KRAS(G12X)-expressing cells exhibit differential sensitivity to panRAS- GEF(OFF)i

Chemical structures of compounds used in this study

Abstract RAS mutations drive over 20% of human cancers and often correlate with poor clinical outcomes. Mutant RAS (RAS-MUT) maintains elevated GTP binding, biasing the protein toward its active (ON) state and driving hyperactivated signaling through effector pathways (MAPK and PI3K, among others), leading to uncontrolled cell growth and resistance to apoptosis. Thus, targeting RAS with small-molecule inhibitors presents a promising therapeutic strategy for RAS-MUT cancers. Recent advances in drug discovery have enabled the development of the pan-RAS(ON) inhibitor RMC6236, which uses a tricomplex strategy, as well as several KRAS-MUT-specific inhibitors (such as Adagrasib, Sotorasib, and MRTX1133) that target the OFF state. However, the antitumor efficacy of these direct RAS inhibitors varies across cancer types and is limited by a “therapeutic ceiling,” marked by stable disease and transient responses for most patients. Thus, a deeper understanding of mechanisms underlying variability in response and development of acquired resistance in RAS-MUT cancers is urgently needed. By analyzing signaling and cell growth inhibition in response to distinct direct RAS inhibitors, we observed similar sensitivity patterns across cell line models, suggesting that the antitumor activity of these drugs is largely confined to a shared subset of RAS-MUT cancers. Notably, we found that a subset of insensitive cells could regain sensitivity to direct RAS inhibitors through serum starvation or cotreatment with an SHP2 inhibitor, indicating hyperactive upstream Receptor Tyrosine Kinase (RTK) signaling contributing to RAS inhibitor resistance. Moreover, we found that resistant cell lines show a greater contribution of wild-type RAS (RAS-WT) to overall RAS activity than sensitive lines, suggesting that resistance may arise from RAS-WT-dependent compensation upon RAS-MUT inhibition. To model acquired resistance, we exposed KRAS-MUT cell line models to high concentrations of various RAS inhibitors individually and derived resistant models that displayed markedly reduced sensitivity compared with parental lines. No evidence of acquired secondary KRAS mutations was observed. However, we detected upregulation of the RTK-RAS-MAPK pathway along with elevated KRAS-MUT protein expression in resistant models. This increased RAS-MUT expression reduced inhibitor effectiveness and impaired suppression of RAS-GTP cycling. We also identified multiple cross-resistance patterns across different direct RAS inhibitors, highlighting the need for better mechanistic understanding to optimize RAS-inhibitor treatments for maximal therapeutic benefit. Together, our results provide mechanistic insight into resistance to direct RAS inhibitors in RAS-MUT cancers and emphasize the urgent need for improved strategies to treat patients who do not respond to current RAS-targeted therapies. Citation Format: Ziyue Kou, Bijaya Gaire, Beau Baars, Mathieu Desaunay, Poulikos I. Poulikakos. Adaptive and acquired mechanisms underlying RAS-mutant tumor response to mutant and state selective RAS inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2951.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is pivotal in development, metabolic homeostasis, and immune responses. While recent research has highlighted AhR's significant role in modulating oxidative stress responses, its mechanistic relationship with ferroptosis—an iron-dependent, non-apoptotic cell death—remains to be fully elucidated. In our study, we discovered that AhR plays a crucial role in ferroptosis, in part by transcriptionally regulating the expression of the solute carrier family 7 member 11 (SLC7A11). Our findings indicate that both pharmacological inactivation and genetic ablation of AhR markedly enhance erastin-induced ferroptosis. This enhancement is achieved by suppressing SLC7A11, leading to increased lipid peroxidation. We also obtained evidence of post-translational modifications of SLC7A11 during ferroptosis. Additionally, we observed that indole 3-pyruvate (I3P), an endogenous ligand of AhR, protects cells from ferroptosis through an AhR-dependent mechanism. Based on these insights, we propose that AhR transcriptionally regulates the expression of SLC family genes, which in turn play a pivotal role in mediating ferroptosis. This underscores AhR's essential role in suppressing lipid oxidation and ensuring cell survival under oxidative stress.

