Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen, progesterone, and HER2 receptors; an aggressive phenotype; high rates of early relapse and metastasis; and the worst mortality rates among all breast cancer subtypes. Currently, there is no effective curative targeted therapy for TNBC and chemotherapy remains the primary treatment for TNBC. Therefore, there is a critical need to develop highly effective, novel therapies to improve patient survival. We previously validated FOXM1, a proto-oncogenic transcription factor, for the first time as a potential molecular target in TNBC through genetic knockdown studies in mice. We show that FOXM1 expression is associated with shorter patient survival and is a marker of poor prognosis. There is no FDA-approved FOXM1 inhibitor. We found that patients with TP53 mutations have dramatically higher FOXM1 expression, indicating that widespread TP53 mutations detected in about 80% of TNBC patients are the major driver of FOXM1 overexpression in TNBC patients. We identified its binding ability using an in silico study, and found it to be a well-known FOXM1 inhibitor that suppresses TNBC cell proliferation, migration, and invasion, and induces apoptosis. In vivo studies in mice bearing TNBC tumors demonstrated that treatment with a novel FOXM1 inhibitor incorporated in single-lipid nanoparticles suppressed the growth of TNBC tumor xenografts. In conclusion, our findings suggest that the novel FOXM1 inhibitor represents a potent and safe therapeutic strategy with significant potential for the treatment of other FOXM1-driven cancers including TNBC that currently have limited treatment options.

IDO1 contributes to tumor-associated immunosuppression by regulating tryptophan catabolism. This study investigated the immunometabolic tryptophan-kynurenine axis in NSCLC by evaluating circulating tryptophan metabolites, the IDO1 rs10089084 genetic variation, IDO1/PD-L1 expression in tumor tissues of NSCLC patients, and soluble sPD-1 in relation to clinicopathological features. Seventy NSCLC patients and 72 healthy controls were included in the study. Plasma TRP, KYN, and KYNA levels were analyzed by high-performance liquid chromatography, IDO1 rs10089084 gene variants by PCR–RFLP, IDO1/PD-L1 expression levels in tumor and adjacent non-tumor tissues by quantitative PCR, and sPD-1 levels by ELISA. Lower TRP levels, higher KYN levels, and increased KYN/TRP ratios were detected in individuals with the IDO1 CC genotype. Correlation analyses supported a possible association between systemic tryptophan-kynurenine metabolism and intratumoral IDO1/PD-L1-related immune-metabolic activity. ROC results demonstrated that decreased TRP and increased KYN/TRP values can differentiate NSCLC patients from healthy controls. Locally advanced disease was associated with lower TRP levels and increased KYN/TRP ratios compared to early-stage disease. Our findings suggest that integrated assessment of TRP-KYN pathway metabolites with IDO1 genetic variation and IDO1/PD-L1 expression may aid in characterizing immunometabolic alterations in NSCLC and support the development of biomarker-based approaches for disease stratification and progression assessment.

Purpose Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by frequent chemoresistance and poor clinical outcomes. Doxorubicin (DOX) is a standard therapeutic agent; however, acquired resistance limits its efficacy. Forkhead box M1 (FOXM1) and nuclear factor-κB (NF-κB) are key regulators of pro-survival signaling pathways implicated in TNBC progression and drug resistance. This study aimed to investigate whether flavopiridol enhances DOX sensitivity in TNBC cells by targeting the FOXM1–NF-κB signaling axis. Methods MDA-MB-231 and BT-549 TNBC cell lines were treated with DOX, flavopiridol, or their combination. Cell viability, clonogenic survival, apoptosis, and cell-cycle distribution were assessed. Protein expression levels of FOXM1, NF-κB, RAS/MAPK, and PARP were analyzed by Western blot. FOXM1 function was further evaluated באמצעות siRNA-mediated gene silencing. Results Combination treatment significantly reduced cell viability and clonogenic potential compared to single-agent treatments. Flavopiridol enhanced DOX-induced apoptosis and induced marked alterations in cell-cycle progression in both TNBC cell lines. At the molecular level, combination therapy suppressed FOXM1 expression and downregulated NF-κB and RAS/MAPK signaling pathways. FOXM1 silencing recapitulated these effects, confirming its central role in pro-survival signaling. Additionally, decreased PARP expression suggested impaired DNA repair capacity. Conclusion The FOXM1–NF-κB–RAS/MAPK axis plays a critical role in DOX resistance in TNBC. Targeting FOXM1 with flavopiridol enhances DOX sensitivity and represents a promising therapeutic strategy to overcome chemoresistance in aggressive TNBC.

