PI3K Inhibitor Research Articles

CD36 inhibition enhances the anti-proliferative effects of PI3K inhibitors in PTEN-loss anti-HER2 resistant breast cancer cells

BackgroundHER2-positive patients comprise approximately 20% of breast cancer cases, with HER2-targeted therapy significantly improving progression-free and overall survival. However, subsequent reprogramed tumor progression due to PI3K signaling pathway activation by PIK3CA mutations and/or PTEN-loss cause anti-HER2 resistance. Previously, alpha isoform-specific PI3K inhibitors were shown to potentiate HER2-targeted therapy in breast cancer cells carrying PI3K pathway alterations with less potent effects on PTEN-loss than PIK3CA-mutant cells. Therefore, seeking for alternative combination therapy needs urgent attentions in PTEN-loss anti-HER2 resistant breast cancer.MethodsSince remodeling of fatty acid (FA) metabolism might contribute to HER-positive breast cancer and is triggered by the PI3K signal pathway, herein, we examined the effects of the inhibition of endogenous FA conversion, SCD-1 or exogenous FA transport, CD36, in combination with PI3K inhibitors (alpelisib and inavolisib) in anti-HER2 resistant PTEN-loss breast cancer cells.ResultsThe activated HER2/PI3K/AKT/mTOR signaling pathway positively correlated with SCD-1 and CD36 expression in PTEN-loss breast cancer cells. PI3K inhibition downregulated SCD-1, and accordingly, the addition of the SCD-1 inhibitor did not augment the antiproliferative effects of the PI3K inhibitors. CD36 was upregulated by blocking the PI3K signal pathway or limited serum supplementation, indicating that suppressing CD36 may decrease the excess transport of exogenous FA. The addition of the CD36 inhibitor synergistically enhanced the anti-proliferative effects of the PI3K inhibitors.ConclusionSimultaneously targeting the PI3K signaling pathway and exogenous FA uptake could potentially be advantageous for patients with PTEN-loss anti-HER2 resistant breast cancer.

A micro-lung chip with macrophages for targeted anti-fibrotic therapy.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. Macrophages are implicated in the fibrotic process, but exhibit remarkable plasticity in the activated immune environment in vivo, presenting significant challenges as therapeutic targets. To explore the influence of macrophages on IPF and develop macrophage-targeted therapies, we engineered a micro-lung chip with a lung epithelium-interstitium tissue unit to establish a controlled immune environment containing only macrophages. We discovered that macrophages exacerbated inflammation and fibrosis by comparing microchips treated with bleomycin in the presence and absence of macrophages. Based on the duration of bleomycin treatment, we established pathological models corresponding to inflammation and fibrosis stages. Transcriptome analysis revealed that activation of the PI3K-AKT signaling pathway facilitates the transition from inflammation to fibrosis. However, LY294002, a PI3K inhibitor, not only suppressed fibrosis and decreased the accumulation of M2 macrophages but also intensified the severity of inflammation. These findings suggest that macrophages play a pivotal role in the potential development at the tissue level. The micro-lung chip cocultured with macrophages holds significant potential for exploring the pathological progression of IPF and elucidating the mechanisms of anti-fibrotic drugs.

Exploring BCL2 regulation and upstream signaling transduction in venetoclax resistance in multiple myeloma: potential avenues for therapeutic intervention

Investigating venetoclax (VTX) resistance in multiple myeloma (MM) is crucial for the development of novel therapeutic strategies to tackle resistance. We conducted a multi-omic characterization of established VTX-resistant isogenic human myeloma cell lines (HMCL) and primary MM patient samples pre- and post-VTX treatment. Transcriptomic and proteomic analysis revealed that resistance was largely associated with BCL-2 family protein dysregulation, including upregulation of anti-apoptotic proteins such as MCL-1, BCL-XL, BCL-2, and downregulation of pro-apoptotic members. Notably, the re-introduction of BIM into resistant cells restored VTX sensitivity and synergized with MCL-1 inhibitors. Upstream signaling pathways, including growth factor receptor tyrosine kinase (RTK) and phosphoinositide-3-kinase (PI3K) were implicated in this dysregulation. Simultaneous inhibition of MCL-1, BCL-XL, and upstream PI3K, RTK (FGF, EGF, and IGF1) mediated signaling enhanced VTX sensitivity. Post-translational modifications of MCL-1, particularly its stabilization via acetylation and phosphorylation, were investigated, although their inhibition only marginally increased VTX sensitivity. Lastly, the inhibition of AURKA and mitochondrial respiration also improved VTX sensitivity in some resistant HMCLs. Our findings suggest that combining VTX with MCL-1 and BCL-XL inhibitors or PIK3/RTK inhibitors holds potential for overcoming resistance. The study illustrates the importance of understanding molecular determinants of resistance to develop tailored therapeutic strategies.

