Trynda, Justyna

Abstract Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of protooncogene cellular myelocytomatosis (c- Myc) and other genes. Because c-Myc cannot be directly targeted by small molecular inhibitors due to disordered alpha helical structure, epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for diseases such as prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. We studied the efficacy of MT1, a novel bivalent BD inhibitor that is 100-fold more potent than the first in class BD inhibitor JQ1, at inhibiting PC growth. We tested the effect on viability by MT-1 on PC cell lines, 3D spheroids derived from clinically annotated drug resistant patient derived xenografts (PDX), mice PDX models and corroborated the molecular mechanism of MT1 down regulation of Myc leading to downstream Myc-dependent up regulation of Protein Kinase D1 (PrKD) substrate phosphorylation by western blot. MT-1 inhibited growth of PC in castration sensitive (LNCaP) and resistant PC cells (PC-3). MT-1 treatment upregulated PrKD expression and phosphorylation of known PrKD substrates: threonine 120 (Thr-120) residues in beta-catenin and the serine 216 in Cell Division Cycle 25 (CDC25C) in PC-3 cells. Moreover, MT-1 was effective in inhibition of 3D spheroids growth at IC 50 between 0.27-0.92µM in Abiraterone, Enzalutamide, Docetaxel, Cabazitaxel metastatic castrate resistant PCa patient-derived tumor 3D spheroids. Additionally, MT1 was effective in inhibiting the tumor growth in PDX mice model. A combined intra-peritoneal administration of MT-1 with another c-Myc inhibitor (3JC48-3), an obligate c-Myc and MYC-associated protein X (MAX) heterodimerization inhibitor, increased the efficacy of inhibiting the PDX growth in mice. This study provides strong pre-clinical in vitro and in vivo evidence for advancing MT- 1 as a novel c-Myc targeting drug in PC. The MT-1 drug development will likely be highly impactful as c-Myc is dysregulated in three fourths of men with advanced PC. Citation Format: Sanjeev Shukla, Mohammed Al-Toubat, Allison H. Feibus, Ahmed Elshafei, Carlos Riveros, Nathalie Meurice, Justyna Gleba, Jonathan Chardon-Robles, Adam M. Kase, John A. Copland III, Joachim L. Petit, Teruko Osumi, K.C. Balaji. Bromodomain inhibitor: MT1 and its potential role in modulation of prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2475.

Abstract Thyroid cancer is the most common endocrine cancer in the US, and its incidence is rising. Most thyroid cancer deaths are attributed to treatment-refractory, metastatic tumors. Thyroid stimulating hormone receptor (TSRH) expression is largely limited to the thyroid gland and is abundantly expressed on thyroid tumor cells, making TSRH a compelling target for advanced thyroid cancer diagnostics and therapeutics. Therefore, we developed a novel TSHR-targeted chimeric antigen receptor (CAR) T cell therapy to treat aggressive thyroid cancers. TSHR-CAR constructs were cloned into a lentiviral CAR construct containing 4-1BB and CD3ζ. First, we demonstrated potent TSHR-CART antigen-specific anti-tumor activity in vitro. Then, NOD-SCID-γ-/- (NSG) mice were inoculated subcutaneously with TSHR+ tumor cells and randomized by tumor volume to treatment with TSHR-CART cells or control Untransduced T cells (UTD). Treatment with TSHR-CART cells resulted in dose-dependent antitumor activity and prolonged survival. De-differentiated anaplastic thyroid cancers (ATC) downregulate TSHR. Our TSHR immunohistochemistry results corroborated these findings and displayed minimal TSHR protein expression, precluding successful TSHR-CART treatment. We therefore sought to sensitize these tumors with MAPK inhibitors, as a strategy to upregulate TSHR expression in patients with metastatic thyroid cancer. TSHR expression was upregulated in patient-derived xenograft (PDX) ATC models after one week of daily administration of the MAPK inhibitors (p=0.0024). After confirming that MAPK inhibition does not dampen TSHR-CART effector functions, we tested sequential and combination therapy of TSHR-CART with MEK and BRAF inhibition in vivo. NSG mice were engrafted with ATC BRAF-mutant PDX tumors and randomized by tumor volume to daily oral treatment with placebo or trametinib (MEK inhibitor) plus dabrafenib (BRAF inhibitor). One week later, mice received either UTD or TSHR-CART. Mice conditioned with trametinib plus dabrafenib (p=0.0018) and subsequently treated with TSHR-CART showed superior antitumor activity. However, the improved antitumor activity in this setting was transient. We therefore tested the durability of TSHR upregulation following MEK/BRAF inhibition and demonstrated that TSHR upregulation lasts less than 48-72 hours after discontinuation. Finally, we tested the combination of TSHR CART cells with MEK/BRAF inhibitors in ATC BRAF-mutant PDX tumors. Here, combining TSHR-CART cells with MEK/BRAF inhibitors result in durable control of the tumors. Collectively, our findings indicate that MEK/BRAF inhibition of de-differentiated thyroid cancers upregulated TSHR expression and enhanced TSHR-CART antitumor activity. This work represents a viable strategy to improve outcomes of patients with aggressive, metastatic thyroid cancers. Citation Format: Claudia Manriquez Roman, Kendall J. Schick, Justyna J. Gleba, Truc N. Huynh, Elizabeth L. Siegler, James L. Miller, Aylin Alasonyalilar Demirer, Matthew L. Pawlush, Ahmet Biligili, Long K. Mai, Erin Tapper, Leo R. Sakemura, Michelle J. Cox, Carli M. Stewart, Ismail Can, Ekene J. Ogbodo, Gaofeng Cui, Georges Mer, Gloria R. Olivier, Yushi Qiu, Robert C. Smallridge, Zubair C. Abba, Han W. Tun, John A. Copland, Saad S. Kenderian. Addition of MAPK inhibitors to prime and sensitize poorly differentiated thyroid cancers as a strategy to improve TSHR-CART cell therapy antitumor activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5074.