In this study, coumarin-3-carboxamide derivatives were designed and evaluated through a comprehensive in silico and in vitro combined approaches to identify cholinesterase inhibitors. Molecular docking, molecular dynamics (MD) simulations, and MM/GBSA analyses revealed strong binding affinities and stable interactions within key catalytic regions of AChE and BChE, with AChE-CM4 (ΔGbind = −60.27 ± 5.88 kcal/mol) and BChE-CM5 (ΔGbind = −54.95 ± 6.90 kcal/mol) emerging as the most promising complexes. ADME predictions indicated generally favorable pharmacokinetic profiles for the compounds; however, the predicted blood-brain barrier permeability levels were low to moderate. In vitro enzyme inhibition studies validated the computational results, showing that CM4 (IC50 = 19.04 ± 1.67 nM) and CM8 (IC50 = 17.73 ± 0.66 nM) exhibited significantly greater AChE inhibition than donepezil (IC50 = 27.27 ± 1.22 nM; p < 0.001), with CM3 (IC50 = 24.84 ± 1.99 nM) comparable to donepezil, while all derivatives (CM1-CM9) significantly outperformed tacrine against BChE (p < 0.001). Moreover, CM6 and CM9 inhibited colony formation in brain, breast, and colon cancer cell lines at concentrations below 5 µM. As a result, the alignment of computational and biological data highlights these coumarin-3-carboxamides as compelling lead candidates with both neuroprotective and anticancer potential for further pharmacological development.

Glioblastoma (GBM) is the most diagnosed primary brain tumor with an extremely poor survival rate. Emerging evidence suggests that miRNAs are involved in GBM tumorigenesis. miR-484 was highly expressed in glioma cells and enhanced cell migration, and invasion. However, the role of miR-484 in GBM and its downstream targets are not well understood. We analyzed miR-484 expression in GBM patient tissue samples (IDH wild-type and IDH-mutant) and cell lines via Real-Time PCR. In GBM cells transfected with inhibitor- and mimic-miR-484, we investigated cell proliferation, migration, invasion, cell cycle, and apoptosis. Additionally, protein expression of FOXM1 and its downstream targets were investigated. RNA-seq analysis was performed on mimic-miR-484-transfected U87-MG and IDH1-mutant-U87 cells. miR-484 expression was higher in IDH-mutant patient tumors compared to IDH1-wild type patient tumors. Ectopic expression of miR-484 suppressed cell proliferation, colony formation, migration and invasion, and induced apoptosis in GBM cells. Furthermore, we found that miR-484 suppresses FOXM1 and its downstream targets including Integrin-β1/FAK/Src and Cyclin-D and PARP, all of which have been shown to be potential therapeutic targets in GBM cells. In addition, expression of miR-484 enhanced TMZ-induced FOXM1 downregulation in GBM. Our findings suggest for the first time that miR-484 act as a tumor suppressor in IDH1-wild type and mutant GBM cells by targeting FOXM1 oncogenic transcription factor and its downstream in GBM cells. Therefore, miR-484-based treatment may provide a new avenue for controlling GMB growth and progression and may enhance the therapeutic efficacy of TMZ. Trial registry: decision no: 2019/247.

Glioblastoma multiforme (GBM) is defined by rapid progression, high invasiveness, and a poor prognosis, with a median survival of only ≅13 months despite current treatments. Its marked genetic heterogeneity, high mutational burden, and cancer stem cell population make GBM exceptionally difficult to treat, highlighting the urgent need for more effective, multitargeted therapies. Non-coding RNAs, particularly tumor suppressor microRNAs (miRNAs), have gained attention for suppressing key oncogenic processes that drive tumorigenesis, metastasis, and drug resistance, positioning them as promising tools for targeting multiple oncogenic pathways. We recently found that FOXM1/AXL-eEF2K collaboratively drive GBM cell proliferation, survival, and invasion through the formation of a signaling hub complex. In this study, we employed miRNA prediction algorithms to identify a specific miRNA, in vitro functional assays and in vivo GBM flank model to target GBM tumorigenesis by distrupting the FOXM1/AXL-eEF2K signaling hub. Our results indicated that FOXM1, AXL, and eEF2K are overexpressed in GBM patient tumors. To target the FOXM1/AXL-eEF2K signaling hub, we identified miR-449b-5p, miR-329-3p, and miR-518c as potential co-inhibitors of FOXM1/AXL-eEF2K and suppressors of cell proliferation, migration-invasion, and spheroid formation. Furthermore, the combination of miR-449b-5p, miR-329-3p, and miR-518c treatments with temozolomide led to synergistic enhancements in cell proliferation suppression and the induction of apoptosis and ferroptosis. More importantly, in vivo miR-329-3p treatment led to remarkable suppression of GBM tumor xenografts. These findings indicate that miR-329-3p-based tumor suppressor therapy may offer a multitargeted approach for GBM treatment.

