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Transforming growth factor-beta (TGF-ß) is a master regulatory cytokine with pleotropic effects important in tumor development. TGF-ß1 binds TGF-ß receptor 2, which recruits and phosphorylates TGF-ß receptor 1 (TGFBR1). TGFBR1 in turn phosphorylates SMAD2 and SMAD3. Phosphorylated SMAD2/3 (pSMAD2/3) complexes with SMAD4 to translocate to the nucleus and enact transcriptional programs. In late-stage tumors, TGF-ß signaling is oncogenic, promoting growth, invasion, immune suppression, tissue remodeling, and epithelial to mesenchymal transition (EMT). In an effort to counter the oncogenic effects of TGF-ß signaling, Sumitomo Pharma Oncology, Inc. is developing TP-6379, an investigational, orally available, small molecule, selective TGFBR1 inhibitor. TP-6379 has a biochemical IC50 of 1.07 nM and has been shown to reduce pSMAD2 in Rhabdomyosarcoma cells with an IC50 of 108 nM. A dose dependent increase of TP-6379 was observed in plasma and tumors of A549 tumor bearing athymic nude mice (10, 50, 75, and 100 mg/kg PO). In another study using the same mouse model, pSMAD2 has shown an inverse correlation with pharmacokinetics showing > 90% inhibition for up to 4 hours (75 mg/kg PO). Pharmacodynamic (PD) biomarkers are critical for determining biological activity, but procuring serial tumor biopsies for PD analysis is challenging for many tumor types. We hypothesize that pSMAD2 can be measured in non-tumor tissues as a surrogate for activity within the tumor. Surrogate tissues, such as peripheral blood mononuclear cells (PBMCs), skin punches, and even circulating tumor cells (CTCs) are much less invasive and can be sampled over multiple timepoints. We observed a measurable decrease of PD biomarkers pSMAD2/3 in PBMCs and skin after treatment with TP-6379 using a bead-based immunoassay (Luminex). Healthy human donor PBMCs treated ex vivo for 2 hours with up to 1 µM TP-6379 showed a 96% reduction in pSMAD2/3 signal. Although TP-6379 was still active in reducing pSMAD2 in PBMCs when treated in whole blood for 2 hours, it required around 30-fold more drug to achieve a similar effect, possibly due to serum protein binding. Skin samples from in vivo dosed tumor bearing mice also show significant inhibition of pSMAD2/3, which correlated with significant pSMAD2/3 inhibition in tumors from both EMT6 and 4T1 syngeneic triple-negative breast cancer mouse tumor models. We confirmed that the Luminex assay is sensitive enough to reliably measure pSMAD2/3 in healthy human doner skin punches down to 3 mm in diameter. Lastly, CTCs from breast cancer patients’ whole blood treated ex vivo for 24 hours with as low as 1 µM TP-6379 showed a decrease in pSMAD2 and EMT marker SNAI1 by as much as 96% and 86%, respectively, when measured by immunofluorescence. In summary, our research has shown that pSMAD2/3 and EMT marker SNAI1 are valid PD biomarkers for TP-6379 therapy. We propose that these biomarkers would be best measured clinically using PBMCs, CTCs and/or skin punches. Citation Format: Richard E. Heinz, Curtis A. Allred, David A. Kircher, Yuta Matsumura, Bettina Franz, Zhizhou Ye, Jinny Lee, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui, Steven L. Warner. Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology [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 2799.

Oncogenic Ras upregulates aerobic glycolysis to meet the bioenergetic and biosynthetic demands of rapidly growing cells. In contrast, Thioredoxin interacting protein (TXNIP) is a potent inhibitor of glucose uptake and is frequently downregulated in human cancers. Our lab previously discovered that Ras activation suppresses TXNIP transcription and translation. In this report, we developed a system to study how Ras affects TXNIP translation in the absence of transcriptional affects. We show that whereas Ras drives a global increase in protein translation, it suppresses TXNIP protein synthesis by reducing the rate at which ribosomes transit the coding region of TXNIP mRNA. To investigate the underlying mechanism(s), we randomized or optimized the codons in the TXNIP message without altering the TXNIP primary amino acid sequence. Translation from these mRNA variants is still repressed by Ras, intimating that mRNA secondary structure, miRNAs, RNA binding proteins, or codon usage do not contribute to the blockade of TXNIP synthesis. Rather, we show that the N-terminus of the growing TXNIP polypeptide is the target for Ras-dependent translational repression. Our work demonstrates how Ras suppresses TXNIP translation elongation in the face of a global upregulation of protein synthesis and provides new insight into Ras-dependent metabolic reprogramming.