PI3K-mediated Kif1a DNA methylation contributes to neuropathic pain: an in vivo study.

Neuropathic pain (NP) is a chronic condition caused by nerve injuries, such as nerve compression. Understanding its underlying neurobiological mechanisms is critical for developing effective treatments. Previous studies have shown that Kinesin family member 1A (Kif1a) heterozygous deficient mice display sensory deficits in response to nociceptive stimuli. PI3K has been found to mitigate these sensory deficits by enhancing Kif1a transcription, highlighting KIF1A's key role in sensory pain. However, the exact mechanism through which PI3K regulates KIF1A expression in relation to pain remains unclear. In this study, we observed a significant increase in PI3K/AKT/CREB (cyclic AMP response element-binding protein) protein levels in the dorsal root ganglia and spinal cord after chronic constriction injury in both male and female C57BL/6 mice. Notably, elevated levels of TET1, as well as Kif1a mRNA and protein, were detected in both male and female mice. Activated (phosphorylated-CREB) p-CREB recruited the DNA demethylase TET1, which interacted with the Kif1a promoter, reducing methylation and increasing Kif1a mRNA and protein expression. PI3K inhibition using wortmannin reversed the demethylation of Kif1a and decreased its expression in male mice. Furthermore, TET1 knockdown or overexpression significantly affected pain-related behaviors, as well as Kif1a methylation and transcription. Female mice given intrathecal injections of PI3K inhibitors exhibited similar molecular and behavioral outcomes as male mice. These findings offer new insights into NP mechanisms, suggesting that targeting the PI3K/KIF1A axis could be a promising therapeutic approach for NP treatment.

Targeted inhibition of the PTEN/PI3K/AKT pathway by YSV induces cell cycle arrest and apoptosis in oral squamous cell carcinoma

BackgroundTyroservatide (YSV), a bioactive tripeptide, holds potential as an anti-tumor agent. However, its specific effects on oral squamous cell carcinoma (OSCC) have not been elucidated. This study aims to investigate the inhibitory effects of YSV on OSCC and explore the underlying molecular mechanisms.MethodsA series of in vitro experiments were conducted to assess the impact of YSV on OSCC cell viability, colony formation, cell cycle, and apoptosis. RNA sequencing (RNA-seq), molecular docking, and western blotting were employed to investigate the molecular mechanisms. Additionally, a subcutaneous tumor model was established to validate the in vitro findings. Furthermore, PI3K inhibitors LY294002 and PI3K-IN-1, were used to confirm the role of the PTEN/PI3K/AKT pathway in YSV-mediated OSCC inhibition. Cell cycle and apoptosis were analyzed to assess the combined effect of YSV and LY294002.ResultsYSV significantly inhibited OSCC proliferation by inducing cell cycle arrest and apoptosis. RNA-seq and molecular docking revealed that YSV regulated the PTEN/PI3K/AKT signaling pathway. Western blotting confirmed the modulation of this pathway both in vitro and in vivo. The use of PI3K inhibitors, LY294002 and PI3K-IN-1, further validated the involvement of the PTEN/PI3K/AKT pathway in YSV-induced anti-tumor effects. Notably, the combination of YSV and LY294002 synergistically enhanced cell cycle arrest and apoptosis, demonstrating effective anti-tumor activity. In vivo experiments also supported these findings.ConclusionYSV inhibited the progression of OSCC by promoting cell cycle arrest and apoptosis through the regulation of the PTEN/PI3K/AKT signaling pathway. The combination of YSV and PI3K inhibitors, such as LY294002, exhibited enhanced anti-tumor activity, suggesting potential therapeutic strategies for OSCC treatment.Graphical abstract

Association between ex vivo pharmacotyping of patient-derived tumor organoids and personalized therapeutic options for patients with biliary tract cancer.