Abstract New effective therapies should be developed for the treatment of hepatocellular carcinoma (HCC). One of the very promising strategies is blocking the activity of stearoyl-CoA desaturase 1 (SCD1). SCD1 is the enzyme responsible for de novo fatty acid (FAs) synthesis by converting saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). In our laboratory, we developed a novel, highly specific SCD1inhibitor - SSI-4. We have tested the biological activity of SSI-4 against different HCC cell lines and patient-derived xenografts (PDX) mouse models. Of the twelve tested HCC cell lines, four were highly sensitive to SSI-4 (IC50 1-50 nM). Other cell lines showed moderate or no sensitivity to SSI-4. We conducted analysis of the enzymatic protein levels involved in de novo fatty acid synthesis. Protein expression levels for these enzymes don’t predict response to therapy. To determine whether lipid metabolites predict response to SSI-4 therapy, lipidomic analysis was investigated. Our studies showed thatSSI-4 sensitive cells when exposed to SSI-4 had a higher baseline level of key lipid metabolites such as phospholipids (PL), diglycerides (DAG), diglycerides (TAG), ceramides (CE), and free fatty acids (FFA),compared to insensitive cells. We are currently validating these results in our in vivo models at the tumor tissue and blood levels. Additionally, mechanistic studies are in progress to determine if SREBP1c, an AKT regulated transcription factor known to regulate each of the fatty acid synthetic enzymes plays a role in HCC fatty acid synthesis and sensitivity to SCD1 inhibition. Citation Format: Justyna Joanna Gleba. Lipid metabolites as potential diagnostic biomarkers for stearoyl-CoA desaturase 1 targeting therapy in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1849.

