Blocked Tumor Growth Research Articles

Matrine inhibits cell proliferation, invasion, and stem cell formation in hepatocellular carcinoma by regulating the ELAVL1/RBM3‐mediated Wnt/β‐catenin signaling pathway

AbstractMatrine has been reported to exert anti‐tumor effects in various cancers, including hepatocellular carcinoma (HCC). Nevertheless, the anti‐tumor molecular mechanism of Martine in HCC is still not fully revealed. Cell proliferative ability, apoptosis, invasion, and sphere formation ability were detected using 3‐(4, 5‐dimethyl‐2‐thiazolyl)‐2, 5‐diphenyl‐2‐H‐tetrazolium bromide (MTT), 5‐ethynyl‐2′‐deoxyuridine (EdU), flow cytometry, transwell, and sphere formation assays. CyclinD1, MMP2, RNA‐binding motif protein 3 (RBM3), embryonic lethal abnormal visual‐like protein 1 (ELAVL1), Wnt3A, and β‐catenin protein levels were assessed using western blot. RBM3 and ELAVL1 expression was measured using real‐time quantitative polymerase chain reaction (RT‐qPCR). After being predicted using Starbase, the possible interaction between ELAVL1 and RBM3 was identified using Co‐Immunoprecipitation (IP) assays. A xenograft model was used to examine how Matrine affects HCC cell growth in vivo. Matrine hindered HCC cell proliferation, invasion, and sphere formation ability, and boosted apoptosis in vitro. ELAVL1 and RBM3 content was decreased in Matrine‐treated HCC cells in a concentration‐dependent manner and was increased in HCC tissues and cell lines. Moreover, ELAVL1 might bind with RBM3 to positively regulate the stability of RBM3 mRNA. Matrine modulated the Wnt/β‐catenin signaling pathway by ELAVL1/RBM3 axis. Matrine blocked tumor growth in xenograft modes in vivo. Matrine suppressed HCC cell progression partly by regulating the ELAVL1/RBM3, providing the possible therapeutic strategy for HCC treatment.

Progress of non-small-cell lung cancer with ROS1 rearrangement.

ROS1 rearrangement is found in 0.9%-2.6% of people with non-small-cell lung cancers (NSCLCs). Tyrosine kinase inhibitors (TKIs) target ROS1 and can block tumor growth and provide clinical benefits to patients. This review summarizes the current knowledge on ROS1 rearrangements in NSCLCs, including the mechanisms of ROS1 oncogenicity, epidemiology of ROS1-positive tumors, methods for detecting rearrangements, molecular characteristics, therapeutic agents, and mechanisms of drug resistance.

Abstract A178: Therapeutic potential of a p14ARF minimal domain peptide in non-small cell lung cancer

Abstract Many current therapeutic efforts in cancer biology focus on inhibiting the function of aberrant oncogenes whose activities are known to increase proliferation and cell growth. Unfortunately, these efforts have had limited success in lung cancer, where the five-year survival rate remains unchanged. We sought a novel approach; rather than inhibiting oncogenes, we worked to reestablish the function of the p14ARF tumor suppressor in non-small cell lung cancer (NSCLC) to block tumor growth and proliferation. The p14ARF tumor suppressor is encoded by the CDKN2A gene and is the second most commonly mutated gene in NSCLC. p14ARF is known to play a fundamental role in preventing cancer progression by regulating senescence, apoptosis, and proliferation. Here, to determine the minimal functional domain of p14ARF, we generated a panel of p14ARF expression mutants, deleting various amino acids within the known functional and conserved domains of p14ARF. These mutants were then evaluated for their ability to function as wild type p14ARF. Through proliferative and transformative studies, we identified a 10-amino acid domain that is required for p14ARF function. New cell-penetrating peptide technology was employed to determine if these resides were sufficient to elicit antiproliferative effects on their own. We now show that this small region of ARF is capable of entering tumor cells when linked to a cell penetrating peptide. Moreover, this small cell penetrating ARF peptide retains all of ARF’s known tumor suppressor functions. Our findings provide evidence that small cell penetrating peptides are capable of inhibiting tumor cell growth and proliferation. These experiments also lay the foundation for more precise therapeutic peptide cancer treatments, ones that could be used to restore p14ARF tumor suppressor function. Citation Format: Anna L Grobelny, Leonard B Maggi, Jason D Weber. Therapeutic potential of a p14ARF minimal domain peptide in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A178.