618 Background: Biliary tract cancers (BTC), including cholangiocarcinomas and gallbladder adenocarcinomas, present significant therapeutic challenges due to limited treatment options and poor prognoses. We report findings from the CLIA-certified PARIS assay, evaluating drug sensitivities of patient-derived tumor organoids (PDTOs) to a panel of oncology drugs. Methods: PDTOs were successfully cultured from 27 out of 46 live tumor samples obtained from 43 BTC patients. The majority of patients presented with advanced metastatic disease (60% stage IV, 13% stage III, 10% stage II, 4.4% stage I, 10% unknown) and had exhausted standard therapeutic options. Each PDTO culture underwent testing against an average of 50 drugs, encompassing chemotherapeutic agents and targeted therapies. Results: Comparative analysis of PDTO drug sensitivities with patients' prior treatment histories revealed non-responsiveness to >80% of drugs associated with clinical progression, including gemcitabine, cisplatin, and targeted therapies for FGFR translocations. In contrast, 100% (26/26) of the samples demonstrated significant sensitivity to one or more targeted agents, particularly inhibitors of EGFR, MEK, ERK, mTOR, PI3K, MDM2, BCL2, and BET families. Despite these shared sensitivities, each individual PDTO culture exhibited unique responses to targeted therapies, highlighting the genetic and phenotypic diversity between BTC patients. Comparison of ex vivo drug sensitivities with tumor genomic profiles validated oncogenic drivers such as HER2 amplification, KRAS and PIK3CA pathogenic mutations, correlating with sensitivities to EGFR/HER2 inhibitors, MEK inhibitors, and PI3K inhibitors, respectively. While mutations in KRAS , BRAF , BRCA1 , BRCA2 , ERBB2 , ERBB3 , or MET are present in less than 8% of cases, their role as biomarkers in BTCs requires further validation. PDTOs offer a promising tool to expedite this validation process. The PARIS assay results guided treatment decisions for five patients with metastatic disease, two of whom maintained treatment for over 4 weeks. Notably, two patients showed clinical benefit with everolimus (5 weeks) and dasatinib (11 weeks), experiencing symptom relief and reduced ascites. Intriguingly, 5 out of 7 PDTOs with FGFR alterations exhibited exceptional PARIS test responses to dasatinib, suggesting potential efficacy linked to FGFR activation. Conclusions: In conclusion, our study demonstrates that ex vivo drug testing of PDTO cultures can inform treatment selection and potentially accelerate drug approvals for BTC, leveraging therapies approved for other cancers. Early integration of such assays in patient management, possibly at diagnosis, holds promise for improving outcomes in this challenging disease.

Preventive vs. therapeutic effects of Shoutai Wan: Maintaining an acidic microenvironment at the maternal-fetal interface to promote angiogenesis and minimize pregnancy loss in RSA mice.

The classic TCM prescription, Shoutai Wan (STW), is extensively used in clinical settings to manage threatened miscarriage and Recurrent spontaneous abortion (RSA). The complexity of pregnancy physiology, coupled with diverse etiologies, and the specificity of energy metabolism for normal embryo attachment and development, pose challenges to clinical diagnosis and treatment. The specific molecular mechanisms of how STW regulates these biological processes and contributes to the treatment of RSA remain to be elucidated. This study aims to investigate the causes of early pregnancy loss in RSA mice and explore how STW mitigates this loss. An RSA mouse model will be established and treated with STW and Dydrogesterone (DYD). Embryo loss will be quantified on the 14th day of pregnancy, and embryos will be collected on the 6th and 10th days to observe the embryonic condition and assess pathological changes. The study will analyze aerobic glycolysis and angiogenesis at the maternal-fetal interface (MFI). Additionally, STW on a knockdown LDHA mouse model and Human Endometrial Microvascular Endothelial Cells (HEMECs) in vitro will also be examined to verify the mechanism. Compared with the control group, the RSA group exhibited significant embryo loss, and reduced levels of aerobic glycolysis at the MFI, the precarious acidic microenvironment (AME), and the PI3K/AKT/mTOR signaling axis downregulated, leading to impaired angiogenesis, which ameliorated following STW treatment. STW treatment enhanced key aerobic glycolysis enzymes-HK2, PKM2, LDHA-and lactate levels, thereby maintaining the AME and upregulating the PI3K/AKT/mTOR axis. This, in turn, promoted the expression of angiogenesis-related factors (VEGFA and VEGFR2) at the MFI, thereby improving angiogenesis, and the same was seen in sh-LDHA mice. In vitro studies confirmed that STW could counteract the glycolysis decline caused by increased oxygen levels, a recovery that was impaired after LDHA knockdown or PI3K inhibition. In RSA mice, disturbances in aerobic glycolysis at the MFI prevent the maintenance of a stable AME, thus impairing angiogenesis and leading to embryo loss, and STW effectively improve early pregnancy outcomes, and laying the foundation for uterine spiral artery remodeling.

