Ubiquitin ligase COP1 coordinates transcriptional programs that control cell type specification in the developing mouse brain.

The E3 ubiquitin ligase CRL4COP1/DET1 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with constitutive photomorphogenesis 1 (Cop1) deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes Vimentin and Gfap was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including Olig1, Olig2, and Sox10. The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several Cop1-deficient mice also lacking c-Jun and Etv5, or lacking Etv5 and heterozygous for Etv1, were viable.

CD8+ T cell infiltration in breast and colon cancer: A histologic and statistical analysis.

The prevalence of cytotoxic tumor infiltrating lymphocytes (TILs) has demonstrated prognostic value in multiple tumor types. In particular, CD8 counts (in combination with CD3 and CD45RO) have been shown to be superior to traditional UICC staging in colon cancer patients and higher total CD8 counts have been associated with better survival in breast cancer patients. However, immune infiltrate heterogeneity can lead to potentially significant misrepresentations of marker prevalence in routine histologic sections. We examined step sections of breast and colorectal cancer samples for CD8+ T cell prevalence by standard chromogenic immunohistochemistry to determine marker variability and inform practice of T cell biomarker assessment in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Stained sections were digitally imaged and CD8+ lymphocytes within defined regions of interest (ROI) including the tumor and surrounding stroma were enumerated. Statistical analyses of CD8+ cell count variability using a linear model/ANOVA framework between patients as well as between levels within a patient sample were performed. Our results show that CD8+ T-cell distribution is highly homogeneous within a standard tissue sample in both colorectal and breast carcinomas. As such, cytotoxic T cell prevalence by immunohistochemistry on a single level or even from a subsample of biopsy fragments taken from that level can be considered representative of cytotoxic T cell infiltration for the entire tumor section within the block. These findings support the technical validity of biomarker strategies relying on CD8 immunohistochemistry.

Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

Cotargeting of MEK and PDGFR/STAT3 Pathways to Treat Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal human diseases and remains largely refractory to available drug treatments. Insufficient targeting of the known oncogenic drivers and activation of compensatory feedback loops and inability to prevent metastatic spread contribute to poor prognosis for this disease. The KRAS-driven MEK pathway is mutationally activated in most pancreatic cancers and is an important target for therapeutics. Using a two-dimensional monolayer culture system as well as three-dimensional spheroid culture system, we conducted a screen of a large panel of anticancer agents and found that MAP2K (MEK) inhibitors were most effective in targeting PDAC spheroids in comparison with monolayer cultures. Combination treatment with an MEK inhibitor and the multikinase inhibitor ponatinib was effective in targeting pancreatic cancer cells both in monolayer and spheroids by effectively blocking signaling via the PDGFRα and MEK kinases, while also preventing the activation of STAT3- and S6-mediated compensatory feedback loops in cancer cells. Furthermore, using xenograft models, we demonstrate that cotreatment with a MEK inhibitor and ponatinib causes significant tumor regression. PDAC patient samples also provided evidence of increased STAT3 activation in PDAC tumors and MAPK1 (ERK) activation in liver metastases, implicating STAT3 and ERK as key drivers in primary tumors and metastases, respectively. These results reveal a combination drug treatment strategy that may be effective in pancreatic cancer. Mol Cancer Ther; 16(9); 1729-38. ©2017 AACR.

The Selective PI3K Inhibitor XL147 (SAR245408) Inhibits Tumor Growth and Survival and Potentiates the Activity of Chemotherapeutic Agents in Preclinical Tumor Models.

Dysregulation of PI3K/PTEN pathway components, resulting in hyperactivated PI3K signaling, is frequently observed in various cancers and correlates with tumor growth and survival. Resistance to a variety of anticancer therapies, including receptor tyrosine kinase (RTK) inhibitors and chemotherapeutic agents, has been attributed to the absence or attenuation of downregulating signals along the PI3K/PTEN pathway. Thus, PI3K inhibitors have therapeutic potential as single agents and in combination with other therapies for a variety of cancer indications. XL147 (SAR245408) is a potent and highly selective inhibitor of class I PI3Ks (α, ß, γ, and δ). Moreover, broad kinase selectivity profiling of >130 protein kinases revealed that XL147 is highly selective for class I PI3Ks over other kinases. In cellular assays, XL147 inhibits the formation of PIP3 in the membrane, and inhibits phosphorylation of AKT, p70S6K, and S6 in multiple tumor cell lines with diverse genetic alterations affecting the PI3K pathway. In a panel of tumor cell lines, XL147 inhibits proliferation with a wide range of potencies, with evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL147 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of at least 24 hours. Repeat-dose administration of XL147 results in significant tumor growth inhibition in multiple human xenograft models in nude mice. Administration of XL147 in combination with chemotherapeutic agents results in antitumor activity in xenograft models that is enhanced over that observed with the corresponding single agents.

Immunohistochemical detection of FLAG-tagged endogenous proteins in knock-in mice.