A ‘golden’ alternative for prevention of cisplatin nephrotoxicity in bladder cancer

Cisplatin (CP) is the first-line standard of care for bladder cancer. However, a significant percentage of advanced bladder cancer patients are ineligible to receive standard CP treatment, due to the drug’s toxicity, and in particular its nephrotoxicity. These patients currently face suboptimal therapeutic options with lower efficacy. To overcome this limitation, here we designed CP-conjugated gold nanoparticles (GNPs) with specific properties that prevent renal toxicity, and concurrently preserve the therapeutic efficacy of CP. Safety and efficacy of the particles were studied in bladder tumor-bearing mice, using clinically-relevant fractionated or non-fractionated dosing regimens. A non-fractionated high dose of CP-GNP showed long-term intratumoral accumulation, blocked tumor growth, and nullified the lethal effect of CP. Treatment with fractionated lower doses of CP-GNP was also superior to an equivalent treatment with free CP, demonstrating both anti-tumor efficacy and prolonged mouse survival. Moreover, as opposed to free drug, CP-conjugated GNPs did not cause fibrosis or necrosis in kidney. These results indicate that conjugating CP to GNPs can serve as an effective, combined anti-cancer and renoprotective approach, and thus has potential to widen the range of patients eligible for CP-based therapy.

Inhibition of FLT3-ITD Kinase in Acute Myeloid Leukemia by New Imidazo[1,2-b]pyridazine Derivatives Identified by Scaffold Hopping.

FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.

Extracellular Matrix Collagen I Differentially Regulates the Metabolic Plasticity of Pancreatic Ductal Adenocarcinoma Parenchymal Cell and Cancer Stem Cell.

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 10 percent largely due to the intense fibrotic desmoplastic reaction, characterized by high levels of extracellular matrix (ECM) collagen I that constitutes a niche for a subset of cancer cells, the cancer stem cells (CSCs). Cancer cells undergo a complex metabolic adaptation characterized by changes in metabolic pathways and biosynthetic processes. The use of the 3D organotypic model in this study allowed us to manipulate the ECM constituents and mimic the progression of PDAC from an early tumor to an ever more advanced tumor stage. To understand the role of desmoplasia on the metabolism of PDAC parenchymal (CPC) and CSC populations, we studied their basic metabolic parameters in organotypic cultures of increasing collagen content to mimic in vivo conditions. We further measured the ability of the bioenergetic modulators (BMs), 2-deoxyglucose, dichloroacetate and phenformin, to modify their metabolic dependence and the therapeutic activity of paclitaxel albumin nanoparticles (NAB-PTX). While all the BMs decreased cell viability and increased cell death in all ECM types, a distinct, collagen I-dependent profile was observed in CSCs. As ECM collagen I content increased (e.g., more aggressive conditions), the CSCs switched from glucose to mostly glutamine metabolism. All three BMs synergistically potentiated the cytotoxicity of NAB-PTX in both cell lines, which, in CSCs, was collagen I-dependent and the strongest when treated with phenformin + NAB-PTX. Metabolic disruption in PDAC can be useful both as monotherapy or combined with conventional drugs to more efficiently block tumor growth.

Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

The synergistic effect of alcohol and HCV mediated through TLR4 signaling transactivates NANOG, a pluripotency transcription factor important for the stemness of tumor-initiating stem-like cells (TICs). NANOG together with the PRC2 complex suppresses expression of oxidative phosphorylation (OXPHOS) genes to generate TICs. The phosphodegron sequence PEST domain of NANOG binds EED to stabilize NANOG protein by blocking E3 ligase recruitment and proteasome-dependent degradation, while the tryptophan-rich domain of NANOG binds EZH2 and SUZ12. Human ARID1A gene loss results in the resistance to combined FAO and PRC2 inhibition therapies due to reduction of mitochondrial ROS levels. CRISPR-Cas9-mediated ARID1A knockout and/or constitutively active CTNNB1 driver mutations promoted tumor development in humanized FRG HCC mouse models, in which use of an interface inhibitor antagonizing PRC2-NANOG binding and/or FAO inhibitor blocked tumor growth. Together, the PRC2-NANOG interaction becomes a new drug target for HCC via inducing differentiation-related genes, destabilizing NANOG protein, and suppressing NANOG activity.