MondoA and its transcriptional target thioredoxin-interacting protein (TXNIP) constitute a regulatory loop that senses glycolytic flux and controls glucose availability. Cellular stress also triggers MondoA activity and TXNIP expression. To understand how MondoA integrates glucose and stress signals, we studied its activation by acidosis. We found that acidosis drives mitochondrial ATP (mtATP) synthesis. The subsequent export of mtATP from mitochondria via adenine-nucleotide transporter and voltage-dependent anion channel, and the enzymatic activity of mitochondria-bound hexokinase results in the production of glucose-6-phosphate (G6P), a known activator of MondoA transcriptional activity. MondoA localizes to the outer-mitochondrial membrane (OMM), and in response to G6P, shuttles to the nucleus and activates transcription. Our data suggests that MondoA is a required feature of a glucose- and mtATP-dependent, OMM-localized signaling center. We propose MondoA functions as a coincidence detector and its ability to sense glucose and cellular stress is coupled to the concerted production of G6P.

Human MondoA requires glucose as well as other modulatory signals to function in transcription. One such signal is acidosis, which increases MondoA activity and also drives a protective gene signature in breast cancer. How low pH controls MondoA transcriptional activity is unknown. We found that low pH medium increases mitochondrial ATP (mtATP), which is subsequently exported from the mitochondrial matrix. Mitochondria-bound hexokinase transfers a phosphate from mtATP to cytoplasmic glucose to generate glucose-6-phosphate (G6P), which is an established MondoA activator. The outer mitochondrial membrane localization of MondoA suggests that it is positioned to coordinate the adaptive transcriptional response to a cell’s most abundant energy sources, cytoplasmic glucose and mtATP. In response to acidosis, MondoA shows preferential binding to just two targets, TXNIP and its paralog ARRDC4. Because these transcriptional targets are suppressors of glucose uptake, we propose that MondoA is critical for restoring metabolic homeostasis in response to high energy charge.

Dynamics of reclaimed coastal wetland soils under cultivation has not been well understood, especially at temporal scales longer than a century. In this study, we analyzed major chemical properties of plow-layer soils extensively sampled under two cropping systems (paddy rice vs. upland cropping) along a 500-year soil chronosequence created by intermittent reclamation of coastal salt marshes. The results suggested a rapid desalinization of soil immediately after reclamation. During 500 years of cultivation, the decalcification process lowered soil pH from >8 to nearly neutral. Soil organic carbon (SOC) contents markedly declined in the initial 16 years, but then rapidly recovered within 30 years and thereafter slowly accumulated with cultivation duration. Meanwhile, the recalcitrance of SOC increased. Soil nutrient status was enhanced after centuries of cultivation as indicated by the improved total nitrogen (TN) and phosphorous (TP). Amorphous Fe oxyhydrates progressively decreased, but the crystallinity of Fe oxyhydrates increased with cultivation time. Cropping system greatly affected plow-layer soil properties, with paddy soils having higher SOC, MBC, NH 4 OAc-extractable Ca 2+ , but lower TP, NH 4 OAc-extractable K + , potentially mineralizable nitrogen and Fe crystallinity than upland soils. Most soil properties revealed clear temporal patterns with more remarkable changes occurring in the first several decades after reclamation than in the following centuries. In conclusion, there was a transition phase of the soil system within the first several decades after reclamation of coastal wetlands, possibly harmful to agricultural production, but centuries of cultivation seemed to have significantly improved overall soil fertility.