Significant genetic heterogeneity has hindered the identification of molecular targets and development of effective targeted therapies for triple negative breast cancer. Currently available targeted therapies are not curative for TNBC patients. Eukaryotic Elongation Factor-2 kinase (eEF2K) is a clinically significant proto-oncogenic therapeutic target linking this atypical alpha kinase to poor patient survival and a key driver of tumor growth and progression in TNBC, positioning it as a critical and emerging molecular target. Development of eEF2K inhibitors for clinical translation has been challenging due to the unknown three-dimensional structure and lack of potent and selective eEF2K inhibitors. Here, we employed a homology modeling, in silico physics-based molecular simulations studies to rationally design, synthesize and in vitro and in vivo identification a novel potent eEF2K inhibitor. The lead compound-2I demonstrated a potential to engage in covalent interactions with eEF2K enzyme, as suggested by in silico covalent docking and static interaction analyses, and significant in vitro inhibitory activity and suppressed primary and multidrug resistant TNBC cell proliferation at submicromolar concentrations, induced ferroptosis and apoptosis, while having no impact on normal breast epithelial cells. In vivo systemic injection of the eEF2K inhibitor encapsulated in single-lipid nanoparticles demonstrated remarkable therapeutic efficacy and suppressing tumor growth in multiple orthotopic TNBC xenograft models in mice with no sign of toxicity. eEF2K inhibition synergistically enhanced the efficacy of standard chemotherapeutics such as paclitaxel. Our results indicate that the novel eEF2K-targeted nanotherapy is safe and has a significant potential for clinical translation as a monotherapy or in combination with chemotherapy for treatment of patients with TNBC or other eEF2K-dependent solid cancers.

AXL is overexpressed in many cancers, including breast, lung, colon, osteosarcoma, kidney, stomach, and gliomas, and associated with poor patient survival and plays an important role in cancer cell proliferation, survival, tumor growth, progression and metastasis. In vivo studies involving genetic targeting of AXL validated it as a promising therapeutic target. In this study, we performed integrated in silico analyses, including virtual screening, molecular docking, molecular dynamics (MD) simulations, and MM/GBSA calculations to search the compound libraries from various small molecule databases to identify potential inhibitors targeting AXL. We identified key amino acid residues such as Met623, Pro621, Asp627, and Asp690 located in the binding pocket of AXL interacting with the potential inhibitors and positive control compounds through with hydrogen and pi-pi bonds. In vitro studies with the three lead compounds (STOCK1N-80636, STOCK1N-66436 and Nebivolol) demonstrated significant antiproliferative effects in four different breast cancer cell lines STOCK1N-80636 was being the most potent. In conclusion, these findings have enabled the identification of potential AXL inhibitors through integrated in silico and in vitro results.

Triple Negative Breast Cancer (TNBC) is one of the most aggressive subtypes of breast cancer (BC), which is associated with a very poor prognosis. It is a broad category of tumors with a variety of biological, clinical, and morphological characteristics. FOXM1 is a pivotal transcription factor that modulates proliferation-associated genes through complex protein-DNA and protein-protein interactions, making it a highly attractive target in cancer therapy. However, existing small-molecule inhibitors often suffer from limited specificity and efficacy. In this study, we designed, synthesized, and evaluated novel series of 2-aminothiazole derivatives (C1-C15) as potential FOXM1 inhibitors. Molecular docking and molecular dynamics (MD) simulations were employed to investigate the binding interactions of these compounds with the FOXM1 DNA-binding domain (FOXM1-DBD). Structural analysis highlighted the importance of crucial residues, including Asn283, His287, and Arg286, in mediating inhibitory activity. Among the synthesized compounds, C11 exhibited remarkable structural alignment and interaction patterns with FOXM1-DBD, comparable to the reference inhibitor FDI-6. In vitro studies using TNBC cell lines (MDA-MB-231, BT-549, and BT-20) demonstrated that compound C11 significantly outperformed FDI-6 in potency. Western blot analysis revealed that C11 effectively suppressed FOXM1 transcriptional activity at concentrations of 10 µM in BT-549 cells and 20 µM in MDA-MB-231 cells. These findings underscore the potential of C11 as a potent FOXM1 inhibitor and highlight its promise for further development in TNBC therapy.