An Aminosteroid Derivative Shows Higher In Vitro and In Vivo Potencies than Gold Standard Drugs in Androgen-Dependent Prostate Cancer Models.

The aminosteroid derivative RM-581 blocks with high potency the growth of androgen-dependent (AR+) prostate cancer VCaP, 22Rv1, and LAPC-4 cells. Notably, RM-581 demonstrated superior antiproliferative activity in LAPC-4 cells compared to enzalutamide and abiraterone, two drugs that exhibited a synergistic effect in combination with RM-581. These findings suggest that RM-581 may have an action that is not directly associated with the hormonal pathway of androgens. Furthermore, RM-581 completely blocks tumor growth in LAPC-4 xenografts when given orally at 3, 10, and 30 mg/kg in non-castrated (intact) nude mice. During this study, an accumulation of RM-581 was observed in tumors compared to plasma (3.3-10 folds). Additionally, the level of fatty acids (FA) increased in the tumors and livers of mice treated with RM-581 but not in plasma. The increase was greater in unsaturated FA (21-28%) than in saturated FA (7-11%). The most affected FA were saturated palmitic acid (+16%), monounsaturated oleic acid (+34%), and di-unsaturated linoleic acid (+56%), i.e., the 3 most abundant FA, with a total of 55% of the 56 FA measured. For cholesterol levels, there was no significant difference in the tumor, liver, or plasma of mice treated or not with RM-581. Another important result was the innocuity of RM-581 in mice during a 28-day xenograft experiment and a 7-week dose-escalation study, suggesting a favorable safety window for this new promising drug candidate when given orally.

Effect of trametinib and anti-PD-1 combo on tumor growth and survival of KRAS-mutant lung cancer by ID1 downregulation.

e14633 Background: Although anti-PD-1/PD-L1 antibodies represent a major breakthrough in NSCLC patients’ survival, this treatment efficacy is highly dependent on tumor PD-L1 expression. We reported the role of ID1 and the efficacy of a combined blockade of PD-1-ID1 in a KRAS mutant NSCLC mouse model (Baraibar et al; Cancers 2020). Trametinib, which is an oral MEK1/2 inhibitor, acts downstream of KRAS. Here we evaluate trametinib as an ID1 inhibitor able to enhance immunotherapy efficacy through PD-L1 overexpression. Methods: To explore whether MEK1/2 inhibition reduces ID1 expression in vitro and in vivo, experiments in NSCLC cells (human and murine) and in murine models were performed. RNAseq analysis was conducted to elucidate the pathways implicated in ID1 inhibition. Trametinib resistant (TR) cells were generated to analyze the effect of ID1 in the acquired resistance to treatment. Apoptosis was measured by flow cytometry. PD-L1 expression was evaluated by flow cytometry in control, ID1 silenced, ID1-overexpressing and TR cell lines. Treatment of trametinib combined with anti-PD1 was performed in KRAS-mutant LUAD models. Results: In vitro experiments in NSCLC cells showed ID1 inhibition at protein level after MEK1/2 blockade. However, we observe an increase in ID1 mRNA levels following treatment. In vivo experiments performed in C57BL6J and in ID1-deficient mice showed no significant differences in tumor growth between ID1 knock-out and ID1 wild-type mice treated with trametinib. IHC analysis of those tumor samples showed reduced ID1 levels (LLC p<0.001; CMT-167 p<0.0001). RNAseq analysis revealed an enrichment of ubiquitin-proteasome pathway after trametinib exposure. Blockade of proteasome activity with a specific inhibitor, restored ID1 levels. TR cells did not show MEKi induced ID1 inhibition, moreover, ID1 blockade rescued the sensitivity to trametinib in human and murine NSCLC TR cells (p<0.001). Flow cytometry analysis showed increased PD-L1 expression (p<0.001) in human and murine NSCLC cell lines after trametinib administration. In contrast, ID1-overexpressing cells and TR cells did not show PD-L1 upregulation after MEK inhibition. In vivo experiments confirmed the therapeutic efficacy of trametinib and anti-PD-1 combo, reducing tumor growth (p<0.05) and increasing mice survival (p<0.05), compared with trametinib monotherapy or immunotherapy alone. Conclusions: Pharmacological inhibition of MEK1/2 decreased ID1 expression in vitro and in vivo, replicating the effect of a specific shRNA against ID1, through proteasome activation. PD-L1 upregulation induced by ID1 blockade after trametinib treatment sensitized KRAS-mutant NSCLC cells to PD-1/PD-L1 immunotherapy, blocking tumor growth and improving overall survival.

