endocrine progenitors and -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 -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 ductalassociated cells and islet -cells. These findings show that Rgs16::GFP and Rgs8::GFP are novel and early reporters of G protein-coupled receptor (GPCR)-stimulated -cell expansion after therapeutic treatment and in diabetes models. Rgs16 and Rgs8 are likely to control aspects of islet progenitor cell activation, differentiation and -cell expansion in embryos and metabolically stressed adults.

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.

Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths. Progress towards effective therapy for PDA has been very limited. We are developing 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 multiple inputs to feedback regulate GPCR signaling. We previously described Rgs16 expression during embryonic and postnatal pancreas development in pancreatic progenitor and endocrine cells (DMM3, 567). Here, we show that the Rgs16::GFP transgene is a KrasG12D dependent marker of all neoplastic stages in the LSL‐KrasG12D; Cdkn2af/f; p48Cre (KIC) mice. Rgs16::GFP expression first emerges in ductal Pancreatic Intraepithelial Neoplasia two weeks after birth. The distribution and intensity of Rgs16::GFP increase proportional to tumor burden and extend to acinar cells of distal lobes after occlusion of proximal ducts. RNA‐seq gene expression analysis of primary PDA cell culture shows characteristics of embryonic progenitors of pancreatic ducts and endocrine cells. The receptor tyrosine kinase Axl is a new target for drug development and overexpressed in PDA cells. In a proof‐of‐principle for drug screens, we find PDA weanling mice treated with gemcitabine and Axl inhibitors for 2 weeks have significantly lower quantitative Rgs16::GFP expression and reduced tumor size and occurrence than gemcitabine alone. Rgs16::GFP is hence an in vivo reporter of PDA progression and sensitivity to new chemotherapeutic drug regimens. Supported by NCI CA161624.

Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer that resists efforts to identify better chemotherapeutics. PDA is associated with chronic pancreatitis and acinar cell dedifferentiation. This reduces enzyme production by the exocrine pancreas, resulting in digestive insufficiencies. Malabsorption of partially digested food causes bloating, overfilled intestines, abdominal pain, excessive feces, steatorrhea, and malnutrition. These maladies affect quality of life and restrict treatment options for pancreatitis and PDA. Here, we characterize health benefits and risks of dietary pancreatic enzymes in three mouse models of PDA-KC, KCR8-16, and KIC. KC expresses oncogenic Kras in pancreatic tissue whereas KCR8-16 also has deletions of the Rgs8 and Rgs16 genes. Rgs proteins inhibit the release of digestive enzymes evoked by G-protein-coupled-receptor agonists. KC and KCR8-16 mice developed dedifferentiated exocrine pancreata within 2 months of age and became malnourished, underweight, hypoglycemic, and hypothermic. KC mice adapted but KCR8-16 mice rapidly transitioned to starvation after mild metabolic challenges. Dietary pancreatic enzyme supplements reversed these symptoms in KC and KCR8-16 animals, and extended survival. Therefore, we tested the benefits of pancreatic enzymes in an aggressive mouse model of PDA (KIC). Median survival improved with dietary pancreatic enzyme supplements and was extended further when combined with warfarin and gemcitabine chemotherapy. However, dietary pancreatic enzymes stimulated tumor growth in the terminal stages of disease progression in KIC mice.