While EZH2 enzymatic activity is well-known, emerging evidence suggests that EZH2 can exert functions in a methyltransferase-independent manner. In this study, we have uncovered a novel mechanism by which EZH2 positively regulates the expression of SKP2, a critical protein involved in cell cycle progression. We demonstrate that depletion of EZH2 significantly reduces SKP2 protein levels in several cell types, while treatment with EPZ-6438, an EZH2 enzymatic inhibitor, has no effect on SKP2 protein levels. Consistently, EZH2 depletion leads to cell cycle arrest, accompanied by elevated expression of CIP/KIP family proteins, including p21, p27, and p57, whereas EPZ-6438 treatment does not modulate their levels. We also provide evidence that EZH2 knockdown, but not enzymatic inhibition, suppresses SKP2 mRNA expression, underscoring the transcriptional regulation of SKP2 by EZH2 in a methyltransferase-independent manner. Supporting this, analysis of the Cancer Genome Atlas database reveals a close association between EZH2 and SKP2 expression in human malignancies. Moreover, EZH2 depletion but not enzymatic inhibition positively regulates the expression of major epithelial-mesenchymal transition (EMT) regulators, such as ZEB1 and SNAIL1, in transformed cells. Our findings shed light on a novel mechanism by which EZH2 exerts regulatory effects on cell proliferation and differentiation through its methyltransferase-independent function, specifically by modulating SKP2 expression.

The Aryl Hydrocarbon Receptor (AhR) is a ligand-activated transcriptional factor pivotal in responding to environmental stress and maintaining cellular homeostasis. Exposure to specific xenobiotics or industrial compounds in the environment activates AhR and its subsequent signaling, inducing oxidative stress and related toxicity. Past research has also identified and characterized several classes of endogenous ligands, particularly some tryptophan (Trp) metabolic/catabolic products, that act as AhR agonists, influencing a variety of physiological and pathological states, including the modulation of immune responses and cell death. Heavy metals, being non-essential elements in the human body, are generally perceived as toxic and hazardous, originating either naturally or from industrial activities. Emerging evidence indicates that heavy metals significantly influence AhR activation and its downstream signaling. This review consolidates current knowledge on the modulation of the AhR signaling pathway by heavy metals, explores the consequences of co-exposure to AhR ligands and heavy metals, and investigates the interplay between oxidative stress and AhR activation, focusing on the regulation of immune responses and ferroptosis.

Reactive oxygen species (ROS) are by-products of metabolism of oxygen and they play an important role in normal homeostasis and cell signaling, as well as in the initiation of diseases including cancer when their production is upregulated. Thus, it is imperative to understand the cellular and molecular basis by which ROS impact on various biological and pathological processes. In this report, we show that human keratinocyte cell line (HaCaT) treated with hydrogen peroxide displayed an increased activity of AhR, leading to enhanced expression of its downstream targets including cytochrome P450 genes. Intriguingly, preincubation of the complete culture medium with hydrogen peroxide accelerated AhR activation and its downstream signaling. Subsequent mass spectrometric analysis reveals that the oxidant elicits the production of oxindole, a tryptophan catabolic product. We further demonstrate that 2-oxindole (a major form of oxindole) is capable of activating AhR, strongly suggesting that ROS may exert a significant impact on AhR signaling. Consistent with this, we also observe that hexavalent chromium [Cr(VI)], a heavy metal known to generate ROS in vivo, enhances AhR protein levels, as well as stimulates expression of CYP1A2 in an AhR-dependent manner. Significantly, we show that hydrogen peroxide and 2-oxindole induce expression of IDO1 and PD-L1, two immune checkpoint proteins. Given the role of IDO1 and PD-L1 in mediating T cell activity and/or differentiation, we postulate that ROS in the tumor microenvironment may play a crucial role in immune suppression via perturbing AhR signaling.