Glioblastoma Multiforme (GBM) is one of the most common brain tumors and is associated with aggressive tumor characteristics and extremely poor patient survival. The median survival time for GBM patients is around 12-15 months. Temozolomide (TMZ) is a key chemotherapeutic drug used in the treatment of GBM. However, at least 50% of GBM patients do not respond to TMZ, necessitating the identification of novel therapeutic strategies sensitizing patients to TMZ. In this study, we aimed to investigate the effects of two different tumor suppressor microRNAs (miR-329 and miR-449b) on cell proliferation and migration of GBM cells, and their potential for sensitizing GBM cells to TMZ. Our findings show that MiR-329/449b treatments suppressed spheroid formation and migration of GBM (LN229 and U87) cells. When miR treatments were combined with Temozolomide (TMZ), we also observed that they synergistically enhanced the suppressive effects of TMZ and inhibited the activity of clinically significant NF-KB and Src/FAK signaling pathways, making the combination therapy a viable option to treat GBM, with greater impact on patient survival.

Triple Negative Breast Cancer (TNBC) is the most aggressive and heterogeneous subtype of breast cancer with the highest mortality rates. TNBC is characterized by a lack of Estrogen (ER), PR, and HER2 receptors and currently, there is no effective targeted therapy for TNBC patients. AXL, a receptor tyrosine kinase (AXL-RTK), is overexpressed in TNBC cells and patient tumors and has been linked to poor prognosis. Despite its potential as a therapeutic target, there are currently no FDA-approved inhibitors for AXL in TNBC. In this study, we aimed to develop AXL- inhibitor-based targeted therapy and evaluate its therapeutic potential for TNBC using a PLGA (poly-lactic-co-glycolic acid)) nanoparticle delivery system. We formulated it into PLGA nanoparticles to improve bioavailability and enable targeted delivery to TNBC tumors. Various dosages of AXL inhibitor-loaded PLGA nanoparticles were tested on MDA-MB-231 and MDA-MB-436 human TNBC cell lines, and significant inhibition of cell growth and colony formation was observed, with a half-maximal inhibitory concentration (IC50) of 5 μM. The size and morphology of the nanoparticles were characterized using Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM), which revealed nanoparticle sizes ranging from 63-99.9 nm. Western blot analysis showed that the AXL inhibitor-loaded PLGA nanoparticles effectively inhibited AXL protein expression and its downstream signaling pathways, leading to superior anti-proliferative effects compared to free AXL inhibitors. These results suggest that AXL inhibitor-loaded PLGA nanoparticles offer a promising strategy for enhancing the therapeutic efficacy of AXL inhibitors in TNBC. In vivo, efficacy studies are currently underway to evaluate their potential for targeted therapy in both primary and metastatic TNBC models. Fatma Kazdal, Nermin Kahraman, Bulent Ozpolat. Development of novel AXL inhibitor-loaded PLGA nanoparticles for targeted therapy in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1860.

Glioblastoma multiforme (GBM) is an aggressive and molecularly heterogeneous brain cancer with a poor prognosis. Despite advancements in standard-of-care therapies, including surgery, radiotherapy, and temozolomide (TMZ), the median survival remains approximately 15 months, with a 5-year survival rate of less than 10%. We and others have demonstrated that FOXM1 is a critical oncogenic driver of GBM cell proliferation. However, the role of FOXM1 and its interaction with other oncogenic signaling pathways in GBM remains incompletely understood. In this study, we identified FOXM1, AXL, and eEF2K as highly upregulated oncogenes in GBM patient tumors. We demonstrated, for the first time, that FOXM1 directly interacts with AXL and eEF2K, regulating their expression and promoting GBM cell proliferation, migration, and invasion. Knockdown of these genes disrupted cell proliferation, spheroid formation, migration, and invasion, and induced apoptosis and ferroptosis. Additionally, inhibiting the FOXM1–AXL/eEF2K signaling axis sensitized GBM cells to TMZ, further enhancing apoptotic and ferroptotic responses. These findings highlight the critical role of the FOXM1–AXL/eEF2K signaling pathway in GBM progression and suggest that targeting this axis may offer a novel multitargeted therapeutic strategy in GBM.

