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.

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 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.

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.

Glioblastoma multiforme (GBM) is a WHO grade IV glioma, that is characterized by its aggressive growth, high invasiveness, and poor clinical outcomes. Despite the current standard of care treatments -surgical resection, radiotherapy, and first-line chemotherapy with temozolomide- the median survival remains only 12–17 months, with a 5-year survival rate of approximately 7%. GBM tumors defined by significant molecular heterogeneity, elevated mutational burden, and presence of therapy-resistant cancer stem cells. Thus, there is an urgent need for innovative, multi-targeted therapeutic strategies. Recent research highlighted the potential of non-coding RNAs, particularly microRNAs, to regulate multiple oncogenic pathways simultaneously. Our analyses of the Rembrandt patient dataset identified three significantly overexpressed key oncogenic signaling molecules (eEF2K, AXL, and FOXM1) in GBM patient tumors and in GBM cell lines. To therapeutically target these drivers, we utilized miRNA-Target gene prediction algorithms and identified three candidate miRNAs with predicted direct binding affinity. We demonstrated, for the first time, that miR-329-5p, miR-449-5p and miR-518c negatively regulate eEF2K, AXL, FOXM1 oncogenes and effectively downregulate their expression. Furthermore, we performed colony formation, wound healing(scratch), invasion(transwell) and 3D spheroid formation assay to evaluate the effects of the candidate miRNAs on cellular proliferation, metastatic potential, and stemness characteristics of GBM. Simultaneously or combined targeting of eEF2K, AXL, FOXM1 oncogenic molecules by each of the miRNAs(miR-329-5p, miR-449-5p, or miR-518c) led to suppression of GBM cell proliferation, migration, invasion, spheroid formation and induced both apoptosis and ferroptosis. Importantly, the combination of these miRNAs with temozolomide produced synergistic anti-proliferative effects in vitro. Moreover, in vivo once a week systemic treatment with miR-329-3p(0.15 mg/kg) incorporated in single lipid nanoparticle significantly suppressed eEF2K, AXL, FOXM1 expression and tumor growth in LN229 GBM xenograft tumor model in mice. These findings support the therapeutic potential of miRNA-based interventions as a multi-targeting strategy, either as monotherapy or in combination with current chemotherapeutics, for more effective management of glioblastoma.

Triple-negative breast cancer (TNBC) accounts for ∼15-20% of breast cancer cases (52, 000 women in the US only), is characterized by lack of estrogen, progesterone, and HER2 receptor expression; aggressive phenotype, high rates of early relapse and metastasis and the worst mortality rates among all breast cancer subtypes. TNBC is insensitive to the available targeted therapies, including anti-estrogens (i.e, tamoxifen, aromatase inhibitors), anti-HER2 and EGFR antibodies due to lack of actionable targets. Unfortunately, about 50-70% of the patients cannot achieve complete pathological remission in response to chemotherapy and die within the first 5 years after diagnosis. Therefore, there is a critical need for the development of highly effective and novel therapies to improve patient survival. We previously validated for the first time FOXM1 protooncogenic transcription factor as a potential molecular target in TNBC through genetic knockdown studies and demonstrated that genetic inhibition of FOXM1 completely blocks TNBC tumor growth in multiple tumor models in mice with no detected toxicity. We also found that FOXM1 expression is associated with dramatically shorter patient survival and identified it as a poor prognostic marker. We demonstrated that FOXM1 drives tumorigenesis, progression by inducing cell proliferation, invasion/metastasis, drug resistance by transactivating expression of multiple oncogenes. We found that FOXM1 is broadly overexpressed in TNBC tumors due to widespread p53 mutations which are detected in about 84% of TNBC patients, suggesting that TP53 mutations are the major driver of FOXM1 overexpression. Overall data suggest that FOXM1 is an excellent potential molecular target for TNBC. However, currently, there is no FDA approved inhibitor of FOXM1. To identify potential inhibitors, utilizing silico docking and molecular dynamics studies we screened the chemical libraries and found identified a novel which interacted with FOXM1 protein based on in silico molecular dynamics studies and demonstrated that this inhibitor suppresses FOXM1 expression and cell proliferation, migration, invasion, and induces apoptosis in TNBC cells and more potent that well known FOXM1 inhibitor (FDI-6). In vivo treatment mice bearing MDA-MB-231 tumors with novel FOXM1 inhibitor (20mg/kg, twice a week) incorporated in single-lipid nanoparticles suppressed growth of TNBC tumor xenografts in mice with no toxicity. In conclusion, the findings indicate that the novel FOXM1 inhibitor represents a potent and safe therapeutic agent with significant potential for the treatment of TNBC and other FOXM1-driven cancers. This discovery offers new hope for improving outcomes in a disease that currently has limited treatment options. Sayra Dilmac, Nermin Kahraman, Ogun Ali Gul, Ferah Comert Onder, Bulent Ozpolat. A novel FOXM1 inhibitor inhibits FOXM1 cell proliferation, migration, and invasion tumor growth 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 1753.