Abstract The tumor suppressor PTEN plays an important role in the regulation of cell survival, proliferation, and angiogenesis. Deregulated expression and/or activities of PTEN frequently result in tumor development. PTEN is a plasma-membrane lipid-phosphatase, functioning to antagonize the PI3K/AKT signaling pathway. It also has a role in the nucleus regulating mitotic progression and suppressing chromosomal instability during cell division. To date, the exact function of nuclear PTEN remains not entirely clear. Several recent studies have revealed that PTEN physically interacts with histones and regulates their post-translational modifications. We previously found that PTEN is heavily phosphorylated by Plk1, which regulates mototic progression and chromosomal instability through its physical interaction with mitotic checkpoint complex (MCC). Therefore, we asked whether PTEN directly interacted with epigenetic regulators and mediated histone modifications and whether PTEN-mediated histone modifications functioned as an important regulatory mechanism during the cell cycle. Here we demonstrated that PTEN was associated with Chromobox Homolog proteins (CBXs) which are part of PRC1 complex that mediates histone modifications. Among CBXs, CBX8 levels were high in mitosis, which were correlated with an increase in PTEN mono-ubiquitination, suggesting its potential involvement in mitotic progression. Supporting this notion, we showed that CBX8 was required for cell proliferation and mitotic progression. Moreover, CBX8 was associated with PTEN/MCC during mitosis and mediated histone H2AK119 mono-ubiquitination. Combined, these results strongly suggest that CBX8/PTEN/MCC plays a significant role in modifying histones and suppressing chromosomal instability during mitotic progression. Citation Format: Byeong Hyeok Choi, Tania Marlyn Colon, Eunji Lee, Ziyue Kou, Wei Dai. PTEN plays a role in histone modifications during mitosis [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 1969.

OBJECTIVES:Besides its role in regulating phosphatidylinositol-3 kinase (PI3K) signalling in the cytosol, PTEN also has a nuclear function. In this study, we attempted to understand the mechanism of chromatin PTEN in suppressing chromosomal instability during cell division.MATERIALS AND METHODS:Immunocoprecipitation, ectopic expression, and deletional analyses were used to identify the physical interaction between Chromobox Homolog protein 8 (CBX8) and PTEN, as well as the functional domain(s) of PTEN mediating the interaction. Cell synchronization followed by immunoblotting was employed to study cell cycle regulation of CBX8 and the functional interaction between chromatin PTEN and CBX8. Small interfering RNAs (siRNAs) were used to study the role of PTEN and CBX8 in modulating histone epigenetic markers during the cell cycle.RESULTS:Polycomb group (PcG) proteins including CBXs function to repress gene expression in a wide range of organisms including mammals. We recently showed that PTEN interacted with CBX8, a component of Polycomb Repressing Complex 1 (PRC1), and that CBX8 co-localized with PTEN in the nucleus. CBX8 levels were high, coinciding with its phosphorylation in mitosis. Phosphorylation of CBX8 was associated with monoubiquitinated PTEN and phosphorylated-BubR1 on chromatin. Moreover, CBX8 played an important role in cell proliferation and mitotic progression. Significantly, downregulation of either PTEN or CBX8 induced H3K27Me3 epigenetic marker in mitotic cells.CONCLUSION:CBX8 is a new component that physically interacts with chromatin PTEN, playing an important role in regulating mitotic progression.

Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anticancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activity or mediate its downstream signaling. To this end, we used a combination of affinity-pulldown and mass spectrometry to search for proteins that are physically associated with KRas. One of the top hits was Radil, a gene product with a Ras-association domain. Radil is known to be a downstream effector of Rap1, inhibiting RhoA signaling to regulate cell adhesion and migration. We demonstrate that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas, although it exhibited the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more efficiently, suggesting a possibility that Radil may be involved in Ras activation. Supporting this, ectopic expression of Radil led to transient activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase; Radil knockdown resulted in weakened activation of Ras downstream signaling components, which was coupled with decreased cell proliferation and invasion, and reduced expression of mesenchymal cell markers. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, molecular processes that facilitate cancer cell migration. Taken together, our present studies strongly suggest that Radil is an important player for regulating Ras signaling, cell adhesion, and the epithelial-mesenchymal transition and may provide new directions for Ras-related anticancer drug development.

Aryl hydrocarbon receptor (AHR) was initially discovered as a cellular protein involved in mediating the detoxification of xenobiotic compounds. Extensive research in the past two decades has identified several families of physiological ligands and uncovered important functions of AHR in normal development and homeostasis. Deficiency in AHR expression disrupts major signaling systems and transcriptional programs, which appear to be responsible for the development of numerous developmental abnormalities including cardiac hypertrophy and epidermal hyperplasia. This mini review primarily summarizes recent advances in our understanding of AHR functions in normal physiology with an emphasis on the cardiovascular, gastrointestinal, integumentary, nervous, and immunomodulatory systems.