POLQ inhibition elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma via cGAS/STING signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination-repair (HR-repair) proteins in 20%-25% of cases. Defects in HR impart a specific vulnerability to poly ADP ribose polymerase inhibitors and platinum-containing chemotherapy in tumor cells. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we found that POLQ knockdown is synthetically lethal in combination with mutations in HR genes such as BRCA1 and BRCA2 and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates signaling of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING), leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to blocking tumor growth while concurrently activating the cGAS-STING signaling pathway to enhance tumor immune infiltration, highlighting what we believe to be a new role for POLQ in the tumor immune environment.

Steroid receptor coactivator 3 is a key modulator of regulatory T cell–mediated tumor evasion

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were "permanently eradicated" in a genetically engineered tamoxifen-inducible Treg-cell-specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the chemokine (C-C motif) ligand (Ccl) 19/Ccl21/chemokine (C-C motif) receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C motif chemokine ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and natural killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish preestablished breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3-deleted Tregs represents an approach to completely block tumor growth and recurrence without the autoimmune side effects that typically accompany immune checkpoint modulators.

Sophflarine A, a novel matrine-derived alkaloid from Sophora flavescens with therapeutic potential for non-small cell lung cancer through ROS-mediated pyroptosis and autophagy

Novel compounds and more efficient treatment options are urgently needed for the treatment of non-small cell lung cancer (NSCLC). The decoction of Sophora flavescens has been used to treat NSCLC in the clinic, and matrine-type alkaloids are generally considered to be the key pharmacodynamic material basis. But the previous study showed that common matrine-type alkaloids exhibit significant cytotoxicity only when at concentrations close to the millimolar (mM) level. The key antitumor alkaloids in S. flavescens seem to have not yet been revealed. The aim of this study was to screen water-soluble matrine alkaloid with novel skeleton and enhanced activity from S. flavescens, and to reveal the pharmacological mechanism of its therapeutic effect on NSCLC. Alkaloid was obtained from S. flavescens by chromatographic separation methods. The structure of alkaloid was determined by spectroscopic methods, and single-crystal X-ray diffraction. The mechanism of anti-NSCLC in vitro with cellular models was evaluated by MTT assay, western blotting, cell migration and invasion assay, plate colony-formation assay, tube formation assay, immunohistochemistry assay, hematoxylin and eosin staining. The antitumor efficacy in vivo was test in NSCLC xenograft models. A novel water-soluble matrine-derived alkaloid incorporating 6/8/6/6 tetracyclic ring system, named sophflarine A (SFA), was isolated from the roots of S. flavescens. SFA had significantly enhanced cytotoxicity compared with the common matrine-type alkaloids, having an IC50 value of 11.3μM in A549 and 11.5μM in H820 cells at 48h. Mechanistically, SFA promoted NSCLC cell death by inducing pyroptosis via activating the NLRP3/caspase-1/GSDMD signaling pathway, and inhibited cancer cell proliferation by increasing the ROS production to activate autophagy via blocking the PI3K/AKT/mTOR signaling pathway. Additionally, SFA also inhibited NSCLC cell migration and invasion by suppressing EMT pathway, and inhibited cancer cell colony formation and human umbilical vein endothelial cell angiogenesis. In concordance with the above results, SFA treatment blocked tumor growth in an A549 cell-bearing orthotopic mouse model. This study revealed a potential therapeutic mechanism of a novel matrine-derived alkaloid, which not only described a rational explanation for the clinical utilization of S. flavescens, but also provided a potential candidate compound for NSCLC treatment.