176 Background: Despite androgen deprivation therapy, prostate cancer will develop resistance known as castrate resistant prostate cancer (CRPC). Androgen signaling is associated with pathways that influence cell proliferation and survival via cell cycle regulation. AR signaling results in inactivation of Rb tumor suppressor by way of AKT and reduction in intrinsic CDK4/6 inhibitors. Since PTEN loss is found in 40% of mCRPC and AKT pathway is a mechanism of CDK4/6 inhibitor resistance, we hypothesize adding an AKT inhibitor to a CDK4/6 inhibitor will have synergistic activity while delaying CDK4/6 inhibitor resistance. Preclinical xenografts have been shown to recapitulate patient response, therefore, can be used to guide clinical trials using novel combinations. Methods: We have established patient derived tumor xenograft models (GUR-017M, TMA-027 and PRJ-88T) from patients with mCRPC with each having a unique genomic alteration profile. In the clinical setting, GUR-017M was resistant to abiraterone, enzalutamide, cabazitaxel and had exposure to docetaxel. TMA-027 and PRJ-88T were resistant to enzalutamide. We combined a CDK4/6 inhibitor with an AKT inhibitor and treated each of these patient tumors in NOD-SCID mice for a total duration of 40 days. Mice were implanted and separated into 4 groups (placebo, CDK4/6 inhibitor, AKT inhibitor and CDK4/6 + AKT inhibitor) with 10-13 mice in each group. Tumor volumes were measured twice a week. For GUR-017M, abemaciclib (50 mg/kg) was combined with MK2206 (120 mg/kg) and was compared to each placebo and each single agent alone. For TMA-027, palbociclib (50 mg/kg) was combined with ipatasertib (50 mg/kg). After 40 days, tumors were harvested for immunohistochemistry analysis. PRJ-88T was grown as 3D spheroids in ultra-low attachment plates and treated with palbociclib + ipatasertib (10 uM-100uM) to evaluate for synergy. Results: GUR-017M and TMA-027 showed significant delayed tumor growth compared to placebo or either single agent alone. In the GUR-017M group, the final mean tumor volume for the combination group was 149 mm3, compared to 377 mm3 (abemaciclib), 358 mm3 (MK2206) and 527 mm3 placebo group, representing a percent decrease compared to control by 72%, 29%, and 32%, respectively. For TMA-027, the final mean tumor volume for the combination group was 102 mm3, compared to 259 mm3 (palbociclib), 416 mm3 (ipatasertib), and 402 mm3 placebo group, representing a percent decrease compared to control by 75%, 36%, and 4%, respectively. PRJ-88T 3D spheroids showed synergy with palbociclib + ipatasertib. In vivo experiments using PRJ-88T are in progress. Conclusions: The preclinical results show combining CDK4/6 inhibitors with AKT inhibitors is a promising treatment strategy and provides the basis for a clinical trial in patients with mCRPC.

Abstract The increase in fatty acid metabolism is a hallmark of cancer. Stearoyl CoA desaturase (SCD) is an endoplasmic reticulum (ER) enzyme that catalyzes D9 cis désaturation of fatty acyl-CoA substrates. One of its isoforms, SCD1, is overexpressed in several cancers including breast cancer. The higher expression of SCD1 in breast cancer patients correlates with significantly shorter relapse-free and overall survival rates, indicating that SCD1 may be a therapeutic target. We have previously developed and published a novel SCD1 inhibitor, SSI4. Metabolic reprogramming facilitates tumor growth by promoting immune suppression in tumors. Fundamental metabolic processes like fatty acid (FA) metabolism are involved in T cell activation and differentiation and their modulation can differentially affect the development of T helper cell lineages. FA synthesis and oxidation are essential for the development and functioning of CD8+ T memory cells and are contributing factors that influence CD4+ T effector and Treg cell development. These associations between metabolism and immune cells lead us to our hypothesis that aberrant de novo lipogenesis is linked to immune suppression. Thus, SCD1 inhibitors should increase anti-tumor immunity. We evaluated the effect of SSI4-mediated SCD1 inhibition in two mouse triple negative breast cancer cell lines. The data showed that SSI4 stimulates calreticulin (CRT) translocation to cell membrane. CRT is an ER resident protein that translocates to the cell surface under ER stress conditions. Cell surface expression of CRT is known to induce immune responses including phagocytosis, translocation of cancer antigen to the cell surface, and apoptosis. We are currently studying the effect of CRT translocation in our model. A major focus of our project is to assess the efficacy of combination treatment of SSI4 and an immune checkpoint blockade. To this end, this combination therapy synergistically blocked E0771 tumor growth in vivo. Further, to understand the immune responses generated, we performed immunohistochemical analysis of the tumors (day 20 post-treatment) indicating that the combination therapy resulted in higher membrane localized CRT, supporting our rationale that CRT translocation is involved in mediating the anti-tumor responses. Flow cytometry analysis indicated that the combination treatment increased immune cell infiltration. Specifically, SSI4 treatment induced infiltration of double negative T cells, natural killer cells and macrophages, suggesting that it promoted a pro-inflammatory immune response. SSI4 treatment down regulated immune suppressive cells including Tregs, dendritic cells and mesenchymal derived stem cells. The response of cancers with low and intermediate tumor mutational burden such as breast cancer to immune checkpoint inhibition is limited. Our data indicates that combination treatment of SSI4 and anti PD1 is a promising therapeutic strategy. Citation Format: Sneha Vivekanandhan, Justyna Trynda, Laura A. Marlow, Barath shreeder, James L. Miller, Adam M. Kase, Winston Tan, Keith L. Knutson, John A. Copland. SCD-1 blockade sensitizes triple negative breast cancer to immune checkpoint inhibition [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 1484.