AIM:In this study, new boron-containing carbamate compounds were synthesized and evaluated as potential acetylcholinesterase (AChE) inhibitors by in vitro and in silico analyses. MATERIALS & METHODS:The structures were characterized by spectroscopic analysis including 1H NMR, 13C NMR, 11B NMR, and MS. The purities of the compounds were determined by HPLC analysis. In vitro and in silico analyses were performed. RESULTS:Based on our findings, compounds (1-4) demonstrated more potent AChE inhibitory activity compared to tacrine, which is an FDA-approved AChE inhibitor. Compound 4 had the highest inhibitory activity with an IC50 of 37.87 ± 0.96 nM and was more effective than tacrine (74.23 ± 0.83 nM). Compounds 1, 2, and 3, respectively, showed 1.78-, 1.73-, and 1.58-fold more potent enzyme inhibition activity compared to tacrine. The strong interactions with critical residues in the binding pocket of AChE were identified between protein and the compounds. Furthermore, compound 4 exerted an antiproliferative activity against various human cancer cell lines (32.91 ± 4.92 µM in HT29 and 42.38 ± 2.73 µM in MCF-7). CONCLUSION:Our study indicates the discovery of new boron-containing AChE inhibitors as potential candidates for the treatment of Alzheimer's disease and cancer.

Goal: In this research, we investigated the changes in elasticity of in vitro glioblastoma multiforme (GBM) spheroids when treated with the gold standard chemotherapy for GBM, Temozolomide (TMZ). Additionally, we aimed to use this alternative biomarker to assess how modifying the tumor microenvironment (TME) with the addition of human astrocytes (HA) would influence treatment efficacy. Methods: Spheroid stiffness was investigated using advanced non-invasive optical techniques, nanobomb optical coherence elastography (nb-OCE) and Brillouin microscopy to obtain new biomechanical insights by assessing local tumor progression or response to therapy using GBM cells (LN229). Results: The treated monocultured GBM groups showed a significant decrease in stiffness and increased sensitivity to treatment with TMZ. Treated HA groups across approaches remained relatively unchanged in stiffness. Treated co-culture groups demonstrated significant resistance to treatment with TMZ, where stiffness decreased less than that of the treated LN229 cells. Conclusions: These results confirm earlier findings using cell viability as a biomarker for treatment efficacy, making nb-OCE and Brillouin promising options to probe 3D tumor models in vitro non-invasively.