Abstract Triple-negative breast cancer (TNBC) represent 15-20% of all breast cancers and is the subtype of breast cancer that is not responsive to targeted breast cancer treatments such as anti-estrogens (tamoxifen) or HER2 targeted antibodies due to the lack of ER, PR, and HER2 receptors. TNBC is associated with poor prognosis, early relapse, and distant metastasis and drug resistance. With the use of standard chemotherapeutics in the neoadjuvant setting, unfortunately the majority of patients (~60%) with TNBC do not achieve complete remission and 40% of the patients with regional (Stage II) and 90% of the patients with distant metastasis (Stage IV) die within 5-years. Currently, there is no effective targeted therapy for TNBC patients for targeting oncogenic pathways. Although recently the FDA approved sacituzumab (govitecan), a Trop-2-receptor directed antibody conjugated with chemotherapeutic agent topoisomerase inhibitor in metastatic TNBC, and response rate was 30% and the median duration of response is 7.7 months, and the majority of patients did not maintain response longer than 12 months. Therefore, identifying novel molecular targets and developing alternative therapeutic strategies are urgently needed. FOXM1 is a protooncogenic transcription factor suppressed by p53 and drives expression of genes, which play a crucial role in promoting cancer cell proliferation, metastasis, progression, tumorigenesis of TNBC. We previously demonstrated for the first time that in vivo targeting of FOXM1 suppresses TNBC tumor growth in mice. Here, analyzing TCGA patient database we found that FOXM1 expression correlates with shorter patient survival and prognosis in TNBC patients. Overall data suggest that FOXM1 is a potential molecular target. However, currently, there is no FDA approved inhibitor of FOXM1. To identify potential inhibitors, utilizing silico docking and molecular dynamics studies we screened the FDA-approved compounds found that flavopiridol interacts with FOXM1. Flavopiridol is a small molecule inhibitor for CDKs and is approved by the FDA for the treatment of Acute Myeloid Leukemia. We demonstrated that flavodilol inhibits FOXM1 expression at nanomolar concentration and cell proliferation, migration, invasion, and induces apoptosis in TNBC cells. We are currently testing favopridol in MDA-MB-231 (human) and 4T1 mouse mammary) TNBC tumor models. In conclusion, our studies suggest that flavopiridol is a highly potent FOXM1 inhibitor and is a promising agent for repurposing to target FOXM1 and for the treatment of TNBC. Citation Format: Sayra Dilmac, Nermin Kahraman, Ferah Comert Onder, Zuhal Hamurcu, Bulent Ozpolat. Flavopiridol inhibits cell proliferation, migration, and invasion via downregulation of FOXM1 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 642.

Abstract Triple-negative Breast cancer (TNBC) is characterized by the absence of ER, PR, and HER-2 receptors and is associated with poor prognosis, early relapse, and distant metastasis. In neoadjuvant setting, most patients (~50-70%) with TNBC do not achieve complete remission. Currently, there is no effective targeted therapy for TNBC patients. Therefore, identifying novel molecular targets and developing alternative therapeutic strategies are urgently needed. PIM3 is a serine/threonine kinase and is upregulated in leukemia and various solid cancers. Although PIM1 has been shown to play a critical role in cell proliferation in cMyc-enriched TNBC tumors, the role of PIM3 in TNBC is not known. To determine the clinical significance of PIM3 in TNBC patient prognosis and progression, we performed a Kaplan-Meier analysis in the TCGA TNBC patient database. We found that PIM3 expression is significantly associated with shorter patient survival in TNBC patients (n=77, p=0.0088). PIM3 protein is highly expressed in TNBC cells compared to normal breast epithelium and other ER+ and HER2+ breast cancer subtypes. Inhibition of PIM3 by siRNA significantly suppressed TNBC cell proliferation, migration, and invasion of TNBC cells including MDA-MB-231, MDA-MB-436, and BT20 cells. Lenti-based PIM3 overexpression induced TNBC cell proliferation, invasion, and migration. Non-biased reverse phase protein array (RPPA) assay and Western blot analysis showed that PIM3 inhibition markedly reduced the expression of EF2 Kinase protein, and we found it to be associated with shorter patient survival and poor prognosis. Immunoprecipitation of PIM3 indicated that it forms a heterodimer with EF2K whose inhibition by siRNA also suppressed TNBC cell proliferation, migration, invasion, and tumor growth. Furthermore, PIM3 inhibition by siRNA significantly enhanced the antiproliferative effect of Doxorubicin, which is one of the first-line chemotherapeutics used in patients. Moreover, in vivo therapeutic delivery of PIM3 siRNA loaded albumin nanoparticles (ALNPs-PIM3 siRNA) markedly delayed tumor growth of two different TNBC tumor xenograft models (MDA-MB-231 and MDA-MB-436) in mice. Analysis of tumors by IHC after 4 weeks of treatment demonstrated that ALNP-PIM3 siRNA treatment led to inhibition of intra-tumoral proliferation and induction of apoptosis. Currently, we are conducting an in vivo study in TNBC tumor models by combining ALNP-PIM3 siRNA therapy with doxorubicin. Overall, our studies suggest that PIM3 is a marker for poor patient survival that drives TNBC tumor growth and progression and a novel potential molecular target in TNBC. Citation Format: Pinar Atalay, Goknur Kara, Rumeysa Ozyurt, Nermin Kahraman, Bulent Ozpolat. Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 650.

