Richard J. Austin

Oncogenic properties of PPM1D located within a breast cancer amplification epicenter at 17q23

We found that PPM1D, encoding a serine/threonine protein phosphatase, lies within an epicenter of the region at 17q23 that is amplified in breast cancer. We show that overexpression of this gene confers two oncogenic phenotypes on cells in culture: attenuation of apoptosis induced by serum starvation and transformation of primary cells in cooperation with RAS.

Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.

Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.

Addressing PXR liabilities of phthalazine-based hedgehog/smoothened antagonists using novel pyridopyridazines

Pyridopyridazine antagonists of the hedgehog signaling pathway are described. Designed to optimize our previously described phthalazine smoothened antagonists, a representative compound eliminates a PXR liability while retaining potency and in vitro metabolic stability. Moreover, the compound has improved efficacy in a hedgehog/smoothened signaling mouse pharmacodynamic model.

Discovery of amide replacements that improve activity and metabolic stability of a bis-amide smoothened antagonist hit

A bis-amide antagonist of Smoothened, a seven-transmembrane receptor in the Hedgehog signaling pathway, was discovered via high throughput screening. In vitro and in vivo experiments demonstrated that the bis-amide was susceptible to N-acyl transferase mediated amide scission. Several bioisosteric replacements of the labile amide that maintained in vitro potency were identified and shown to be metabolically stable in vitro and in vivo.

Abstract 1773: HPN424, a half-life extended, PSMA/CD3-specific TriTAC for the treatment of metastatic prostate cancer

Metastatic, castrate-resistant prostate cancer (mCRPC) is diagnosed in up to 50,000 patients each year in the US alone, and roughly 27,000 patients will succumb to it every year. Once metastasized beyond regional lymph nodes, the 5-year survival rate is 30%. While novel therapeutics like abiraterone and enzalutamide have improved the treatment options for mCRPC, no curative treatment is available, and new therapies are urgently needed. HPN424 is a ~50-kDa antibody derivative called TriTAC (Tri-specific T cell Activating Construct) under development for the treatment of mCRPC. It is designed to simultaneously bind to CD3ϵ on T cells and to prostate specific membrane antigen (PSMA, FOLH1) on prostate cancer cells. A third domain of HPN424 binds non-covalently to serum albumin for extension of serum half-life. PSMA is expressed in >90% of malignant lesions of patients, and outside the central nervous system, its expression on normal tissue is largely restricted to the prostate. HPN424 binds human PSMA with sub-nanomolar affinity. When incubated in co-cultures with resting, human T cells and prostate cancer cells, it activates T cells and induces cytokine production, proliferation and redirected target cell killing with EC 50 values in the single digit picomolar range. When administered to mice bearing human prostate cancer xenografts and human T cells, HPN424 eradicates subcutaneous tumors. The affinities of HPN424 for human and cynomolgus monkey CD3 and albumin are comparable, while HPN424 binds only marginally to cynomolgus PSMA. HPN424 is very well tolerated in non-human primates, even at high doses, indicating that CD3-binding has little if any pharmacological effect in the absence of target binding. Pharmacokinetic analysis supports weekly administration in humans. Our preclinical data suggest that HPN424 will be highly efficacious, safe and convenient for the treatment of patients with mCRPC. Citation Format: Bryan Lemon, Wade Aaron, Richard Austin, Patrick Baeuerle, Manasi Barath, Adrie Jones, Susan D. Jones, Kathryn Kwant, Che-Leung Law, Anna Muchnik, Kenneth Sexton, Laurie Tatalick, Holger Wesche, Timothy Yu. HPN424, a half-life extended, PSMA/CD3-specific TriTAC for the treatment of metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1773.