Cancer cells reprogram their metabolism toward aerobic glycolysis, which produces lactate to generate an acidic microenvironment. Acidosis-driven activation of the MondoA transcription factor results in its activation of thioredoxin-interacting protein (TXNIP), which is a potent repressor of aerobic glycolysis. We propose a checkpoint function for the MondoA–TXNIP axis that normalizes glycolytic flux in response to a low pH microenvironment.

Background: Mammalian cell growth and proliferation is tightly controlled by a coordinated response to nutrient, growth factor, and intracellular energy status. AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are prototypical metabolic or stress “sensors” that regulate cellular homeostasis in response to the changing environment. Our lab recently defined the MondoA-TXNIP axis as a metabolic checkpoint that responds to the hypermetabolic state common to transformed cells. MondoA is a basic helix loop helix leucine zipper transcription factor. TXNIP is a potent negative regulator of glucose uptake and a direct transcriptional target of MondoA. Upon glucose stimulation, the glycolytic intermediate glucose-6-phosphate (G6P) drives nuclear accumulation of MondoA where it binds the TXNIP promoter and activates its expression. We propose that MondoA's G6P-dependent induction of TXNIP constitutes a negative feedback mechanism that restores glucose homeostasis in response to high glycolytic flux. The MondoA-TXNIP checkpoint also acts as a sensor in response to other metabolic stresses such as lactic acidosis (LA), a characteristic feature of the tumor microenvironment. LA-induced TXNIP expression restricts glucose uptake and aerobic glycolysis. Underscoring the clinical significance of this finding, TXNIP is a critical component of a LA-induced gene signature that correlates with good clinical outcome in breast cancer. Like many metabolic checkpoints, the MondoA-TXNIP checkpoint is inactivated by common oncogenic lesions such as c-Myc, PI3K and activated Ras. Based on these findings, we propose that oncogene inactivation of the MondoA-TXNIP checkpoint is a common route to aerobic glycolysis, which is required for tumor progression. Results: In this study, we investigated the mechanisms by which the MondoA-TXNIP checkpoint is activated by metabolic stress and suppressed by oncogenic signaling. We used Hank's balanced salt solution (HBSS) as a TXNIP inducing stress condition that recapitulates characteristics of the tumor microenvironment, e.g. low pH, low growth factors and nutrients. We used constitutively active RasG12V to inactivate the MondoA-TXNIP checkpoint. Our results demonstrated that HBSS induced TXNIP transcription (primarily) and translation. The low pH of HBSS was responsible for the transcriptional upregulation through increased MondoA occupancy at the TXNIP promoter. The highly-conserved histidines at MondoA N-terminus acted as pH sensors to regulate MondoA nuclear accumulation and DNA binding. We mapped the secondary translational component of HBSS induction to the 5' and 3' untranslated regions of the TXNIP mRNA. Serum stimulation and RasG12V suppressed TXNIP induction by HBSS. The PI3K-AKT and Ras-MEK-p42/44MAPK pathways were responsible for the transcriptional and translational downregulation, respectively. The TXNIP coding region was sufficient for translational repression and we showed that growth factor signaling stalled elongating ribosomes on the TXNIP message. Combined with the very short half-live of the TXNIP protein, these regulatory mechanisms ensure a rapid metabolic response to extra and intracellular growth signals. Conclusions: Our studies establish that a mimic of the nutrient-poor microenvironment, i.e. HBSS, triggers the MondoA-TXNIP checkpoint in a pH-dependent manner with highly conserved histidines playing a critical role. Growth factor signaling pathways restrict transcription of the TXNIP gene (PI3K) and translation of TXNIP mRNA (Ras-MAPK). Given the prevalence of PI3K and Ras pathway dysregulation in cancer, our findings suggest that their repression of the MondoA-TXNIP checkpoint restricts the normal homeostatic response to microenvironmental signals. Citation Format: Zhizhou Ye. MondoA-TXNIP acts as a metabolic checkpoint for coordinated cell growth. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A49.