Oscar B. Goodman

THE ALPHA CHAIN OF THE AP-2 ADAPTOR IS A CLATHRIN BINDING SUBUNIT

We have utilized a rabbit reticulocyte lysate coupled transcription-translation system to express the large subunits of the clathrin associated protein-2 (AP-2) complex so that their individual functions may be studied separately. Appropriate folding of each subunit into N-terminal core and C-terminal appendage domains was confirmed by limited proteolysis. Translated β2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated αa exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of αa bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a ≈29-kDa αa clathrin-binding fragment that remained tightly cage-associated. Translated αa also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated β2 subunit. Clathrin binding by both the α and β subunits is consistent with a bivalent cross-linking model for lattice assembly (Keen, J. H.(1987) Cell Biol. 105, 1989). It also [Abstract] raises the possibility that the α-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other α functions.

Abstract B001: CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance

Purpose: Androgen receptor (AR) plays a major role in promoting growth and progression of prostate cancer and thus has been successfully targeted therapeutically. Therapeutic resistance to androgen deprivation therapy (ADT) accounts for much of the morbidity and mortality attributable to prostate cancer. Therapeutic resistance is typically accompanied by insufficient blockade of AR activation, whose initial step involves translocation of AR to the nucleus. In recent studies we demonstrated that CYP3A5 facilitates the nuclear translocation of AR, promoting prostate cancer growth. This work focuses on understanding the role of CYP3A5 in regulating AR nuclear translocation and therapeutic resistance. Method: To understand how CYP3A5 effects AR downstream signaling, we used RT2 profiler PCR arrays to help identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy and cell fractionation experiments were used to further confirm the effect of CYP3A5 modulation of AR signaling. As concomitant CYP3A5 inducer/inhibitor medications can affect intratumoral CYP3A5 activity and thus may modify AR downstream signaling, we utilized cell fractionation and confocal microscopy to analyze the effect of these medications on AR nuclear translocation. Further, to decipher the role of CYP3A5 in the AR translocation process we performed a series of co-immunoprecipitation (co-IP) experiments with CYP3A5 and components of HSP90-AR complex. We used extracts of LNCaP transfected with a CYP3A5-Flag-tagged construct for IP, with either Flag-agarose or HSP90 and AR antibodies with A/G beads. Finally, to decipher CYP3A5’s role in therapeutic resistance we analyzed cDNA after CYP3A5 siRNA treatment using drug resistance RT2 profiler PCR array. Results: The q-PCR based profiler assay revealed a set of nine AR regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005. MYC and PIK3R3 are known to be involved in regulating cell growth by either affecting cell cycle progression and/or apoptosis. PSA, known to aid in the cell growth, is regulated by SPDEF, a transcription factor. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. Concomitantly prescribed CYP3A inhibitors amiodarone and ritonavir blocked AR nuclear migration and decreased PSA expression whereas inducers phenytoin and rifampicin promoted increased AR nuclear migration and increased PSA production, supporting our hypothesis. Co-immunoprecipitation experiments revealed that CYP3A5 is a part of the HSP90-AR complex. Both Flag-immunoprecipitation and indirect immunoprecipitation revealed that CYP3A5 associates with the HSP90-AR complex. The CYP3A5 siRNA also downregulated 11 genes (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB, and HPRT1) known to confer drug resistance, with a p value of ≤0.005. The role of TOP2A, MYC, and BRCA is well known in prostate cancer drug resistance; thus, their downregulation with CYP3A5 siRNA treatment may predict better outcome. Conclusion: Our results indicate that CYP3A5 is a part of the HSP90-AR complex and positively regulates AR nuclear translocation promoting AR downstream signaling and therapeutic resistance. Therefore, targeting CYP3A5 to block AR nuclear translocation may be a therapeutic adjunctive approach in advanced prostate cancer. As CYP3A5 siRNA also downregulates drug-resistance genes, targeting CYP3A5 may also help overcome therapeutic resistance. As many concomitant medications can alter CYP3A5 activity, thus they can modify AR signaling. We hypothesize that AR directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors. These observations may play a significant role in African Americans who express high level of wild-type CYP3A5 and often have aggressive prostate cancer. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B001.

Abstract CT045: Phase 1, open-label, first-in-human study of AMG 900, an orally administered pan-aurora kinase inhibitor, in adult patients (pts) with advanced solid tumors

Purpose: Aurora kinases are associated with high proliferation, poor prognosis, and therapeutic resistance in several human tumor types. AMG 900 is an investigational, oral, highly potent, selective, pan-aurora kinase inhibitor. We evaluated the safety, tolerability, pharmacokinetics, and clinical activity of AMG 900 in pts with advanced solid tumors (NCT00858377). Methods: In the dose escalation phase, eligible pts were ? 18 years old, with advanced solid tumors that were refractory to standard treatment, measurable disease per RECIST, ECOG ? 2, and life expectancy > 3 months. In the dose expansion phase, eligible pts had taxane- and platinum-resistant epithelial ovarian cancer (OC), taxane-resistant triple-negative breast cancer (TNBC), or castration-resistant and taxane- or cisplatin-etoposide-resistant stage IV prostate cancer (CRPC). AMG 900 was administered 4 days on/10 days off at doses of 1 to 50 mg/day (dose escalation), and at the maximum tolerated dose of 40 mg/day with G-CSF support (dose-expansion). The primary objective was safety. Tumor response was determined per RECIST for all pts and additionally by GCIG CA-125 for pts with OC. Results: Treatment-related AEs were reported by 98 of 105 pts (93.3%). Myelotoxicities were the most common grade ? 3 treatment-related AEs. In the dose escalation (N = 50), 1 pt with OC (30-mg cohort) had a partial response (PR) by RECIST and GCIG. In the dose expansion (N = 55), 3 of 29 pts (10.3%) with OC had a PR by RECIST, and 7 of 29 pts (24.1%) had a PR by GCIG; 72.4% of pts with OC had stable disease (SD), and the disease control rate (PR + SD) was 82.8%. Median (95% CI) duration of response in pts with OC per RECIST was 24.1 (16.1, 34.1) weeks, and median (80% CI) PFS was 31.1 (23.6, 34.1) weeks. See table for additional results. Conclusions: AMG 900 had manageable toxicity with G-CSF support and promising single-agent activity in pts with heavily pretreated taxane- and platinum-resistant OC. Citation Format: Montasser Shaheen, Ben Markman, Michael Carducci, Sara Hurvitz, Daruka Mahadevan, Dusan Kotasek, Oscar Goodman, Erick Gamelin, Vincent Chow, Gloria Juan, Erik Rasmussen, Gregory R. Friberg, Florian D. Vogl, Jayesh Desai. Phase 1, open-label, first-in-human study of AMG 900, an orally administered pan-aurora kinase inhibitor, in adult patients (pts) with advanced solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT045.