Gregory F. Hollis

A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance.

The ability to counterselect, as well as to select for, a genetic marker has numerous applications in microbial genetics. Described here is the use of 5‐fluoroanthranilic acid for the counterselection of TRP1, a commonly used genetic marker in the yeast Saccharomyces cerevisiae. Counterselection using 5‐fluoroanthranilic acid involves antimetabolism by the enzymes of the tryptophan biosynthetic pathway, such that trp1, trp3, trp4 or trp5 strains, which lack enzymes required for the conversion of anthranilic acid to tryptophan, are resistant to 5‐fluoroanthranilic acid. Commonly used genetic procedures, such as selection for loss of a chromosomally integrated plasmid, and a replica‐plating method to rapidly assess genetic linkage in self‐replicating shuttle vectors, can now be carried out using the TRP1 marker gene. In addition, novel tryptophan auxotrophs can be selected using 5‐fluoroanthranilic acid. Copyright

Aspartyl beta-hydroxylase (Asph)and an evolutionarily conserved isoform of Asph missing the catalytic domain share exons with junctin

The mouse aspartyl β-hydroxylase gene (Asph, BAH) has been cloned and characterized. The mouseBAH gene spans 200 kilobase pairs of genomic DNA and contains 24 exons. Of three major BAH-related transcripts, the two largest (6,629 and 4,419 base pairs) encode full-length protein and differ only in the use of alternative polyadenylation signals. The smallest BAH-related transcript (2,789 base pairs) uses an alternative 3′ terminal exon, resulting in a protein lacking a catalytic domain. Evolutionary conservation of this noncatalytic isoform of BAH (humbug) is demonstrated in mouse, man, andDrosophila. Monoclonal antibody reagents were generated, epitope-mapped, and used to definitively correlate RNA bands on Northern blots with protein species on Western blots. The gene for mouse junctin, a calsequestrin-binding protein, was cloned and characterized and shown to be encoded from the same locus. When expressed in heart tissue, BAH/humbug preferably use the first exon and often the fourth exon of junctin while preserving the reading frame. Thus, three individual genes share common exons and open reading frames and use separate promoters to achieve differential expression, splicing, and function in a variety of tissues. This unusual form of exon sharing suggests that the functions of junctin, BAH, and humbug may be linked.

A yeast genetic assay for caspase cleavage of the amyloid-β precursor protein

A functional assay for proteolytic processing of the amyloid precursor protein (APP) was set up in yeast. This consisted of a membrane-bound chimeric protein containing the β-secretase cleaved C-terminal fragment of APP fused to the Gal4 transcription factor. Using this chimera in a GAL-reporter yeast strain, an expression library of human cDNAs was screened for clones that could activate the GAL-reporter genes by proteolytic processing of the membrane-bound APP-Gal4. Two human proteases, caspase-3 and caspase-8, were identified and confirmed to act by a mechanism that involved proteolysis at the site in the APP-Gal4 chimera that corresponded to the natural caspase cleavage site in APP, thus linking a readily scorable phenotype to proteolytic processing of APP. The activation of caspase-3 involved a mechanism that was independent of aspartic acid residue 175 at the cleavage site normally required for processing of caspase-3.

Abstract 2100: Selective inhibition of FGFR4 by INCB062079 is efficacious in models of FGF19- and FGFR4-dependent cancers

Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. FGFR4 signaling contributes to the development and progression of subsets of cancer: in approximately 10 percent of hepatocellular carcinoma (HCC), genetic amplification of FGF19, encoding an endocrine FGF ligand that activates FGFR4-KLB receptors, has been reported. In models with this alteration, FGF19-FGFR4 signaling is oncogenic and antagonism of the FGF19-FGFR4 axis has been shown to be efficacious suggesting that selective targeting of FGFR4 may be an effective strategy for malignancies with FGFR4 activation. We describe the preclinical characterization of INCB062079 a potent and selective inhibitor of the FGFR4 kinase. In biochemical assays INCB062079 inhibited FGFR4 with low nM potency and exhibited at least 250-fold selectivity against other FGFR kinases and greater than 800-fold selectivity against a large kinase panel. This selectivity derives from the ability of INCB062079 to bind irreversibly to Cys552, a residue within the active site of FGFR4 that is non-conserved among other FGFR receptors. Covalent binding of INCB062079 to Cys552 was demonstrated using a LC/MS/MS-based proteomic analysis that confirmed specificity for the target Cys. In assays using HCC cells with autocrine production of FGF19, INCB062079 inhibited the autophosphorylation of FGFR4 and blocked signal transduction by FGFR4 to downstream markers of pathway activation. Cancer cell lines that have amplification and expression of FGF19 are uniquely sensitive to growth inhibition by INCB062079 (EC50 less than 200 nM) compared with HCC cell lines or normal cells without FGF19-FGFR4 dependence (EC50 > 5000 nM) confirming selectivity for FGFR4. In vivo, oral administration of INCB062079 inhibited the growth and induced significant regressions of subcutaneous xenograft tumors dependent upon FGFR4 activity at doses that were well-tolerated (10-30 mg/kg BID) and did not result in a significant increase in serum phosphate levels which is observed with FGFR1/2/3 inhibition. Suppression of tumor growth correlated with pharmacodynamic inhibition of FGFR4 signaling. Collectively, these preclinical studies demonstrate that INCB062079 potently and selectively inhibits models of FGF19-FGFR4-dependent cancers in vitro and in vivo, supporting clinical evaluation in patients harboring oncogenic FGFR4 activation. Citation Format: Phillip C.C. Liu, Liang Lu, Kevin Bowman, Matthew C. Stubbs, Liangxing Wu, Darlise DiMatteo, Sindy Condon, Ronald Klabe, Ding-Quan Qian, Xiaoming Wen, Paul Collier, Karen Gallagher, Michael Hansbury, Xin He, Bruce Ruggeri, Yan-ou Yang, Maryanne Covington, Timothy C. Burn, Sharon Diamond-Fosbenner, Richard Wynn, Reid Huber, Wenqing Yao, Swamy Yeleswaram, Peggy Scherle, Gregory Hollis. Selective inhibition of FGFR4 by INCB062079 is efficacious in models of FGF19- and FGFR4-dependent cancers [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 2100. doi:10.1158/1538-7445.AM2017-2100

Abstract 5397: Characterization of INCB053914, a novel pan-PIM kinase inhibitor

The PIM (Proviral Integration sites for Moloney virus) family of constitutively active serine/threonine kinases consists of three members (PIM1, PIM2 and PIM3) that phosphorylate substrates involved in both apoptosis and cellular metabolism. As downstream targets of the JAK/STAT pathway and by having overlapping substrates with the PI3K/AKT pathway, PIM kinases integrate signals from multiple pathways important for the survival and proliferation of malignant cells. Overexpression of PIM kinases has been reported in human hematological cancers with each isoform showing a distinct expression pattern among the various malignancy subtypes, including MM, AML, ALL and NHL. Although the three family members possess overlapping functions that are mutually compensatory, mice deficient for all three PIM kinases exhibit minor phenotypic abnormalities, with smaller overall size at maturity and decreased cellular responses to cytokines. These data suggest that pan-inhibition of the PIM kinase family will have a favorable toxicity profile and may be required for therapeutic benefit in hematological malignancies. INCB053914 is a novel, ATP-competitive, small molecule, pan-inhibitor of PIM kinases. In biochemical assays, INCB053914 is potent against PIM1, PIM2 and PIM3 and highly selective. In cell proliferation assays, INCB053914 is active as a single agent in the majority of cell lines derived from different hematological malignancies, including MM, AML, DLBCL, MCL and T-ALL, with IC50 values ranging from 3 - 300 nM. INCB053914 synergizes with a variety of cytotoxic and targeted agents, reducing the viability of a panel of hematological tumor cell lines. In pharmacodynamic assays, INCB053914 inhibits phosphorylation of S6RP, P70S6K, 4E-BP-1 and BAD, known PIM kinase targets. Following treatment with INCB053914, induction of PIM kinase protein levels was noted in human cancer cell lines. Importantly, this compensatory response does not reduce the activity of the molecule, as effects on downstream signaling and efficacy remain intact when PIM kinase levels are elevated. Induction of PIM kinases after INCB53914 treatment was also evident in whole blood from healthy volunteers and in primary blast cells isolated from patients diagnosed with AML, further supporting the need for pan-PIM kinase inhibition. Taken together, these results indicate that INCB053914 has potent and selective activity against PIM kinases and may be useful as monotherapy or in combination with other agents for treating hematologic malignancies that are dependent on PIM kinase activity for growth and survival. Citation Format: Niu Shin, Maryanne Covington, Richard Wynn, Yanlong Li, Alexander Margulis, Qian Wang, Kathy Wang, Cindy Marando, Patricia Feldman, Lynn Leffet, Karen Gallagher, Xin He, Hong Chang, He Zhang, Hao Feng, Yun-Long Li, Chu-Biao Xue, Wenqing Yao, Timothy Burn, Kris Vaddi, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Gregory Hollis, Robert Newton, Reid Huber, Peggy Scherle, Holly Koblish. Characterization of INCB053914, a novel pan-PIM kinase inhibitor. [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 5397. doi:10.1158/1538-7445.AM2015-5397