With recent advances in immunohistochemical (IHC) techniques, immunohistochemistry now plays a more important role in research, especially in mouse models where characterization of cellular patterns of protein expression has become critical. Even with these recent advances, a paucity of IHC quality antibodies for some proteins still exists. To address this, we have developed a novel IHC assay that utilizes a commercially available goat anti-DDDDK peptide polyclonal antibody on paraffin-embedded tissues from knock-in mice expressing proteins of interest tagged with a 3 × FLAG epitope at physiologically relevant levels. Focusing on two 3 × FLAG-tagged proteins for which specific antibodies were available, USP48 and RIPK3, we were able to validate our anti-DDDDK assay by comparing the IHC directed against the actual proteins to the anti-DDDDK IHC assay, which recognizes the FLAG epitope. We were also able to detect a third 3 × FLAG-tagged protein, BAP1, for which quality reagents were not available. This universal IHC method will enable researchers to characterize the expression patterns of proteins of interest when specific antibodies are lacking.

Characterization of the activity of the PI3K/mTOR inhibitor XL765 (SAR245409) in tumor models with diverse genetic alterations affecting the PI3K pathway.

Activation of the PI3K (phosphoinositide 3-kinase) pathway is a frequent occurrence in human tumors and is thought to promote growth, survival, and resistance to diverse therapies. Here, we report pharmacologic characterization of the pyridopyrimidinone derivative XL765 (SAR245409), a potent and highly selective pan inhibitor of class I PI3Ks (α, ß, γ, and δ) with activity against mTOR. Broad kinase selectivity profiling of >130 protein kinases revealed that XL765 is highly selective for class I PI3Ks and mTOR over other kinases. In cellular assays, XL765 inhibits the formation of PIP(3) in the membrane, and inhibits phosphorylation of AKT, p70S6K, and S6 phosphorylation in multiple tumor cell lines with different genetic alterations affecting the PI3K pathway. In a panel of tumor cell lines, XL765 inhibits proliferation with a wide range of potencies, with evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL765 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of approximately 24 hours. Repeat dose administration of XL765 results in significant tumor growth inhibition in multiple human xenograft models in nude mice that is associated with antiproliferative, antiangiogenic, and proapoptotic effects.

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG.

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This "panel-specific" feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.

In vitro and in vivo activity of cabozantinib (XL184), an inhibitor of RET, MET, and VEGFR2, in a model of medullary thyroid cancer.

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.

Cabozantinib (XL184), a novel MET and VEGFR2 inhibitor, simultaneously suppresses metastasis, angiogenesis, and tumor growth.

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.

A phase I study of foretinib, a multi-targeted inhibitor of c-Met and vascular endothelial growth factor receptor 2.

PURPOSE: Foretinib is an oral multikinase inhibitor targeting Met, RON, Axl, and vascular endothelial growth factor receptor. We conducted a phase I, first-time-in-human, clinical trial using escalating doses of oral foretinib. The primary objectives are to identify a maximum tolerated dose and determine the safety profile of foretinib. Secondary objectives included evaluation of plasma pharmacokinetics, long-term safety after repeated administration, preliminary antitumor activity, and pharmacodynamic activity. EXPERIMENTAL DESIGN: Patients had histologically confirmed metastatic or unresectable solid tumors for which no standard measures exist. All patients received foretinib orally for 5 consecutive days every 14 days. Dose escalation followed a conventional "3+3" design. RESULTS: Forty patients were treated in eight dose cohorts. The maximum tolerated dose was defined as 3.6 mg/kg, with a maximum administered dose of 4.5 mg/kg. Dose-limiting toxicities included grade 3 elevations in aspartate aminotransferase and lipase. Additional non-dose-limiting adverse events included hypertension, fatigue, diarrhea, vomiting, proteinuria, and hematuria. Responses were observed in two patients with papillary renal cell cancer and one patient with medullary thyroid cancer. Stable disease was identified in 22 patients. Foretinib pharmacokinetics increased linearly with dose. Pharmacodynamic evaluation indicated inhibition of MET phosphorylation and decreased proliferation in select tumor biopsies at submaximal doses. CONCLUSIONS: The recommended dose of foretinib was determined to be 240 mg, given on the first 5 days of a 14-day cycle. This dose and schedule were identified as having acceptable safety and pharmacokinetics, and will be the dose used in subsequent phase II trials.

Inhibition of the T790M gatekeeper mutant of the epidermal growth factor receptor by EXEL-7647.

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 WT and 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 and T790M 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.

Cerebrospinal fluid rhinorrhea: diagnosis and management.

Advances in imaging and endoscopic techniques have improved the ability to diagnose, localize, and treat in a less morbid fashion CSF leaks of the anterior skull base. An appreciation for the mechanism of leak and of the relationship between CSF production and absorption must be kept in mind when individualizing a repair. Increased CSF pressure caused by overproduction or underabsorption may result in persistence of a leak despite one's best efforts. Numerous advances in dural replacement grafts and tissue sealants have improved the ability to achieve watertight closure of the cranial vault. Microvascular techniques have allowed larger defects previously not reconstructable to be handled with relative ease by trained personnel. With expanded reconstructive techniques, the ability to handle larger disease processes of the skull base continues to expand.