Isoniazid and nicotinic hydrazide hybrids mitigate trehalose-6,6'-dimycolate-induced inflammatory responses and pulmonary granulomas via Syk/PI3K pathways: A promising host-directed therapy for tuberculosis.

Granulomas, dense clusters of immune cells and bacteria, are critical barriers in tuberculosis (TB) treatment. Recent advancements in TB management have highlighted granuloma control as a potential host-directed therapy (HDT) strategy. Although isoniazid (INH) is the first-line drug for TB therapy, its efficacy is limited to non-replicating Mycobacterium tuberculosis (Mtb) under granulomatous conditions, necessitating the development of more effective derivatives. In this study, hybrid compounds of isoniazid, designated as INH-D1 and INH-D2, were synthesized and evaluated for their effects on controlling inflammatory responses and pulmonary granuloma lesions induced by trehalose-6,6'-dimycolate (TDM), a glycolipid of Mtb. Both INH-D1 and INH-D2 demonstrated stronger inhibitory effects on inflammatory mediators (TNF-α, interleukin-6, co-stimulatory molecules, and MHC class I) in TDM-stimulated macrophages compared to original INH. These anti-inflammatory effects were mediated by the inhibition of Syk, p38, PI3K, and NF-κB transcription. INH-D1 and INH-D2 exhibited stronger binding energies to Syk and PI3Kα/β than INH, which are known as proximal kinases and key mediator in TDM-mediated inflammatory responses. Oral administration of INH-D2 successfully relieved TDM-induced pulmonary granuloma pathology by reducing innate immune cell infiltration, hypoxic conditions in the lungs, and systemic inflammation by decreasing serum cytokines and chemokines. In contrast, original INH and INH-D1 did not effectively alleviate pulmonary granuloma pathology. These findings demonstrate that the novel molecule INH-D2 is effective in treating pulmonary granulomas owing to its strong anti-inflammatory effects, highlighting it as a promising HDT candidate for the management of pulmonary tuberculosis, thereby providing a strategic alternative to standard anti-TB antibiotics.

Mediation of the liver protective effect of the anti-cancer drug candidate namodenoson via adiponectin.

604 Background: Namodenoson (CF102) is a small, orally available, anti-cancer drug candidate, currently in pivotal phase 3 clinical study for the treatment of hepatocellular carcinoma. Adiponectin is a positive cytokine released by adipocytes and endothelial cells, known to induce liver-, cardio-, and neuro-protective effects. We evaluated the potential role of adiponectin as the mediator of the observed namodenoson-induced protective effects. Methods: Proteins extracted fromN1S1 tumor-bearing mice and from murine adipocyte culture treated with namodenoson or vehicle were analyzed using Western blot. Apoptosis was determined in the N1S1 tumor-bearing mice using the TUNEL assay. The effect of namodenoson on hepatic ischemia/reperfusion (IR) was evaluated in Wistar rats by temporarily clamping the hepatic vasculature; the effect on hepatic inflammation was evaluated in Con. A-induced hepatitis in mice. The STAM murine in-vivo model was used to study the effect of namodenoson on metabolic dysfunction-associated steatohepatitis (MASH). Adiponectin levels were determined in the serum of patients in the phase 2 trial evaluating namodenoson vs placebo in patients with metabolic dysfunction-associated steatotic liver disease (MASLD)/MASH. Results: The anti-cancer effect of namodenoson was attributed to its capability to bind to the A3 adenosine receptor, inducing inhibition of PI3K and subsequent de-regulation of the Wnt/β-catenin and NF-kB signal transduction pathways, resulting in apoptosis of liver tumor cells. At the same time, namodenoson demonstrated a liver protective effect manifested by prevention of hepatic IR injury, suppression of liver inflammation, and induction of anti-fibrotic and anti-steatotic effects. In adipocyte cell culture, namodenoson stimulated adiponectin production. An increase in adiponectin levels was also shown in the murine MASH model (STAM). These in-vitro and in-vivo results were consistent with translational data from the phase 2 study of namodenoson in MASLD/MASH where upregulation of adiponectin was observed with namodenoson (mean change from baseline of 539 ng/mL for namodenoson 12.5 mg BID vs -78 ng/mL for placebo, P = 0.032). Conclusions: The anti-cancer effect of namodenoson combined with its protective effects, position it as a unique drug inducing a dual effect in patients with advanced liver cancer.