Abstract Triple-negative breast cancer (TNBC) represents 20% of breast cancer (BC) patients (51,000 cases/year in the US). It is associated with a highly aggressive course, early metastasis and relapses, and drug-resistant phenotype. About 40% of the patients die within 5 years despite available therapies. If the disease is metastatic, patients survive only 13 months. Since TNBC does not express targetable receptors such as ER, PR, and HER2, and it is a highly heterogeneous cancer with six genetically defined sub-types, currently, there are no common actionable molecular targets and no effective targeted therapies for TNBC. microRNAs (miRNAs) are 18-20 nt long non-coding RNAs and are often dysregulated in cancer cells. Tumor-suppressive miRNAs specifically silence their protooncogenic target genes (mRNAs) by binding specifically to their 3’-UTRs located in their target mRNAs, leading to inhibition of oncogenes that play critical roles in cell proliferation, cell cycle, migration, invasion, angiogenesis, drug resistance, tumor growth, and progression. We have previously demonstrated that Eukaryotic Elongation factor-2 kinase (eEF2K) and AXL are oncogenic kinases, and their higher expression is associated with poor survival in patients with TNBC. However, currently, there is no FDA-approved eEF2K and AXL-targeted therapeutics. To effectively co-target these kinases, we extensively analyzed miRNA databases using miRNA target prediction algorithms and recently, we discovered that miR-329-3p has a specific binding site at both 3’-UTRs of eEF2K and AXL mRNAs. Analyzing the TCGA TNBC database, we found that low expression of miR-329-3p is correlated with shorter overall survival in TNBC patients, and the expression of miR-329-3p is commonly reduced or lost in TNBC patient tumors. Since the successful clinical application of miRNA-based therapeutics requires safe and effective nano-delivery systems, we have developed magnetic iron-oxide-based nanoparticles (MNPs) for the delivery of miR-329-3p. We showed that miR-329-3p significantly suppresses cell proliferation, invasion, and migration by targeting both eEF2K and AXL and their downstream mediators such as SRC/FAK and Cyclin D1 in TNBC cell lines. In vivo systemic treatment with MNPs-miR-329 completely blocks tumor growth in orthotopic MDA-MB-231 and MDA-MB-436 TNBC xenograft models in mice. Analysis of tumors shows remarkable inhibition in eEF2K and AXL expression, and clinically significant downstream targets including SRC, FAK, and Cyclin D1. We also demonstrated that miR-329 nanotherapy induces apoptosis and inhibits intratumoral proliferation in TNBC tumors in mice. Toxicity markers in mouse blood samples show that miR-329 nanotherapy is safe and not toxic to mice. Taken together, our study suggests that miR-329-3p nanotherapy may be used as a novel potential therapeutic approach in TNBC patients. Citation Format: Goknur Kara, Pinar Atalay Dundar, Nermin Kahraman, Emir Baki Denkbas, Bulent Ozpolat. Dual-kinase targeted miRNA nanotherapy for the treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 461.

Abstract Purpose: Zoledronic acid is being increasingly recognized for its antitumor properties, but the underlying functions are not well understood. In this study, we hypothesized that zoledronic acid inhibits ovarian cancer angiogenesis preventing Rac1 activation. Experimental Design: The biologic effects of zoledronic acid were examined using a series of in vitro [cell invasion, cytokine production, Rac1 activation, reverse-phase protein array, and in vivo (orthotopic mouse models)] experiments. Results: There was significant inhibition of ovarian cancer (HeyA8-MDR and OVCAR-5) cell invasion as well as reduced production of proangiogenic cytokines in response to zoledronic acid treatment. Furthermore, zoledronic acid inactivated Rac1 and decreased the levels of Pak1/p38/matrix metalloproteinase-2 in ovarian cancer cells. In vivo, zoledronic acid reduced tumor growth, angiogenesis, and cell proliferation and inactivated Rac1 in both HeyA8-MDR and OVCAR-5 models. These in vivo antitumor effects were enhanced in both models when zoledronic acid was combined with nab-paclitaxel. Conclusions: Zoledronic acid has robust antitumor and antiangiogenic activity and merits further clinical development as ovarian cancer treatment. Clin Cancer Res; 21(9); 2127–37. ©2015 AACR.

The effects of miR-34a expression on eEF2K and its downstream targets in TNBC cells.

Ectopic expression of miR-34a increases relative miR-34a expression levels in control lentiviral and FOXM1 lentiviral transduced TNBC cells.

Schematic model of the regulatory pathway involving miR-34a in triple-negative breast cancer.