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SUMOylation has been implicated in many cellular processes that are important for cancer cell survival, including cell cycle, chromosome structure and segregation, nuclear and subnuclear organization, transcription and DNA damage repair. However, a potent and selective inhibitor to target the SUMO pathway has been lacking. The SUMO-activating enzyme (SAE) is an essential enzyme in the pathway that initiates the SUMOylation process. Here we report the identification of the first mechanism-based SAE inhibitors with nanomolar potency in cellular assays. These inhibitors selectively block SAE enzyme activity and total SUMOylation in cells, which leads to reduced cancer cell proliferation. Moreover, SAE inhibition resulted in disruption of PML nuclear bodies and redistribution of DAXX. In vivo administration of SAE inhibitor into tumor bearing mice results in modulation of several biomarkers including a significant reduction in SUMO-conjugates and E2 Ubc9 thioesters, demonstrating SUMO pathway inhibition. Our results demonstrate the feasibility of inhibiting the SUMO pathway with small molecule inhibitors and provide tools to study the SUMO biology in cancer. Citation Format: Sai M Pulukuri, Xingyue He, Stephen Grossman, Jessica Riceberg, Erik Koenig, Teresa Soucy, Keli Song, Anna Kreshock, Dylan England, Hirotake Mizutani, Larry Dick, James Brownell, John Newcomb, Steve Langston, Eric Lightcap, Katherine Galvin. Probing the roles of SUMOylation in cancer cell biology using a selective SUMO activating enzyme inhibitor [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 1147. doi:10.1158/1538-7445.AM2017-1147

Background: The ubiquitin-proteasome system (UPS) has been validated as a target in multiple myeloma (MM) through the success of proteasome inhibitors such as bortezomib, but drug resistance is an emerging challenge. Targeting some of the upstream components of the UPS, such as the E1 ubiquitin activating enzyme (UAE), could therefore be a promising alternative. TAK-243 (MLN7243) specifically blocks the ubiquitin conjugation cascade through the formation of a TAK-243-ubiquitin adduct, thereby inhibiting the UAE. Our aim was to explore the effectiveness of TAK-243 against pre-clinical myeloma models, and to understand some its mechanisms of action. Methods: We performed pre-clinical studies in myeloma cell lines and mouse models using TAK-243. Downstream effects were evaluated using viability, apoptosis assays, western blotting, gene expression profiling (GEP), and Reverse Phase Protein Array (RPPA) techniques. Results: MM1.S and MOLP-8 TP53 wild-type cell lines were sensitive to TAK-243, with median inhibitory concentrations (IC50) of 25 nM at 24 hours based on viability assays. In otherwise isogenic cell lines in which TP53 was suppressed using genome editing techniques, the IC50 was ~40 nM, but higher TAK-243 concentrations of 100 nM overcame resistance due to TP53 inactivation. Similarly, TAK-243 was able to overcome resistance to both conventional (dexamethasone) and novel (bortezomib, lenalidomide) drugs in paired sensitive and resistant cell line models. After treatment with TAK-243, Annexin V and TO-PRO3 staining determined that viable MM1.S cells were induced into early or late apoptosis. This was accompanied by a significant increase in cleaved caspase-3, -8, and -9 as detected by flow cytometry, and in cleaved caspase-7 detected by RPPA and western blot. Exposure to TAK-243 reduced the cellular content of ubiquitin-protein conjugates, and did not enhance expression levels of a fusion protein degraded by the proteasome in a ubiquitin-independent manner, indicating the lack of direct proteasome inhibition. GEP analysis and RPPA detected enhanced expression of p53-pathway related proteins, including MDM2, TP53, and p21 in TAK-243 treated MM1.S cells. Several mRNAs and proteins in the ER stress pathway, including ATF6, ATF4, IRE1a and XBP1 were also elevated, as were many non-coding RNAs and DNA-damage related genes. Combination experiments in MM cell lines demonstrated synergy between TAK-243 and lenalidomide, pomalidomide, panobinostat, melphalan and doxorubicin. Finally, TAK-243 demonstrated in vivo antitumor activity against MM1.S and MOLP-8 xenograft models when dosed at 12.5 mg/kg IV twice-weekly for 2 weeks (tumor growth inhibition of 60% and 73%, respectively). Elevation of BiP, ATF4, XBP1s and cleaved-caspase 3 was detected within the first 24 hrs after dosing in the sensitive MM1.S xenografts. In contrast, RPMI 8226 cells, which showed a 2000 nM IC50 in cell culture, were also resistant to TAK-243 in vivo, with no tumor growth inhibition detected. Conclusions: TAK-243 is a UAE inhibitor that is active against myeloma cells in vitro and in xenograft models in vivo, overcomes conventional and novel drug resistance, and its action is associated with stimulation of the TP53 and ER stress pathways. Thus, it may deserve further evaluation as an anti-myeloma agent. Disclosures Berger: Takeda Pharmaceuticals: Employment. Hyer:Takeda Pharmaceuticals: Employment. Chattopadhyay:Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Shi:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Shinde:Takeda Pharmaceuticals: Employment. Kreshock:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Menon:Takeda Pharmaceuticals: Employment. Orlowski:Takeda Pharmaceuticals: Research Funding.