Blocking p85β nuclear translocation by importazole enhances Alpelisib efficacy against PIK3CA-helical-domain-mutant tumors.

PIK3CA, which encodes protein p110α, is one of the most frequently mutated oncogenes and a promising drug-target for human cancer. Previously, we demonstrate that p85β is released from PI3K complex which contain PIK3CA helical domain mutations and translocates into nucleus to regulate tri-methylation of H3K27, thereby promoting tumorigenicity. Here, we identify DIRAS2 and SOWAHB as target genes of nuclear p85β in PIK3CA-helical-domain-mutant tumors. DIRAS2 and SOWAHB are tumor suppressive genes, whose expression are repressed by nuclear p85β through histone methyltransferase EZH2. More importantly, combination of PI3K inhibitor and importin-β inhibitor effectively inhibits the growth of PIK3CA-helical-domain-mutant tumors by synchronously blocking both AKT signaling and nuclear p85β/DIRAS2 and SOWAHB axis. In this study, we evaluate the combination effect of Alpelisib and Importazole for PIK3CA helical domain mutant tumors and demonstrate its underlying mechanism.

Molecular mechanism underlying cardioprotective effect of dehydroepiandrosterone on endoplasmic reticulum stress induced apoptosis in human vascular smooth muscle cells and human umbilical vein endothelial cells

IntroductionThis study aimed to investigate the underlying mechanisms involved in the cardioprotective effects of dehydroepiandrosterone (DHEA) on endoplasmic reticulum stress (ERS) -mediated apoptosis in human vascular smooth muscle cells (HVSMCs) and human umbilical vein endothelial cells (HUVECs).Material and methodsVarious concentrations of Dithiothreitol (DTT) were used to induce ERS-mediated apoptosis. DHEA was utilized to inhibit the apoptotic effects of DTT, while estrogen receptor (ER) antagonists ICI 182,780 and G15, the androgen receptor (AR) antagonist flutamide and the aromatase inhibitor letrozole were used to identify the receptors activated during DHEA treatment in HVSMCs and HUVECs. Flow cytometry assessed the apoptotic rate, and Western blotting analysis evaluated the expression levels of ERS-related proteins GRP78 and PERK, and the apoptotic protein marker CHOP. Furthermore, the primary receptor signaling pathways were identified using signaling pathway blockers: LY294002 (PI3K blocker), SP600125 (JNK blocker), and U0126 (ERK1/2 blocker).ResultsIn the DTT pretreatment group (0.8 mmol/L, for 8 h), the expressions of GRP78 and CHOP were significantly up regulated, indicating that an optimal ERS model was successfully established. Additionally, 10-4 mmol/L DHEA significantly inhibited the DTT-induced upregulation of GRP78 and CHOP. The results also demonstrated that the apoptotic rate in the DTT group was increased, while DHEA significantly reduced this rate. The addition of ER antagonists ICI 182,780 and G15 to HVSMCs reversed the effects of DHEA; however, the aromatase inhibitor letrozole and the AR antagonist flutamide did not reverse this effect. Notably, the use of the JNK inhibitor SP600125, the PI3K inhibitor LY294002, and the ERK1/2 inhibitor U0126 antagonized the protective effects of DHEA, with SP600125 showing the most significant impact on both HVSMCs and HUVECs.ConclusionOur study has identified a novel mechanism underlying the cardioprotective effects of DHEA. Specifically, DHEA may mitigate ERS-induced cell apoptosis by activating estrogen receptors ERα, ERβ, and GPER via the activated JNK pathway.

Evaluation of co‑inhibition of ErbB family kinases and PI3K for HPV‑negative head and neck squamous cell carcinoma.

The ErbB/HER family of protein‑tyrosine kinases and PI3K represent crucial targets in the treatment of head and neck squamous cell carcinoma (HNSCC). A combination therapy of afatinib (ErbB inhibitor) and copanlisib (PI3K inhibitor), both Food and Drug Administration‑approved kinase inhibitors, can suppress the growth of human papillomavirus (HPV)‑positive HNSCC. The current study further evaluated the efficacy and clinical potential of this combination therapy for the treatment of HPV‑negative HNSCC in vitro and in vivo. Sulforhodamine B cell viability assay and Annexin V/propidium iodide staining demonstrated that this combination treatment markedly enhanced inhibition of cell viability and reduced cell survival when compared with treatment with either inhibitor alone in two HPV‑negative HNSCC cell lines. Notably, this combination also led to significant inhibition of xenograft tumor growth in mice, without any apparent effects on body weight. Western blot analysis found that copanlisib alone effectively blocked PI3K/Akt signaling but caused upregulation of HER2 and HER3 phosphorylation, as reported in other types of cancer. However, the combination of copanlisib and afatinib completely blocked phosphorylation of the ErbB family (including HER3) and Akt, while also increasing apoptosis. In conclusion, these results suggested that co‑targeting the ErbB family kinases and PI3K using a combination treatment of afatinib and copanlisib may have clinical potential for patients with HPV‑negative HNSCC.