Background Adenomyosis is a common gynaecological condition where ectopic endometrial glands and stroma grow within the myometrium. This condition has a high clinical burden impacting those afflicted with debilitating symptoms including heavy painful periods. Simvastatin is an oral hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, typically used to treat hyperlipidaemia. Simvastatin has recently shown promise for treating gynaecological conditions such as endometriosis and uterine fibroids with nanoliposomal formulations demonstrating improved efficacy. In this pilot study, we tested simvastatin-loaded liposomal nanoparticles on xenografted adenomyosis tissues in a patient-derived mouse model.Methods We surgically inserted oestrogen/progesterone pellets into mice, followed by adenomyosis tissue xenografts 15 days later. Mice were then randomised into three groups: control, simvastatin, and simvastatin-loaded liposomal nanoparticles (simvastatin-NP). We quantified the changes in adenomyosis xenograft size weekly using a calliper as well as ultrasound imaging 28 days after treatment, prior to sacrifice. We also measured the proliferation of biomarker Ki67 in the xenografted tissues using immunohistochemistry after animal sacrifice.Results Treatment with simvastatin-NP significantly reduced volume and weight of adenomyosis xenografts while attenuating Ki67 expression when compared to the control and simvastatin groups. Conclusions: This pilot study demonstrates promising improved efficacy of simvastatin delivered via liposomal nanoparticles. However, larger studies are needed to fully explore the potential of simvastatin-NP in adenomyosis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with an average survival time of only six months following diagnosis, even with currently available therapies. Thus, PDAC represents a significant therapeutic challenge, necessitating a deeper understanding of its biology and tumor microenvironment (TME) to develop more effective treatments and improve patient outcomes. Here, we report that the expression of Eukaryotic Elongation Factor-2 Kinase (eEF2K) is associated with shorter patient survival and demonstrate that eEF2K signaling is critical for the PDAC tumor growth and regulated by the TME. Furthermore, in vivo targeted genetic inhibition of eEF2K suppressed tumor growth in two different PDAC mouse models, reduced tumor-associated macrophages (TAMs), and induced marked apoptosis in tumor tissues without any signs of toxicity. Our data suggest that eEF2K knockdown diminishes the activity of the AXL receptor tyrosine kinase and reduces the expression of macrophage-derived factors, such as Monocyte Chemoattractant Protein-1 (MCP1), along with the Gas6/AXL signaling pathway in PDAC cells. Additionally, analysis of the NCI-TCGA PDAC patient database further showed that eEF2K expression, in the presence of TAM markers, correlates with even shorter patient survival. TAM-released factors, such as MCP1, Gas6, and exosomes, induce eEF2K expression in PDAC cells, as well as the activity of AXL, SRC, VEGF, Snail, and MMP2, contributing to epithelial-to-mesenchymal transition (EMT), invasion, metastasis, and angiogenesis. In conclusion, our findings reveal for the first time that eEF2K is a critical oncogenic driver of PDAC tumor growth and thus targeting eEF2K represents a promising and novel therapeutic strategy for PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most challenging cancers to treat, with current therapies providing a median survival of just six months. The intrinsic chemoresistance and genetic heterogeneity of PDAC tumors complicate the development of effective targeted therapies. Approximately 90% of PDAC cases harbor KRAS mutations, which drive tumor initiation and progression, making KRAS a critical therapeutic target. Although the FDA has approved two KRAS inhibitors (Sotorasib and Adagrasib) for lung cancer, these drugs specifically target the KRAS-G12C mutation, found in only 2% of PDAC cases. Most PDAC tumors carry other KRAS mutations, particularly KRAS-G12D, leaving patients with limited targeted treatment options. To address this, we synthesized 21 compounds and identified 3E as a novel pan-KRAS inhibitor targeting KRAS-G12D, KRAS-G12C, and KRAS-G12S. Kaplan-Meier analysis of the TCGA PDAC database (n=177, p=0.00036) showed that elevated KRAS expression correlates with shorter survival, highlighting KRAS's role in PDAC progression. In vitro, treatment with 3E inhibited KRAS signaling in PDAC cells (PANC1, Miapaca-2) and KPC transgenic cells, as indicated by p-ERK suppression. 3E significantly reduced cell proliferation, migration, and invasion. Clonogenic assays demonstrated that 3E was more effective than Adagrasib and Salirasib (a non-specific RAS inhibitor in Phase II trials) at inhibiting PDAC cell growth. Moreover, 3E showed synergy with gemcitabine, a first-line chemotherapeutic agent for PDAC, enhancing its antiproliferative effects. In vivo, systemic delivery of 3E encapsulated in single lipid nanoparticles (SLNP) delayed tumor growth in PDAC xenograft and KPC transgenic mouse models without toxicity, as shown by blood chemistry markers. Tumor analysis after 3E treatment revealed reduced intra-tumoral proliferation (Ki-67 by IHC) and increased apoptosis (TUNEL assay). These findings suggest that 3E is a promising therapeutic candidate for KRAS-driven PDAC and other cancers. Its superior efficacy over FDA-approved KRAS inhibitors and synergy with gemcitabine support its clinical potential as a novel treatment approach for this aggressive cancer. Pinar Atalay, Asli Ture, Nermin Kahraman, Izabela Fokt, Waldemar Priebe, Erdem Buyukbingol, Bulent Ozpolat. Identification of novel pan-KRAS mutation inhibitor shows significant efficacy in pancreatic cancer with no toxicity, offering hope beyond current therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6390.

