D'Andrea, Alan

Abstract DUB-2 is a T-cell specific de-ubiquitinating enzyme whose expression is induced by IL-2. Expression of DUB-2 results in prolonged STAT5 phosphorylation, enhanced IL-2 induced gene expression, and resistance to apoptosis resulting from cytokine withdrawal. The molecular basis of DUB-2s effects, however, is not understood. In order to better understand the role of DUB-2 in the IL-2 response, we performed a systematic analysis of DUB-2 catalytic activity, subcellular localization, and domain organization. Subcellular localization of DUB-2 was examined using a C-terminal DUB-2-Cherry (enhanced red fluorescence) fusion and live-cell imaging. When expressed in HeLa cells, wild-type DUB-2-Cherry is predominantly localized to the cytoplasm, although 8–10% of cells have a strong nuclear accumulation. Strikingly, the nuclear localization is entirely eliminated by a mutation in the catalytic cysteine (c60s). This effect on DUB-2s localization is not due to changes in cell cycle progression, as we observe identical cell cycle profiles (DNA content) in wt or c60s expressing cells. Conversely, enhanced DUB-2 nuclear localization is seen following nocodazole-induced cell cycle synchronization and with mutant DUB-2-Cherry fusions lacking a duplicated 8-residue sequence within the C-terminal domain. We next analyzed the catalytic activity of DUB-2 toward ubiquitin and other ubiquitin-like (UbL) molecules using a series of semi-synthetic activity-based probes. These probes consist of full-length Ub or UbL molecules equipped with a C-terminal electrophilic group. These probes act as suicide substrates, forming a covalent bond with the active site cysteine of catalytically active (but not inactive) Ub(L) isopeptidases. Unexpectedly, DUB-2 forms adducts not only with the Ub-based probe, but also the probe derivative of the UbL molecule ISG15, which suggests that it has dual-specificity. The activity towards Ub and ISG15 is abrogated by a mutation in the catalytic cysteine (c60s) of DUB-2, but is not affected by deletions of the poly-lysine or hypervariable domains. Taken together, these results suggest that DUB-2 is a bi-specific (Ub and ISG15) isopeptidase which shuttles between the cytoplasm and the nucleus in a manner dependent on its catalytic activity. This work, and the ultimate elucidation of the molecular target of DUB-2, will further increase our understanding of DUB-2’s role in IL-2-dependent T-cell proliferation and immunologic tolerance.

Abstract Background: In vivo synergy with concurrent PI3-Kinase inhibition and PARP inhibition has been observed in BRCA-deficient and BRCA-proficient preclinical models of triple negative breast cancer (TNBC) and ovarian cancer (OC). A phase I trial of the oral pan-class I PI3-Kinase inhibitor BKM120 and the PARP inhibitor olaparib demonstrated anti-cancer activity in TNBC and OC, both in patients with and without germline BRCA1 and BRCA2 (BRCA) mutations. However, CNS toxicity (depression) and liver function test abnormalities limited dose escalation of BKM120 prompting evaluation of the alpha specific PI3-Kinase inhibitor BYL719 (which has no CNS toxicity) in combination with olaparib. Methods: Olaparib was administered twice daily (tablet formulation) and BYL719 daily on a 28-day cycle, both orally. A 3 + 3 dose-escalation design was employed with primary objectives of defining the maximum tolerated dose (MTD) and recommended phase 2 dose of the combination of BYL719 and olaparib, and secondary objectives of defining toxicity, activity, and pharmacokinetic profiles of both agents. Eligibility included recurrent TNBC or high grade serous (HGS) OC, or any histology OC or breast cancer (BC) with presence of a known germline BRCA mutation, performance status of 0-1 and measurable/evaluable cancer. Patients with platinum sensitive or resistant or refractory OC were eligible and prior PARP inhibitor use was allowed. Dose-expansion cohorts at the MTD were enrolled for both BC and OC. Results: 46 patients (16 BC and 30 OC) have been enrolled in the study; 28 patients participated in the dose escalation portion of the study (4 BC and 24 OC). Two patients with OC did not receive study drugs because of ineligibility. MTD was defined as BYL719 200mg once daily and olaparib 200mg twice daily. Dose limiting toxicities included hyperglycemia, rash and fever with decreased neutrophil count. Four patients (3 OC and 1 BC) discontinued protocol therapy because of toxicity (2 for hyperglycemia, 1 for nausea and 1 for allergic reaction). Most common toxicities included nausea, hyperglycemia, fatigue, diarrhea and vomiting. At the MTD, 6 patients with OC and 12 patients with BC were enrolled into a dose expansion cohort. The OC expansion cohort has completed enrollment, while the BC cohort is still enrolling. Among patients with OC who received study drugs (28 patients, 26 (93%) with platinum resistant disease), objective response rate (ORR) by RECIST 1.1 was 36% (10/28 patients, all partial responses (PRs)). Median duration of response was 167 days (range 16-398 days); 5 of 10 patients with PR remain on treatment. ORR was 33% for patients with germline BRCA mutations and 31% for patients without germline BRCA mutations. Among patients without germline BRCA mutations with platinum resistant OC, ORR was 29%. Conclusions: Combined BYL719 and olaparib is feasible, and similar clinical benefit was observed in patients with and without germline BRCA mutations. The activity of this combination in OC patients without germline BRCA mutations and with platinum resistant disease was higher than expected from olaparib monotherapy and warrants further investigation. This work was funded in part by the Stand Up To Cancer Ovarian Dream Team. Clinical trial: NCT01623349. Citation Format: Panagiotis A. Konstantinopoulos, William T. Barry, Michael Birrer, Shannon N. Westin, Sarah Farooq, Karen Cadoo, Christin Whalen, Weixiu Luo, Hui Liu, Carol Aghajanian, David B. Solit, Gordon B. Mills, Barry S. Taylor, Helen Won, Michael F. Berger, Sangeetha Palakurthi, Joyce F. Liu, Lew Cantley, Scott H. Kaufmann, Elizabeth M. Swisher, Alan D. D'Andrea, Eric Winer, Gerburg M. Wulf, Ursula A. Matulonis. Phase I study of the alpha specific PI3-Kinase inhibitor BYL719 and the poly (ADP-Ribose) polymerase (PARP) inhibitor olaparib in recurrent ovarian and breast cancer: Analysis of the dose escalation and ovarian cancer expansion cohort [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 CT008. doi:10.1158/1538-7445.AM2017-CT008