Background: Immunohistochemistry provides visual context to protein modulation and is relied upon to elucidate mechanism of action, marker translocation and pharmacodynamics (PD) in drug discovery. Markers of the PI3K-MTOR pathway have been monitored in this way to aid in the clinical development of various inhibitors targeting this pathway. Here, assays were developed using preclinical xenograft PD studies to explore the feasibility of automated staining of pAKT 473, pS6, p4EBP1 and pRb. In preparation for clinical assessment of these markers in human skin samples; these markers were also examined in normal mouse skin. p4EBP1 was selected to examine the reproducibility of these automated assays and to explore the correlation between pathway inhibition in PD xenograft tissues and murine skin tissues. The automated staining technique was coupled with whole slide scanning and digital image analysis for quantitative comparison. Methods: Assays were chosen for evaluation based on known biology. FFPE PTEN-null breast cancer primary human tumor xenografts with high, medium and low biomarker expression, with companion skin samples, were selected for feasibility and reproducibility testing. Whole slide scanning and reproducibility analysis was executed using Aperio Scanscope XT and Spectrum software. Statistical analysis methods of mixed effect linear models and two sample t-tests were applied. Xenograft and skin samples from NCI-H1048 tumor bearing Nude mice treated with MTOR (MLN0128)and PI3Kα (MLN1117) investigational inhibitors were immunohistochemically stained for p4EBP1. Definiens object-based imaging software was used to isolate and analyze viable tumor area and exclude necrotic and stromal components in xenografts. A customized Definiens® algorithm was used on skin to identify the epithelial layer for analysis. Results: After evaluation, baseline biomarker expression in the xenograft and skin samples determined marker selection for future steps. Baseline p4EBP1 levels were high in both xenograft and skin. The reproducibility testing yielded consistent results in both xenograft and skin, validating autostaining use for PD studies. Phosphomarker p4EBP1 expression was inhibited after MLN0128 administration, with increased reduction after combination with MLN1117. Development of a novel analysis solution using Definiens software for skin samples permitted the area of interest to be restricted to the epithelial layer and excluded other epidermal features with similar morphometric characteristics. The p4EBP1 modulation was concordant between skin and tumor though more pronounced in skin, indicating that skin is a suitable surrogate tissue for determining modulation of this PI3K-MTOR pathway marker. Conclusions: Autostaining platforms provide reproducible results and stain consistently for PI3K-MTOR phosphomarkers on both xenografts and mouse skin samples. Utilizing p4EBP1, target effect was observed after MTOR and PI3Kα administration. This development and evaluation of the phosphobiomarker using automated staining technology and advanced image analysis supports preclinical PD assessment and combination clinical trials as well as influenced timepoint selection for clinical assessment. Citation Format: Anna Kreshock, Natalia Iartchouk, Yueying Cao, Jeffrey Szwaya, Feng Gao, Christopher Simpson, Evan Luongo, Kristine Burke, Esha Gangolli, Keisuke Kuida, Rachael Brake, Stephen Tirrell, Vaishali Shinde. Automated immunohistochemistry of phosphobiomarkers: Case study of MTOR (MLN0128) and PI3Kα (MLN1117) investigational inhibitors, single agent and in combination, on xenografts and mouse skin. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A41.