Co-targeting CK1ɛ and PIK3CB/p110β as a Therapeutic Approach for Glioblastoma. (P11-13.005)

<h3>Objective:</h3> To determine whether co-inhibition of CK1ɛ and PIK3CB/p110β is an effective therapeutic treatment for Glioblastoma (GBM). <h3>Background:</h3> GBM remains the most common and malignant brain cancer in adults with dismal prognosis, largely due to lack of effective treatments. CK1ɛ and PIK3CB/p110β have been identified as GBM survival kinase genes. CK1ɛ promotes growth of GBM cells by inhibiting β-catenin. However, blocking CK1ɛ alone fails to completely block tumor growth perhaps because β-catenin can be regulated by other kinases, including PIK3CB/p110β. It is possible that blocking either CK1ɛ or PIK3CB/p110β alone does not substantially activate β-catenin to induce massive GBM cell death, thus limiting their therapeutic efficacy as a single agent. It is therefore imperative to investigate if co-targeting CK1ɛ and PIK3CB/p110β yields synergistic inhibitory effects on GBM cell growth. <h3>Design/Methods:</h3> GBM cells were treated with either DMSO (control), IC261 (chemical inhibitor of CK1ɛ), GSK2636771 (chemical inhibitor of PIK3CB/p110β), or both IC261 and GSK2636771. Cell viability was measured using the MTS viability assay. <h3>Results:</h3> Cell viability for the treatment groups were as follows: GSK2636771 (95.76%), IC261 (61.84%), and combination of GSK2636771+IC261 (41.70%). Statistical analysis of drug synergy using the Bliss independence model yielded an Excess Over Bliss score greater than 0%, indicating a synergistic effect. <h3>Conclusions:</h3> Preliminary results show that co-inhibition of CK1ɛ and PIK3CB/p110β with chemical inhibitors yields synergistic GBM cell death. Future directions include a dose response experiment to determine the most effective dose of GSK2636771 and IC261 in order to yield stronger synergistic effects. To further elucidate the mechanism of GBM cell death, immunoblotting will also be performed to detect β-catenin activity after inhibition of CK1ɛ and PIK3CB/p110β. <b>Disclosure:</b> Miss Xu has nothing to disclose. Zhi Sheng, 1800 has nothing to disclose.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

Stearoyl-CoA Desaturases1 Accelerates Non-Small Cell Lung Cancer Metastasis by Promoting Aromatase Expression to Improve Estrogen Synthesis.

Metastases contribute to the low survival rate of non-small cell lung cancer (NSCLC) patients. Targeting lipid metabolism for anticancer therapies is attractive. Accumulative evidence shows that stearoyl-CoA desaturases1 (SCD1), a key enzyme in lipid metabolism, enables tumor metastasis and the underlying mechanism remains unknown. In this study, immunohistochemical staining of 96 clinical specimens showed that the expression of SCD1 was increased in tumor tissues (p < 0.001). SCD1 knockdown reduced the migration and invasion of HCC827 and PC9 cells in transwell and wound healing assays. Aromatase (CYP19A1) knockdown eliminated cell migration and invasion caused by SCD1 overexpression. Western blotting assays demonstrated that CYP19A1, along with β-catenin protein levels, was reduced in SCD1 knocked-down cells, and estrogen concentration was reduced (p < 0.05) in cell culture medium measured by enzyme-linked immunosorbent assay. SCD1 overexpression preserving β-catenin protein stability was evaluated by coimmunoprecipitation and Western blotting. The SCD1 inhibitor A939572, and a potential SCD1 inhibitor, grape seed extract (GSE), significantly inhibited cell migration and invasion by blocking SCD1 and its downstream β-catenin, CYP19A1 expression, and estrogen concentration. In vivo tumor formation assay and a tail vein metastasis model indicated that knockdown of SCD1 blocked tumor growth and metastasis. In conclusion, SCD1 could accelerate metastasis by maintaining the protein stability of β-catenin and then promoting CYP19A1 transcription to improve estrogen synthesis. SCD1 is expected to be a promised therapeutic target, and its novel inhibitor, GSE, has great therapeutic potential in NSCLC.

