Aim:A novel strategy for prostate cancer (PrCa) biomarker discovery is described.Materials & methods:In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens.Results:Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated.Conclusion:These data support the strategy and the potential use of the biomarkers for PrCa.

Tazemetostat (EZM6438) is a potent, orally bioavailable small molecule inhibitor of EZH2, the enzymatic subunit of the polycomb repressive complex 2, which has been approved for treatment of epithelioid sarcoma and relapsed/refractory follicular lymphoma. EZH2 has been shown to play a key role in B-cell maturation and multiple B-cell malignancies are dependent on EZH2 for survival. Mantle cell lymphoma (MCL) is a rare subtype of mature B cell non-Hodgkin lymphoma characterized by the t(11;14)(q13;q32) translocation leading to overexpression of cyclin D1, which plays a significant role in tumor cell proliferation via cell cycle dysregulation, chromosomal instability, and epigenetic regulation. This disease most often presents at an advanced stage and while initial responses occur, most cases relapse to frontline therapy, including Bruton's Tyrosine Kinase (BTK) inhibitors. The rate of intrinsic and acquired resistance to these treatments results in a high unmet medical need. We previously reported that treatment with EZH2 inhibitors, as monotherapy or in combination with BTK inhibitors, elicited in vitro anti-proliferative activity and in vivo tumor growth inhibition in MCL models, demonstrating that EZH2 may be a promising therapeutic target in this indication. To extend our previous in vitro combination studies, we demonstrated that the recently approved BTK inhibitor, zanubrutinib synergized with tazemetostat and demonstrated that tazemetostat resensitized a subset of cell lines intrinsically resistant to BTK inhibitors. In vivo studies confirmed these findings, showing a significant tumor growth delay when tazemetostat was combined with zanubrutinib in the MCL MINO cell line-derived murine xenograft model compared to the single agent treatments. To understand if EZH2 inhibition offered potential therapeutic benefit to the MCL BTK inhibitor-resistant population, we generated in vitro MCL models of acquired resistance to the BTK inhibitors ibrutinib and zanubrutinib. These cell lines retained the in vitro sensitivity to tazemetostat observed in the parental cell line, suggesting a potential therapeutic option for tazemetostat in the setting of acquired resistance to BTK inhibitors. Additionally, we showed that tazemetostat induced anti-proliferative effects ex vivo in samples derived from MCL patients that were relapsed or refractory to one or more current standard of care agents, including the BTK inhibitor ibrutinib. Subsequent studies aim to identify the mechanisms of growth inhibition driven by tazemetostat in MCL models sensitive and resistant to BTK inhibitors. In summary, these data suggest that tazemetostat treatment (alone or in combination with a BTK inhibitor) could be a potential therapeutic option in the treatment of the MCL patient population that is relapsed/refractory to BTK inhibitor therapy. Citation Format: Jeffrey A. Keats, Arleide Lee, Jeremy C. Cunniff, Weiqing Chen, Revonda Mehovic, Vania Estanek, Crag Markwood, Cuyue Tang, Daniel T. Dransfield, Veronica Gibaja, Alejandra Raimondi. EZH2 inhibitor tazemetostat demonstrates activity in preclinical models of Bruton's tyrosine kinase inhibitor-resistant relapsed/refractory mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1161.

Cell-cell communication in the tumor microenvironment drives tumor growth and facilitates neovessel recruitment from the adjacent vasculature. The aim of these experiments were to identify novel cancer derived factors that modulate the vascular endothelial response in a paracrine manner. An array of cancer cell lines from multiple tissue types (Panc1; Hep2G; LnCap; Skmel; Hep3B; PL45; SCC25; CaCo2; PaCa2; SKBR3) were grown under normoxic or hypoxic (1% oxygen) conditions for a total of 20 conditions. After 48 hours, conditioned media was collected and filtered prior to testing on endothelial cells. The effects of these media on endothelial apoptosis, proliferation and tube formation were investigated. In tandem with these endothelial cell fate assays, the proteomic and lipidomic profile of these 20 conditions were profiled and assessed using the Berg Interrogative Biology™ platform. Our results reveal that conditioned media from all cancer cells tested can alter endothelial cell apoptosis, proliferation rate and tube formation in 3D matrigel assays i.e. critical cell fate decisions that enable formation of nascent vessel formation. Interestingly, the predominant effect of tumor-derived conditioned media on endothelial apoptosis was a protective anti-apoptotic effect, with the discernable exception of normoxic Panc1 derived media. An unexpected finding was the general anti-proliferative effects of tumor-derived conditioned media on endothelial cell growth, with the exception of Hep2G, LnCap and PaCa2 cell derived media. Using a media fractionation strategy and the Berg Interrogative Biology™ platform, we identified several endothelial anti-apoptotic candidates from Panc1 cells within a unique molecular weight range. Upon application of ASO gene knockdown in Panc1 cells, these targets were verified for apoptotic and proliferation effects on vascular endothelial cells.Specific factors that determine paracrine cell-cell interaction in the tumor microenvironment have been identified and validated using the Berg Interrogative Biology™ platform. Citation Format: Tony Walshe, Justin J. Bourdelais, Arleide Lee, Rakib Ouro-Djobo, Vivek Vishnudas, Michael Kiebish, Rangaprasad Sarangarajan, Niven R. Narain. Identification and characterization of novel cancer cell derived factors that dictate vascular endothelial cell biology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3463. doi:10.1158/1538-7445.AM2014-3463