Synergistic antitumor effect of MK-1775 and CUDC-907 against prostate cancer.

Due to the emergence of drug resistance, androgen receptor (AR)-targeted drugs still pose great challenges in the treatment of prostate cancer, and it is urgent to explore an innovative therapeutic strategy. MK-1775, a highly selective WEE1 inhibitor, is shown to have favorable therapeutic benefits in several solid tumor models. Recent evidence suggests that the combination of MK-1775 with DNA-damaging agents could lead to enhanced antitumor efficacy. Here, our results demonstrate that MK-1775 alone could indeed inhibit proliferation and induce apoptosis in prostate cancer. Moreover, the combination of MK-1775 and a dual PI3K and HDAC inhibitor, CUDC-907, can synergistically inhibit cell proliferation and dramatically induces apoptosis in prostate cancer cells. This effect is partially mediated by DNA damage, resulting from the downregulation of DNA damage response (DDR) proteins such as CDK, CHK, and RRM1/2. Notably, the combination of MK-1775 and CUDC-907 leads to significant antitumor effects in vivo. Our findings provide a strong basis for a promising combination strategy against prostate cancer.

Lipid Emulsion Mitigates the Cardiotoxic Effects of Labetalol in Rat Cardiomyoblasts

Lipid emulsion has recently emerged as an effective agent for improving the cardiotoxicity of highly lipophilic drugs. However, its effect on cardiotoxicity induced by labetalol, a nonselective beta-blocker, remains unknown. In this study, we investigated the effects of lipid emulsion on the cardiotoxicity of labetalol in rat cardiomyoblasts and tried to decipher the underlying mechanisms. The effects of lipid emulsion on labetalol-induced changes in cell viability, expression of Bax/Bcl-2, cleaved caspase-3, and cleaved caspase-9, and phosphorylation of GSK-3β, Akt, and PI3K were examined. Lipid emulsion inhibited labetalol-induced decrease in cell viability, whereas LY294002, MK2206, and SB216763, the inhibitors of phosphoinositide 3-kinase (PI3K), Akt, glycogen synthase kinase-3β (GSK-3β), respectively, partially attenuated this restoration of cell viability. Lipid emulsion reversed the increase in expression of cleaved caspase-3, cleaved caspase-9, and Bax/Bcl-2 and decrease in the phosphorylation of GSK-3β, Akt, and PI3K by labetalol. Lipid emulsion and cyclosporin, a mitochondrial permeability transition pore (MPTP) inhibitor, reduced the labetalol-induced increase in the number of TUNEL-positive cells and promoted late-stage apoptosis. Overall, lipid emulsion inhibited apoptotic cell death caused by labetalol toxicity via the inhibition of intrinsic apoptotic pathway and MPTP in rat cardiomyoblasts, which appears to involve PI3K, Akt, and GSK-3β signaling pathways.

Abstract A013: Shikonin, a strong inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen-Activated Protein (MAP) Kinase pathways, induces cell death in UV-B irradiated B16F10 melanoma cells