Abstract 1474: Hippo-mediated control of the mutant TERT promoter via MYC and GABP

Abstract Most cancers achieve replicative immortality through reactivation of telomerase reverse transcriptase (TERT), the rate limiting subunit in telomerase activity. Tumors from more than 50 distinct cancer types reactivate TERT expression via mutations in the TERT promoter. Understanding how the mutations lead to activation and immortality is a central question in cancer research. Oncogenic signaling pathways including EGFR/AMPK, MAPK, and C-MYC/ASL, have been linked to telomere maintenance via their regulation of the mutant TERT promoter. Two mutually exclusive point mutations in the TERT promoter generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression (Bell et al, Science 2015). Recruitment of GABP to the mutant TERT promoter facilitates the recruitment of C-MYC to the promoter (Shi et al, Mol. Cell 2022), which aids in transcriptional activation. The Hippo pathway plays a key role in development, organ size determination, and homeostasis, and has been found dysregulated in numerous cancer types. GABP is a member of the Hippo pathway, with GABP activating the promoter of the Hippo transcriptional effector Yes-associated protein (YAP1) and the Hippo pathway core kinases stimulating GABP nuclear exit (Wu et al., Cell Reports 2013). We have found that YAP and its paralogue transcriptional coactivator with PDZ-binding motif (TAZ) are linked to mutant TERT promoter activation in glioblastoma via transcriptional activation of both GABPA and MYC. These observations suggest the Hippo pathway supports tumor immortality in glioblastoma. While immortality has proven to be an elusive target, further investigations of the signaling networks that converge on the activation of the mutant TERT promoter may reveal therapeutic targets that block tumor growth and have secondary benefit by their impact on tumor immortality. Citation Format: Nicholas O. Stevers, Andrew Mancini, Joseph F. Costello. Hippo-mediated control of the mutant TERT promoter via MYC and GABP [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 1474.

Abstract 1081: Co-targeting CK1epsilon and PIK3CB/p110beta to treat glioblastoma

Abstract The purpose of this study is to determine whether co-inhibition of CK1epsilon and PIK3CB/p110β is an effective therapeutic treatment for Glioblastoma (GBM). GBM is the most common and malignant primary brain cancer in adults. GBM has a dismal prognosis of death within 6 months if left untreated, thus there is a clinically unmet need for new and effective treatments. CK1epsilon has been identified as a GBM survival kinase gene. CK1epsilon promotes growth of GBM cells by inhibiting β-catenin. However, blocking CK1epsilon alone fails to completely block tumor growth perhaps because β-catenin can be regulated by other kinases. PIK3CB/p110β is another GBM survival kinase gene which also acts upstream of β-catenin. As seen with CK1epsilon, blocking PIK3CB/p110β alone also fails to completely block tumor growth. It is possible that blocking either CK1epsilon or PIK3CB/p110β alone does not substantially activate β-catenin to induce massive GBM cell death, thus limiting their therapeutic efficacy as a single agent. It is therefore imperative to investigate if co-targeting CK1epsilon and PIK3CB/p110β yields a synergistic inhibitory effect on GBM cell growth. GBM cells were treated with either DMSO (control), IC261 (chemical inhibitor of CK1epsilon), AZD6482 (chemical inhibitor of PIK3CB/p110β), or both IC261 and AZD6482. Cell viability was measured using the MTS viability assay. Cell viability for the treatment groups were as follows: AZD6482 (88.81%), IC261 (66.78%), and combination of AZD6482+IC261 (42.59%). Statistical analysis of drug synergy using the Bliss independence model yielded an Excess Over Bliss score greater than 0%, indicating a synergistic effect. To further elucidate the mechanism of GBM cell death, immunoblotting will also be performed to detect β-catenin activity after inhibition of CK1epsilon and PIK3CB/p110β. Overall, preliminary results show that co-inhibition of CK1epsilon and PIK3CB/p110β with chemical inhibitors yields synergistic GBM cell death. Citation Format: Eileen Xu. Co-targeting CK1epsilon and PIK3CB/p110beta to treat glioblastoma [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 1081.