Aryl hydrocarbon receptor (AhR) was initially discovered as a cellular protein involved in mediating the detoxification of xenobiotic compounds including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Subsequent studies have identified several families of metabolic products as endogenous AhR agonists such as 6-Formylindolo[3,2-b] carbazole (FICZ) and kynurenine and revealed important physiological roles of AhR in the regulation of metabolic homeostasis and immune responses. Recently, we found that treatment with DNA damage agents including etoposide, doxorubicin, and hexavalent chromium resulted in elevated levels of AhR and CYP1A2, the latter being an AhR transcriptional target. Moreover, we found that treatment with the oxidant hydrogen peroxide stimulated AhR expression, which was also accompanied by enhanced expression of CYP1A2. It has been shown that hydrogen peroxide is capable of converting tryptophan into FICZ in vitro. Thus, we speculated that oxidant-induced activation of the AhR axis was mediated through the generation of AhR ligand(s) in vivo. Supporting this, we showed that pre-incubation of tryptophan that was dissolved in PBS with hydrogen peroxide elicited a rapid induction of CYP1A2 expression with an kinetics similar to that of FICZ. Given that a common feature of treatment with etoposide, doxorubicin, and hexavalent chromium is the generation of reactive oxygen species in vivo, we propose that enhanced expression of AhR by these DNA damage agents is primarily due to an elevated level of tryptophan metabolic products including FICZ and that the AhR signaling axis may play a significant role in limiting the extent of DNA damage caused by these compounds.

Ras proteins regulate a variety of cellular processes including cell survival, proliferation, and differentiation. Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anti‐cancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activities or mediate their downstream signaling pathways. Recently, we have identified a series of proteins that are associated with KRas using a combination of immunoprecipitation and mass spectrometry. We have initially focused on Radil, a gene product with Ras‐association (RA) and DIL domains. Radil is known to be a downstream effector of RAP1, inhibiting RhoA signaling in the regulation of cell survival, adhesion, and migration. We have demonstrated that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas although it exhibits the strongest interaction with the latter. Radil knockdown does not seem to affect MEK phosphorylation induced by KRasV12, suggesting that it may lie upstream of KRas regulatory pathway. Intriguingly, ectopic expression of Radil only transiently activates MEK and ERK whereas high levels of Radil are correlated with downregulation of KRas downstream components, suggesting a biphasic regulation of KRas signaling. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, which was associated with reduced FAK activity, deceased cell proliferation, and downregulation of Vimentin and Snail (two mesenchymal cell markers). Taken together, our current studies strongly suggest that Radil may be important for regulating Ras signaling and the epithelial‐mesenchymal transition.

RAS proteins regulate a variety of cellular processes including cell survival, proliferation, and differentiation. RAS genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anti-cancer drugs in the clinic that target RAS proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate RAS activity or mediates its downstream signaling. Recently, we identified a series of proteins that are associated with HRAS using a combination of immunoprecipitation and mass spectrometry. We first focused on RADIL, a gene product with Ras-Association (RA) and DIL domains. RADIL is known to be a downstream effector of RAP1, inhibiting RhoA signaling in the regulation of cell survival, adhesion, and migration. We demonstrated that RADIL interacted with all three isoforms of RAS, including H, N, and KRAS. Interestingly, constitutively active HRAS (HRASG12V) was strongly associated with RADIL whereas dominant negative HRAS (HRASS17N) displayed a compromised association with RADIL, suggesting that RADIL may be required for RAS activation. We also found that Radil Knockout cells had weakened ERK phosphorylation whereas induced expression of RADIL promoted ERK phosphorylation, suggesting that RADIL is required for the RAS-MEK-ERK signaling cascade in vitro. Taken together, our current studies strongly suggest that RADIL may be important for regulating RAS signaling.Citation Format: Byeong Hyeok Choi, Ziyue Kou, Mark R. Philips, Wei Dai. RADIL regulates RAS downstream signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1791.