Abstract Ultraviolet (UV) radiation-induced skin damage plays a pivotal role in the pathogenesis of melanoma, a highly aggressive and fatal form of skin cancer. Shikonin, naturally occurring nathoquinone , is well-known for its inhibitory effects on phosphoinositide 3-kinase (PI3K) and mitogen-activated protein (MAP) kinase signaling pathways. It has previously been demonstrated to exert effects against various non-melanoma skin cancers. In this study, we investigated the effects of Shikonin treatment on UVB-irradiated B16F10 melanoma cells, revealing a dose-dependent reduction in cell viability and an enhancement of apoptosis. Invitro findings indicate that the pro-apoptotic effects of shikonin in UVB-exposed B16F10 cells are mediated by several mechanisms, including the generation of reactive oxygen species (ROS), modulation of the antioxidant defense response, and mitochondrial membrane potential (ΔΨM) depolarization. Further supporting the apoptotic role of Shikonin, we observed DNA fragmentation, caspase activation, poly(ADP-ribose) polymerase (PARP) cleavage, and an increase in sub-G2 cell populations. Shikonin treatment also significantly attenuated MEK-ERK signaling, influenced the PI3K/Akt pathway, and potentially inhibited UVB-induced nuclear translocation of NF-κB. Additionally, co-treatment with UVB and Shikonin led to a decrease in the Bcl-2/Bax ratio and upregulated the expression of pro-apoptotic proteins Bax and caspases. Collectively, these data suggest that Shikonin may act as a potential therapeutic agent for melanoma when combined with UVB exposure, providing a novel avenue for future treatment strategies. Citation Format: Aalim Maqsood Bhat, Sheikh Tasduq Abdullah. Shikonin, a strong inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen-Activated Protein (MAP) Kinase pathways, induces cell death in UV-B irradiated B16F10 melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr A013.

Processed Products of Aconitum soongaricum Stapf. Inhibit the Growth of Ovarian Cancer Cells In vivo via Regulating the PI3K/AKT Signal Pathway.

The alkaloids of songorine, aconitine, and benzoylaconitine, as the processed products of Aconitum soongaricum Stapf., can significantly inhibit the migration and invasion of ovarian cancer cells in vitro. Herein, we studied the in vivo role and mechanism of these natural products in processed A. soongaricum Stapf. A xenograft tumor model was constructed. Tumor volumes and weights were calculated. HE staining assessed the histopathological changes of tumors. Inflammatory factors were detected using ELISA. Gene and protein expressions of E-cadherin, N-cadherin, PIK3CA, and AKT1 proteins were measured using RT-qPCR and immunohistochemistry. Protein expressions of E-cadherin, N-cadherin, PIK3CA, AKT1, p-PIK3CA, and p- AKT1 proteins were detected using western blot analysis. Songorine, aconitine, and benzoylaconine significantly inhibited the growth of tumors as evidenced by decreased tumor volume and weight. The extent and scope of tumor cell necrosis were less in the songorine group compared to the vehicle group. Songorine, aconitine, and benzoylaconine significantly reduced IL-6, IL-1β, and TNF-α levels. Furthermore, songorine, aconitine, and benzoylecgonine induced down-regulation of N-cadherin and AKT1 mRNA in comparison to the vehicle group. Meanwhile, songorine, aconitine, and benzoylaconine also significantly reduced N-cadherin, p-PIK3CA, and p-AKT1 proteins, while upregulating E-cadherin protein expression in comparison to the vehicle group. These effects were further enhanced when combined with the PI3K inhibitor LY294002. Songorine, aconitine, and benzoylaconine may inhibit ovarian cancer growth in vivo by blocking the PI3K/AKT signaling pathway. Our findings may provide evidence for the clinical application of the processed products of Aconitum soongaricum Stapf. in ovarian cancer treatment.

Targeting TTK Inhibits Tumorigenesis of T-Cell Lymphoma Through Dephosphorylating p38α and Activating AMPK/mTOR Pathway.

T-cell lymphoma (TCL) is a group of non-Hodgkin's lymphoma with high heterogeneity and unfavorable prognosis. Current standard treatments have demonstrated limited efficacy in improving the outcomes for TCL patients. Therefore, identification of novel drug targets is urgently needed to improve the prognosis of TCL patients. Through multi-omics analysis, aberrant expression of threonine tyrosine kinase (TTK) in TCL is identified. High expression of TTK is closely associated with poor prognosis in TCL patients. Targeting TTK through gene knockdown exerts anti-tumor effects in vitro and in vivo, including inhibiting the cell proliferation, inducing G2/M phase arrest, enhancing DNA damage and cell apoptosis. Mechanically, p38α is identified as the potential phosphorylation substrate of TTK through phosphoproteomic quantification and motif prediction. Furthermore, inhibition of TTK suppresses activation of p38α through dephosphorylating it at Thr180/Tyr182, thereby promoting the activation of AMPK/mTOR pathway. In addition, targeting TTK enhances the autophagy in TCL cells through dephosphorylating p38α. CFI-402257, a specific inhibitor of TTK, is found to exhibit anti-tumor effects and exerted synergistic efficacy with PI3K inhibitor, Duvelisib, in TCL. The study shows that TTK contributes to the development of TCL by regulating p38α-mediated AMPK/mTOR pathway. CFI-402257 is expected to be a promising strategy for TCL treatment.

