Pyruvate kinase is a crucial enzyme responsible for the last step of glycolysis. Cancer cells can use the M2 isoform of pyruvate kinase (PKM2), to better balance respiration and biosynthesis due to allosteric switching between the less active dimeric and fully active tetrameric forms. Additionally, the dimeric form of PKM2 can translocate to the nucleus, altering transcription to enhance cancer cells' ability to grow and evade immune detection. Inducing tetramerization presents an opportunity to target PKM2 resulting in the metabolic reprogramming of tumor-immune microenvironment (TME). TP-1454 is a potent PKM2 activator with low nanomolar PKM2 activation in biochemical assays (AC50 = 10 nM) and multiple cell types (AC < 50 nM), tolerated in mice, rats and dogs after repeat doses as high as 1000 mg/kg/day and has recently entered a Phase I clinical trial (NCT04328740). We hypothesize that PKM2 activation may reverse the immune-suppressive TME. To test this hypothesis, we examined the activity of TP-1454 combination with immunotherapy (I/O) in multiple mouse syngeneic tumor models. TP-1454 and anti-PD-1 combination therapy in colorectal cancer models resulted in tumor growth inhibition versus vehicle (53% in CT26; 99% in MC38, P < 0.001). We observed decreases in multiple glycolytic intermediates in TP-1454-treated tumors versus vehicle. We conducted immunophenotyping of the TME in multiple models to identify targets of PKM2 activation. TP-1454 treatment reduced the CD4+ Foxp3+ T-regulatory (Treg) population in MC38, 4T1, RENCA models. Further, we assayed TP-1454 induced PKM2 activation in different immune cell types. To confirm the effect of PKM2 activation on Treg cells we conducted an in vitro assay to explore TP-1454 treatment response on polarization of Tregs and/or toxicity and proliferation. We further utilized LCMS to explore metabolic intermediates that play a critical role in Treg regulation, including regulation of the O-linked β-N-acetylglucosamine (O-GlcNac) post-translational modification, which is reported to stabilize Foxp3 in CD4+ cells. We are currently exploring the effect of TP-1454 treatment on O-GlcNac of Foxp3 and its stability in HEK293 cells, to support the link between PKM2 activation and stabilization of Foxp3. TP-1454 effects on tumor-specific immunity were validated using tumor rechallenge studies. The results of a tumor rechallenge study will be presented using murine MC38 or RENCA xenograft models that are treated with TP-1454 and I/O combination therapies that exhibited a complete response (CR) and were re-implanted. These preclinical studies indicate a unique mechanism modulating tumor metabolism and the TME to improve the response of cancer patients to immunotherapy. Citation Format: Salah Sommakia, Satya Pathi, Yuta Matsumura, Curtis Allred, Ethika Tyagi, Matthew Lalonde, Jason Foulks, Adam Siddiqui, Clifford Whatcott, David Bearss, Steven Warner. Pkm2 activation modulates the tumor-immune microenvironment and enhances response to checkpoint inhibitors in preclinical solid tumor models [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 606.
Antibodies and peptides directed against the switch regions of RAS can disrupt interactions with its downstream effector proteins, blocking downstream protumorigenic signaling. However, these biologic therapies are unable to reach the intracellular environment. We have overcome this problem using peptidic molecular guidance system (MGSs) that have the unique ability to bind specifically to epithelial-derived cancer cells, and upon binding, trigger rapid internalization and trafficking to a specific subcellular location. Using an unbiased phage display approach, we have identified a series of cancer-specific MGSs that deliver cargo to discrete subcellular locations. Chemical optimization results in MGSs with 1-10 nM affinity for their cellular target, serum stability >48 hours, and 50- to 1000-fold specificity for cancer cells compared to normal control cells. The rate of MGS internalization is rapid with t1/2 of 10-30 minutes, achieving intracellular concentrations up to 1.5 μM. Using these MGSs as delivery agents, we have delivered a function-blocking RAS antibody (Y13-259), resulting in a reduction of p-ERK and apoptosis in KRAS mutant cell lines. The effect is MGS dependent, and confocal microscopy indicates co-localization of KRAS and the antibody indicating target engagement. The MGS-antibody homes to tumors in an animal model, resulting in significant suppression in tumor growth compared to untreated animals. Free MAb does not impact tumor growth. Using MGSs to delivery MAb for intracellular targets is a paradigm change for immunotherapy and represents a new approach for controlling intracellular protein-protein interactions. Combination of these cancer-specific delivery agents with effectors of RAS signaling, such as antibodies and other biologics, has therapeutic potential for KRAS-driven cancers. Citation Format: Michael J. McGuire, Susan Li, Indu Venugopal, Claire Gormley, Kevin J. Luebke, Curtis A. Allred, Kathlynn C. Brown. Cancer-specific intracellular delivery of therapeutic antibodies against KRAS [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B21.