Abstract 4879: Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid

Abstract Tranexamic Acid (TA) is an anti-fibrinolytic agent that inhibits Plasminogen activation and is used to control bleeding. Past studies have suggested that the ability of TA to block Plasminogen activation may be useful for ablating the growth or invasion of cancer. Based on the similarity of TA to the amino acid Lysine we hypothesize that TA might exhibit novel mechanisms of action independent of blocking Plasminogen activation. Analysis of a series of signaling pathways indicated that TA inhibits phosphorylation of S6K1 and STAT3 on sites required for their activation and reduces expression of the MYC oncogene in a concentration-dependent manner. Interestingly, MYC transformed MCF10A human mammary epithelial cells exhibited enhanced sensitivity to TA-mediated reduction of cell viability, suggesting that TA may exhibit selectivity for MYC overexpressing breast cancers. Treatment of breast tumor bearing mice with TA strongly blocked tumor growth and was associated with extensive cancer cell death compared with control tumors. Analysis of extracts from TA or vehicle treated tumors showed that TA blocked S6K1 phosphorylation in vivo in a statistically significant manner. Since S6K1 phosphorylation is regulated by amino acid levels, we hypothesized that due to the structural similarity between TA and Lysine and Arginine, TA may block Lysine or Arginine uptake by blocking cationic amino acid transporters (CATs). Molecular docking simulations utilizing the structure of the bacterial CAT predicted that TA binds to CAT with affinity similar to that of Lysine. Amino acid uptake experiments performed with radiolabeled Arginine and Lysine showed that TA blocked both Arginine and Lysine absorption by cancer cells in a concentration-dependent manner. Together, these results indicate a novel mechanism by which TA inhibits uptake of Arginine and Lysine, triggering loss of S6K1 activity in parallel with tumor growth inhibition. Initial studies suggest the feasibility of designing TA analogs with significantly improved potency for amino acid transporters, and with corresponding increases in selective toxicity to breast cancer cells. Based on these novel mechanisms of TA action, we expect TA to exhibit anti-cancer activity against a broad range of human malignancies. Citation Format: Brian K. Law, Mary E. Law, Amanda F. Ghilardi, Elham Yaaghubi, Brad J. Davis, Zaafir M. Dulloo, Mengxiong Wang, Olga A. Guryanova, Coy D. Heldermon, Stephan C. Jahn, Ronald K. Castellano. Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid. [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 4879.

Abstract 3489: TNK1 is a ubiquitin sensing kinase that can be targeted in vivo to block tumor growth

Abstract Thirty eight negative kinase 1 (TNK1) is an understudied non-receptor tyrosine kinase with no established function or mechanism of regulation. Our recent publication reported TNK1 as the primary kinase driver of cell survival in subsets of patient blood cancer samples. In an effort to understand TNK1 function and regulation, we discovered that it has an unusual C-terminal ubiquitin-association (UBA) domain that binds with high affinity to multiple poly-ubiquitin linkages. A 1.5 angstrom crystal structure of the TNK1 UBA shows an atypical five helix UBA domain with two ubiquitin-binding interfaces. Our data suggest that TNK1 relies on its UBA domain to localize to phase-separated ubiquitin-rich condensates and becomes fully active at these sites. To our knowledge, this feature of TNK1 (a bona-fide ubiquitin-association domain that promotes kinase activation) makes TNK1 unique across the human kinome. We also show that the phospho-binding protein 14-3-3 interacts with a MARK-mediated phosphorylation at S502 within the proline-rich domain of TNK1 to sequester TNK1 away from ubiquitin condensates and inhibit kinase activation. In this way, TNK1 toggles between poly-ubiquitin bound (active) and 14-3-3-bound (inactive) states. Our preliminary data suggest that the interaction with poly-ubiquitin at condensates tethers TNK1 to substrates involved in the lysosomal degradation of the condensates via aggrephagy. Furthermore, we found that patient mutations that truncate the 14-3-3 binding site and UBA domain convert TNK1 into a cancer driver with an altered substrate profile, skewed toward pro-growth substrates like STAT3. Finally, we show that mutant TNK1-driven tumors can be targeted in vivo with an anti-TNK1 small molecule. Together, our data provide a first mechanism of regulation and role for TNK1 as a ubiquitin sensor and implicate TNK1 as a candidate for targeted cancer therapy. 1. Chan TY*, Egbert CM*, Maxson JE, Siddiqui A, Larsen LJ, Kohler K, Balasooriya ER, Pennington KL, Tsang TM, Frey M, Soderblom EJ, Geng H, Müschen M, Forostyan TV, Free S, Mercenne G, Banks CJ, Valdoz J, Whatcott CJ, Foulks JM, Bearss DJ, O'Hare T, Huang DCS, Christensen KA, Moody J, Warner SL, Tyner JW, Andersen JL. (2021) TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth. Nature Communications, Sept 9;12(1):5337, PMID: 34504101, *co-first authors Citation Format: Tania Lopez-Palacios, Tsz-Yin Chan, Christina M. Egbert, Spencer Ashworth, Alec Vaughan, Tanya V. Forostyan, Adam Siddiqui, Jason M. Foulks, James Moody, Julia E. Maxson, Jeffrey W. Tyner, Steven L. Warner, Joshua Lyon Andersen. TNK1 is a ubiquitin sensing kinase that can be targeted in vivo to block tumor growth [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 3489.