Isolation of proteins on chromatin (iPOC) reveals signaling pathway-dependent alterations in the DNA-bound proteome.

Signaling pathways often convergence on transcription factors (TFs) and other DNA-binding proteins (DBPs) that regulate chromatin structure and gene expression, thereby governing a broad range of essential cellular functions. However, the repertoire of DBPs is incompletely understood even for the best-characterized pathways. Here, we optimized a strategy for the isolation of Proteins on Chromatin (iPOC) exploiting tagged nucleoside analogues to label the DNA and capture associated proteins, thus enabling the comprehensive, sensitive, and unbiased characterization of the DNA-bound proteome. We then applied iPOC to investigate chromatome changes upon perturbation of the cancer-relevant PI3K/AKT/mTOR pathway. Our results show distinct dynamics of the DNA-bound proteome upon selective inhibition of PI3K, AKT, or mTOR, and we provide evidence how this signaling cascade regulates the DNA-bound status of SUZ12, thereby modulating H3K27me3 levels. Collectively, iPOC is a powerful approach to study the composition of the DNA-bound proteome operating downstream of signaling cascades, thereby both expanding our knowledge of the mechanism of action of the pathway, and unveiling novel chromatin modulators that can potentially be targeted pharmacologically.

Rationalizing Predictions of Isoform-Selective Phosphoinositide 3-Kinase Inhibitors Using MolAnchor Analysis.

Explaining the predictions of machine learning models is of critical importance for integrating predictive modeling in drug discovery projects. We have generated a test system for predicting isoform selectivity of phosphoinositide 3-kinase (PI3K) inhibitors and systematically analyzed correct predictions of selective inhibitors using a new methodology termed MolAnchor, which is based on the "anchors" concept from explainable artificial intelligence. The approach is designed to generate chemically intuitive explanations of compound predictions. For nearly all correctly predicted isoform-selective inhibitors, well-defined structural fragments determining the predictions were identified, and in most cases, an individual substructure was responsible for the prediction outcome. For inhibitors with different isoform selectivities, recurrent substructures determining the predictions were distinct. The comparison of newly identified anchor substructures with independent explanations based on calculated feature importance values supported the superior interpretability of MolAnchor explanations. Two highly recurrent substructures determining correct predictions were found to be directly implicated in isoform selectivity of PI3K inhibitors, thus indicating a causal relationship between decisive substructures and selectivity determinants.

Bypassing cisplatin resistance in Nrf2 hyperactivated head and neck cancer through effective PI3Kinase targeting.

For patients with head and neck squamous cell carcinoma (HNSCC), failure of definitive radiation combined with cisplatin nearly universally results in death. Although hyperactivation of the Nrf2 pathway can drive radiation and cisplatin resistance along with suppressed anti-tumor immunity, treatment-refractory HNSCC tumors may retain sensitivity to targeted agents secondary to synergistic lethality with other oncogenic drivers (e.g., NOTCH1 mutations). We evaluated the efficacy of PI3K inhibitors (PI3Ki) in bypassing Nrf2-mediated cisplatin resistance in HNSCC. We measured transcriptomic, metabolomic and signaling changes driven by PI3Kis in cisplatin-resistant HNSCCs in vitro and tested efficacy in vivo in subcutaneous, orthotopic and metastatic xenograft models using immunodeficient and humanized murine models of HNSCC coupled with spatial transcriptomics. The PI3K pathway is activated in Nrf2-driven cisplatin-resistant HNSCC and is suitable for blockade as demonstrated in an in vivo shRNA screen. The PI3Ki gedatolisib inhibits cisplatin-resistant HNSCC proliferation, induces G2M arrest and potentiates cisplatin effectiveness through activation of autophagy, senescence and disruption of fatty acid metabolism. Gedatolisib suppresses HNSCC tumor growth in orthotopic and metastatic settings and demonstrates profound anti-tumor activity in humanized murine models of HNSCC, coupled with a reduction in hypoxia-rich regions and reduced infiltration by regulatory T lymphocytes. Our findings emphasize the critical role of the PI3K-AKT-mTOR pathway in cisplatin-resistant HNSCC and highlight the therapeutic potential of PI3K inhibitors. Gedatolisib induced metabolic regulation and substantial re-sensitization of resistant cells to cisplatin, positioning it as a promising candidate for combination therapies aimed at overcoming primary chemo-radiation failure in HNSCC.