This study demonstrates that BPM 31510, a Coenzyme Q10 containing proprietary formulation to influences cellular metabolism that is deregulated in cancers and is now considered to be a “hallmark of cancer”. These studies demonstrate that BPM 31510 alone or in combination with standard of care significantly improved survival of triple negative breast cancer (TNBC) animal model. To further tease out beneficial effects of BPM31510, human breast cancer cells of varying receptor status (SKBR3, MDA-MB231) were subjected to either (a) pretreatment with BPM 31510 (6 h) followed co-incubation with chemotherapeutic agents for 48-72 h or (b) co-treatment with BPM 31510 and chemotherapeutic agents, and cancer cell responses were compared to non-tumorigenic mammary cells (MCF12A). Cellular bioenergetics profiling revealed that BPM 31510 shifted cellular metabolism from glycolysis to mitochondrial metabolism, and this metabolic shift was associated with significant increases in reactive oxygen species (ROS). Both BPM 31510 alone or pretreatment and cotreatment strategies with BPM 31510 plus standard of care resulted in significant decreases in viable breast cancer cells when compared to chemotherapeutic agents; however, minimal effects were observed in MCF12A cells. In contrast, BPM 31510 in combination with chemotherapeutic agents amplified caspase 3 activation and apoptotic cell death, indicating BPM 31510 enhances apoptotic signaling. Taken together, these data demonstrate that BPM 31510 is a novel agent that reengages the cellular metabolic and apoptotic machinery of cancer cells independent of genetic make-up underlying malignancy. In addition, BPM 31510 enhances the cytotoxicity of standard-of-care chemotherapeutic agents in breast cancer cells through regulation of mitochondrial metabolism and oxidative stress. These findings confirm that BPM 31510 is a novel, paradigm shifting agent with multiple utility (as a single agent or in combination) in breast cancer including TNBCs that otherwise have poor prognosis and limited therapeutic options. Citation Format: Niven R. Narain, Anne R. Diers, Tony E. Walshe, Arleide Lee, Rakib Ouro-Djobo, Vivek K. Vishnudas, Ely Benaim, Rangaprasad Sarangarajan. Significant increase in survival of triple negative breast cancer animal model in response to BPM31510 alone or in combination with standard of care: BPM31510 mediated dynamic metabolic (Warburg) shift in breast cancer as potential mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3358. doi:10.1158/1538-7445.AM2014-3358

Despite global advances in cancer detection and treatment in some indications, the early diagnosis and overall survival rate for pancreatic cancer (PanCa) remains dismal. Thus, there is a critical need for novel therapeutics that may combine well with standard-of-care therapy or work through novel mechanisms. Given that most pancreatic tumors exhibit a highly metabolic phenotype, we examined the effects of BPM 31510 employing in vitro and in vivo PanCa models. BPM 31510 is a metabolic-modulating agent that reverses the Warburg effect and is currently in clinical development for solid tumors alone and in combination with chemotherapy. Determination of BPM 31510 IC50 values in vitro demonstrated the PanCa cell lines MIA PaCa-2 and Panc-1 cells were significantly more sensitive to BPM 31510 (IC50 = 137 and 455 μM, respectively) compared to primary fibroblasts (IC50 = 1537 μM). IC50 and IC90 doses of BPM 31510 also decreased the viable cell population while concomitantly increasing Annexin V- and PI-positive populations in both PanCa cell types, indicating BPM 31510 induces programmed cell death. Furthermore, in combination with gemcitabine (0.1-5 μM), BPM 31510 (100 μM) decreased cell viability by more than 75% compared to either treatment alone. In vivo, treatment of MIA PaCa-2 tumor-bearing mice with increasing doses of BPM 31510 (0.5-50 mg/kg IP, 3X/week) significantly improved median survival in a dose-dependent manner, with the highest dose extending median survival by more than 36 days compared to saline control. Moreover, while median survival of MIA PaCa-2 tumor-bearing mice treated with BPM 31510 (50 mg/kg IP, 1X/day) or gemcitabine (150 mg/kg IV, 1X/week, given on cycles, 3 weeks on 1 week) monotherapy was 77 and 63 days, respectively, combination treatment resulted in median survival improvement to 113.5 days. Examination of alternative dosing regimens revealed that more frequent dosing of BPM 31510 (2X or 3X/day) alone and in combination with gemcitabine further extended median survival in this model. The preliminary mechanistic insight into additive efficacy of combination treatment was explored in vitro. BPM 31510 treatment alone significantly altered multiple aspects of mitochondrial function in MIA PaCa-2 cells, indicating that BPM 31510-driven bioenergetic alterations are separate from the effects of gemcitabine. Hence, these data demonstrate that BPM 31510 has a potent anti-cancer activity alone and in combination with standard-of-care chemotherapy in preclinical PanCa models. Citation Format: Tulin Dadali, Anne R. Diers, Arleide Lee, Rakibou Ouro-Djobo, Justin Bourdelais, Ezer Benaim, Bianca Jambhekar, Tony E. Walshe, Joaquin J. Jimenez, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM 31510 enhances efficacy of gemcitabine through orthogonal mechanisms in a preclinical model of pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C117.