Breast cancer remains the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among women. Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers, is a highly aggressive and lethal form of breast cancer (BC) and characterized by the lack of ER, PR, and HER2 receptor expression, early relapses, drug resistance, and the poorest survival outcomes among all BC subtypes. Currently, there are no effective targeted therapies for TNBC, highlighting the urgent need for novel treatment strategies. eEF2K is an atypical alpha kinase that has been shown to promote TNBC cell proliferation, migration, invasion, and tumor growth. We previously validated eEF2K for the first time as a potential molecular target in TNBC and demonstrated that genetic inhibition of eEF2K completely suppresses in vivo TNBC tumor growth in mice. Overall data suggests that eEF2K is a critical oncogenic driver of TNBC tumorigenesis. Cancer stem cells (CSCs), a small but specific group of cancer cells, play a significant role in relapses and resistance to therapeutics including chemo-drugs. CSCs initiate tumor formation, promote tumor growth and cause relapses with their self-renewal and self-proliferation properties. While TNBC is known for the high number of CSCs population, the role of eEF2K in CSCs is unknown. In this study, we report the critical role of eEF2K in TNBC CSCs survival and tumorigenesis and found that the inhibition of eEF2K by siRNA and a small molecule inhibitor that we developed dramatically suppressed CSCs growth, migration, invasion and spheroid formation as well as TNBC cell proliferation and tumor growth in mice multiple TNBC models. Conversely, eEF2K overexpression promoted sphere formation. Our Kaplan-Meier analysis of the Cancer Genome Atlas (TCGA) revealed that high expression of eEF2K and ALDH1a3, a stemness marker, is associated with shorter overall and relapse-free survival. We also found that eEF2K is highly overexpressed in the ALDH-positive TNBC stem cell population. Genetic knockdown and pharmacological inhibition of eEF2K significantly suppressed the expression of key stemness signaling molecules, including ALDH1a3, Notch1, and Notch2. Currently, we are targeting eEF2K in MDA-MB-231 and MDA-MB-436 TNBC orthotopic xenograft and 4T1 mouse mammary tumor models in syngeneic mice to further assess its impact on CSCs-driven tumorigenesis. Our findings suggest that eEF2K plays a pivotal role in promoting TNBC stemness through the modulation of key stemness pathways, including ALDH1a3, Notch1, and Notch2 and tumor growth. Targeting eEF2K may therefore be a promising therapeutic strategy to eradicate TNBC by targeting primary and CSCs populations. Ezgi Biltekin, Nermin Kahraman, Yasemin M.Akay, Gulperi Oktem, Metin Akay, Bulent Ozpolat. Targeting eukaryotic elongation factor 2 kinase (eEF2K) to combat cancer stem cells and tumorigenesis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer with 1.9 million new cases and the second leading cause of cancer-related deaths, with 935, 000 deaths reported in 2023. Despite advances in therapy, the 5-year survival rate for metastatic CRC remains as low as 14%, emphasizing the need for novel treatment strategies. Previously, we have validated that Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical alfa kinase as a potential molecular target that is commonly expressed in highly aggressive solid cancers such as triple-negative breast cancer, pancreatic, ovarian, and lung cancers and demonstrated that it acts as a key player in the survival, proliferation, invasion, and tumor growth of cancer cells and promoting cell. In this study, we investigated the role of eEF2K in colon cancer tumorigenesis and progression and its potential as a therapeutic target using a novel eEF2K small molecule inhibitor and siRNA-based knockdown studies. We found that eEF2K expression is highly expressed in CRC cell lines and eEF2K is associated with shorter patient survival and poor prognostic marker in CRC patients (NCI-TCGA patient database), inhibition of eEF2K in various CRC cell lines (HCT116, RKO, COLO320, and with different genetic alterations leads to suppression on cell proliferation, migration, and invasion. Western blot analysis confirmed that treatment with the siRNA and the eEF2K inhibitor reduced the activity of clinically significant signaling molecules including Src, FAK, and Cyclin D1 which are crucial for tumor growth and progression. In vivo, targeting of eEF2K by eEF2K siRNA encapsulated in iron oxide nanoparticles (IONPS) and our novel water soluble small-molecule eEF2K inhibitor suppressed CRC tumor growth of HTC116 and RKO tumors (flank) CRC tumor models in nude mice, with no observed toxicity. Analysis of tumor tissues by immunohistochemistry showed significant inhibition of the Ki-67 proliferation marker and induction of apoptosis by TUNEL staining. Our study provides the first evidence that eEF2K is a clinically significant potential molecular target and contributes to tumorigenesis and progression of CRC. Its effective inhibition is safe and can suppress tumor growth in preclinical CRC models. These promising results lay the foundation for further developing eEF2K-targeted therapies, offering a potential new approach for treating CRC. Nermin Kahraman, Ezgi Biltekin, Ogun Ali Gul, Goknur Kara, Izabela Fokt, M. Akay Yasemin, Waldemar Priebe, Metin Akay, Bulent Ozpolat. Inhibition of Eukaryotic elongation factor 2 kinase (eEF2K) disrupts key processes in colorectal cancer progression and tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6953.