Abstract 2559: Loss of AKT2 correlates with activation of β-catenin signaling in immortalized mouse hepatocytes

Abstract Background: Primary liver cancer is the second most common cause of cancer-related mortality. Abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/AKT and Wnt/β-catenin signaling pathways, which act as key regulators in cell proliferation and growth, is frequently observed in liver cancer. This study investigated the role of AKT2, the major AKT isoform expressed in the liver, on the activation of β-catenin signaling in immortalized mouse hepatocytes. Methods: Regulation of β-catenin signaling by AKT2 was examined in hepatocytes lacking the negative regulator of PI3K/AKT signaling, PTEN (phosphatase and tensin homolog deleted on chromosome 10). Immortalized hepatocytes were established from mice with liver specific deletions of Pten alone (PtenloxP/loxP; albumin [Alb]-Cre+) or in combination with Akt2 (PtenloxP/loxP; Akt2-/-; Alb-Cre+). Control mice were PtenloxP/loxP; Alb-Cre-. Immunoblotting was used to assess protein expression of AKT and β-catenin. Gene expression of β-catenin targets cyclin D1, CD44, and Jag1 was determined with quantitative PCR. Cell growth was evaluated with hemacytometer count. Results: Pharmacological inhibition of PI3K using LY294002 resulted in decreased phosphorylation levels of AKT (Ser473 and Thr308) and β-catenin (Ser552), suggesting crosstalk between the PI3K/AKT and β-catenin pathways. When compared to wild-type hepatocytes, Pten null and Pten;Akt2 double mutant (DM) expressed significantly higher protein levels of β-catenin (p≤0.0327), with no difference observed in Pten null vs. Pten;Akt2 double mutant groups (p&amp;gt;0.05). Phosphorylation of β-catenin at Ser552, a residue that has been previously shown to be phosphorylated by AKT, was also significantly enhanced in both Pten null and Pten;Akt2 double mutant groups, when compared to wild-type cells. Enhanced phosphorylation of β-catenin in these groups correlated with increased expression of β-catenin transcriptional targets CD44 and Jag1 (p≤0.0041), with no significant difference in mRNA levels of these targets observed in Pten null vs. DM group (p&amp;gt;0.05). Activation of the β-catenin signaling pathway in Pten null and DM groups correlated with enhanced cell growth and significantly increased mRNA levels of cyclin D1 (p&amp;lt;0.0001). Interestingly, when compared to the control group, DM group showed enhanced expression of Akt1 and total phosphorylated AKT. Conclusions: Pten loss alone or accompanied with Akt2 deletion resulted in enhanced activation of β-catenin signaling and cell proliferation. These changes correlated with increased expression of AKT1 and total phosphorylated AKT in the Pten;Akt2 DM group. These findings suggest the need to further evaluate the role of AKT isoforms in the PI3K/AKT/β-catenin signaling axis and a promise for evaluation of isoform-specific inhibitors targeted at AKT to block tumor growth. Citation Format: Ielyzaveta Slarve, Bangyan Stiles, Lina He. Loss of AKT2 correlates with activation of β-catenin signaling in immortalized mouse hepatocytes [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 2559.