Adult granulosa cell tumor (AGCT) is a subtype of sex-cord stromal tumors and accounts for ~5% of all ovarian neoplasms. Nearly 100% of AGCT cases are caused by an oncogenic point mutation in the Forkhead Box L2 (FOXL2) transcription factor. Weis-Banke et al. found this gain-of-function mutation (FOXL2C134W) allows FOXL2 to hijack the nuclear SMAD2/3/4 complex, the downstream effector of transforming growth factor- ß (TGF-ß), and redirect to novel transcription sites, inducing transcription of epithelial to mesenchymal transition (EMT) and other oncogenes. We hypothesized that FOXL2 mutant AGCT would be sensitive to TGF-ß inhibition. To test this hypothesis, we treated two GCT cell lines with TP-6379, an orally available, investigational small molecule kinase inhibitor of TGFBR1 that has been shown to block the phosphorylation and nuclear translocation of SMAD2 and SMAD3 in cells. TP-6379 was tested in the KGN cell line, derived from an AGCT patient and heterozygous for the FOXL2C134W mutation, and the COV434 cell line, derived from a juvenile GCT patient, which is FOXL2 wild type (WT). KGN cells (IC50 = 135 nM) were observed to be more than 70-fold more sensitive to TP-6379 treatment than COV434 (IC50 = >10,000 nM), after a 6- and 7- day treatment, respectively. KGN cells that were edited to remove the WT FOXL2 or both the WT and FOXL2C134W alleles were observed to be 1.6-fold more sensitive to TP-6379 and 22-fold less sensitive than the parental KGN cells. In vivo testing using KGN cells is ongoing. Viably cryopreserved dissociated tumor cells (DTCs) from two AGCT patients and one JGCT patient, which contain a mixture of tumor, immune, endothelial, and other stromal cells, were also tested in proliferation assays with TP-6379. All three ex vivo samples were positive for the FOXL2C134W mutation as detected by a qPCR genotyping assay and were sensitive to TP-6379 (IC50 = 555-1600 nM) after 6-day treatment. Xenograft models using these patient samples are currently under development. TGF-ß signaling is also a master regulator of the tumor microenvironment (TME) and immune evasion by modulating deposition of extracellular matrix and suppression of immune cells. We performed an immunophenotyping assay in tissue microarrays of thin-needle biopsy cores of multiple cancer types by looking at the distribution of CD8 T cells within tumor and stroma. AGCT showed the strongest excluded and desert phenotype among the tested cancer types, where CD8 T cells were confined to the stroma or absent entirely. TP-6379 treatment was observed to increase expression and reverse TGF-ß induced suppression of HLA class I in KGN cells. These data suggest that TGF-ß may play a significant role in the TME of AGCT. In conclusion, preclinical data shows inhibition of TGFß signaling with TP-6379 in FOXL2C134W mutant AGCT is active at blocking cell growth and may prove to be a potential therapy in this rare disease. Citation Format: Curtis A. Allred, Richard E. Heinz, Yuta Matsumura, Tetyana V. Forostyan, David Kircher, Salah Sommakia, Thomas Welte, Veena Vuttaradhi, Jason M. Foulks, Steven L. Warner, R Tyler Hillman. TGFBR1 as a novel therapeutic target in adult granulosa cell tumors [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 1617.
Antibodies and peptides directed against the switch regions of RAS can disrupt interactions with its downstream effector proteins, blocking downstream pro-tumorigenic signaling. However, these biologic therapies are unable to reach the intracellular environment. We have overcome this problem using peptidic molecular guidance system (MGSs) that have the unique ability to bind specifically to epithelial-derived cancer cells, and upon binding, trigger rapid internalization and trafficking to a specific subcellular location. Using an unbiased phage display approach, we have identified a series of cancer specific MGSs that deliver cargo to discrete subcellular locations. Chemical optimization results in MGSs with 1-10 nM affinity for their cellular target, serum stability >48 hours, and 50-1000-fold specificity for cancer cells compared to normal control cells. The rate of MGS internalization is rapid with t1/2 of 10-30 minutes, achieving intracellular concentrations up to 1.5 µM. Using these MGSs as delivery agents, we have delivered a function blocking RAS antibody (Y13-259), resulting in a reduction of p-ERK and apoptosis in KRAS mutant cell lines. The effect is MGS dependent. The MGS-Antibody homes to tumors in an animal model resulting in significant suppression in tumor growth compared to untreated animals. Free MAb does not impact tumor growth. Using MGSs to delivery MAb for intracellular targets is a paradigm change for immunotherapy and represents a new approach for controlling intracellular protein-protein interactions. Combination of these cancer-specific delivery agents with effectors of RAS signaling, such as antibodies and other biologics, has therapeutic potential for KRAS-driven cancers. Citation Format: Michael J McGuire, Susan Li, Curtis A Allred, Indu Venugopal, Claire Gormley, Kathlynn C Brown. Cancer-specific intracellular delivery of therapeutic antibodies against RAS [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C034. doi:10.1158/1535-7163.TARG-19-C034
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