The signaling pathway of the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) is important for cell growth, survival, and motility and is functionally linked to the signaling pathway of VEGF, which is widely recognized as a key effector in angiogenesis and cancer progression. Dysregulation of the MET/VEGF axis is found in a number of human malignancies and has been associated with tumorigenesis. Cabozantinib (XL184) is a small-molecule kinase inhibitor with potent activity toward MET and VEGF receptor 2 (VEGFR2), as well as a number of other receptor tyrosine kinases that have also been implicated in tumor pathobiology, including RET, KIT, AXL, and FLT3. Treatment with cabozantinib inhibited MET and VEGFR2 phosphorylation in vitro and in tumor models in vivo and led to significant reductions in cell invasion in vitro. In mouse models, cabozantinib dramatically altered tumor pathology, resulting in decreased tumor and endothelial cell proliferation coupled with increased apoptosis and dose-dependent inhibition of tumor growth in breast, lung, and glioma tumor models. Importantly, treatment with cabozantinib did not increase lung tumor burden in an experimental model of metastasis, which has been observed with inhibitors of VEGF signaling that do not target MET. Collectively, these data suggest that cabozantinib is a promising agent for inhibiting tumor angiogenesis and metastasis in cancers with dysregulated MET and VEGFR signaling.

Highlights d Antibody blockade of MerTK prevents apoptotic cell clearance by macrophages d MerTK blockade induces tumor-cGAS-and host-STINGdependent type I IFN response d Extracellular ATP facilitates transfer of tumor-derived cGAMP to TAMs via P2X7R d MerTK blockade increases tumor immunogenicity and enhances anti-PD-1/PD-L1 therapy

Background: A limited number of approved therapeutic options are available to metastatic medullary thyroid cancer (MTC) patients, and the response to conventional chemotherapy and/or radiotherapy strategies is inadequate. Sporadic and inherited mutations in the tyrosine kinase RET result in oncogenic activation that is associated with the pathogenesis of MTC. Cabozantinib is a potent inhibitor of MET, RET, and vascular endothelial factor receptor 2 (VEGFR2), as well as other tyrosine kinases that have been implicated in tumor development and progression. The object of this study was to determine the in vitro biochemical and cellular inhibitory profile of cabozantinib against RET, and in vivo antitumor efficacy using a xenograft model of MTC. Methods: Cabozantinib was evaluated in biochemical and cell-based assays that determined the potency of the compound against wild type and activating mutant forms of RET. Additionally, the pharmacodynamic modulation of RET and MET and in vivo antitumor activity of cabozantinib was examined in a MTC tumor model following subchronic oral administration. Results: In biochemical assays, cabozantinib inhibited multiple forms of oncogenic RET kinase activity, including M918T and Y791F mutants. Additionally, it inhibited proliferation of TT tumor cells that harbor a C634W activating mutation of RET that is most often associated with MEN2A and familial MTC. In these same cells grown as xenograft tumors in nude mice, oral administration of cabozantinib resulted in dose-dependent tumor growth inhibition that correlated with a reduction in circulating plasma calcitonin levels. Moreover, immunohistochemical analyses of tumors revealed that cabozantinib reduced levels of phosphorylated MET and RET, and decreased tumor cellularity, proliferation, and vascularization. Conclusions: Cabozantinib is a potent inhibitor of RET and prevalent mutationally activated forms of RET known to be associated with MTC, and effectively inhibits the growth of a MTC tumor cell model in vitro and in vivo.

Purpose: Agents inhibiting the epidermal growth factor receptor (EGFR) have shown clinical benefit in a subset of non^small cell lung cancer patients expressing amplified or mutationally activated EGFR. However, responsive patients can relapse as a result of selection for EGFR gene mutations that confer resistance to ATP competitive EGFR inhibitors, such as erlotinib and gefitinib.We describe here the activity of EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, against both wild-type (WT) and mutant EGFR in vitro and in vivo. Experimental Design: The activity of EGFR inhibitors against WTand mutant EGFRs and their effect on downstream signal transduction was examined in cellular assays and in vivo using A431 and MDA-MB-231 (WT EGFR) and H1975 (L858R andT790M mutant EGFR) xenograft tumors.Results: EXEL-7647 shows potent and long-lived inhibition of the WT EGFR in vivo. In addition, EXEL-7647 inhibits cellular proliferation and EGFR pathway activation in the erlotinib-resistant H1975 cell line that harbors a double mutation (L858R and T790M) in the EGFR gene. In vivo efficacy studies show that EXEL-7647 substantially inhibited the growth of H1975 xenograft tumors and reduced both tumor EGFR signaling and tumor vessel density. Additionally, EXEL-7647, in contrast to erlotinib, substantially inhibited the growth and vascularization of MDA-MB-231 xenografts, a model which is more reliant on signaling through vascular endothelial growth factor receptors. Conclusions: These studies provide a preclinical basis for clinical trials of XL647 in solid tumors and in patients bearing tumors that are resistant to existing EGFR-targeted therapies.

Purpose: GDC-0973 is a potent and selective mitogen-activated protein (MAP)/extracellular signalregulated kinase (ERK) kinase (MEK) inhibitor. Pharmacokinetic-pharmacodynamic (PK-PD) modeling was used to relate GDC-0973 plasma and tumor concentrations, tumor pharmacodynamics and antitumor efficacy to establish pharmacokinetic endpoints and predict active doses in the clinic.Experimental Design: A PK-PD model was used to characterize GDC-0973 tumor disposition and in vivo potency in WM-266-4 xenograft mice. Simulations were conducted using the PK-PD model along with human pharmacokinetics to identify a target plasma concentration and predict active doses. In vivo potency and antitumor efficacy were characterized in A375 melanoma xenograft mice, and a population-based integrated PK-PD-efficacy model was used to relate tumor pharmacodynamics (%pERK decrease) to antitumor activity.Results: GDC-0973 showed a sustained tumor pharmacodynamic response due to longer residence in tumor than in plasma. Following single doses of GDC-0973, estimated in vivo IC 50 values of %pERK decrease based on tumor concentrations in xenograft mice were 0.78 (WM-266-4) and 0.52 mmol/L (A375). Following multiple doses of GDC-0973, the estimated in vivo IC 50 value in WM-266-4 increased (3.89 mmol/L). Human simulations predicted a minimum target plasma concentration of 83 nmol/L and an active dose range of 28 to 112 mg. The steep relationship between tumor pharmacodynamics (%pERK decrease) and antitumor efficacy suggests a pathway modulation threshold beyond which antitumor efficacy switches on.Conclusions: Clinical observations of %pERK decrease and antitumor activity were consistent with model predictions. This article illustrates how PK-PD modeling can improve the translation of preclinical data to humans by providing a means to integrate preclinical and early clinical data.

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrP M ) to the pathogenic isoform (PrP Sc ). While most familial cases are heterozygous and thus express the normal (PrP C ) and mutant alleles of PrP, the role of PrP C in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrP M is detected, whereas in others both PrP C and PrP M are transformed to PrP Sc . To understand if the coaggregation of PrP C is governed by PrP mutations or is a consequence of the cellular compartment of PrP M aggregation, we coexpressed PrP M and PrP C in neuroblastoma cells, the latter tagged with green fluorescent protein (PrP C-GFP ) for differentiation. Two PrP M forms (PrP 231T , PrP 217R/231T ) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrP C-GFP aggregates when coexpressed with PrP 231T or PrP 217R/231T , regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP 231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP ( Ctm PrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrP M from the ER is blocked by the cellular quality control.

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